Journal articles on the topic 'Site-specific labeling of antigens and antibodies'
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1
Sapozhnikova, Ksenia A., Evgeny L. Gulyak, Vsevolod A. Misyurin, Maria A. Simonova, Ekaterina V. Ryabukhina, Anastasiya V. Alexeeva, Nataliya A. Tikhonova, et al. "Branched Linkers for Site-Specific Fluorescent Labeling of Antibodies." Molecules 28, no. 1 (January 3, 2023): 425. http://dx.doi.org/10.3390/molecules28010425.
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Fluorescent antibodies have proved to be an invaluable tool for molecular biology and diagnostics. They are routinely produced by modification of lysine residues, which leads to high heterogeneity. As such, their affinity may be compromised if the antigen-binding site is affected, the probability of which increases along with the degree of labeling. In this work, we propose a methodology for the synthesis of site-specific antibody-dye conjugates with a high degree of labeling. To this end, we synthesized two oxyamine-based branched triazide linkers and coupled them with a periodate-oxidized anti-PRAME antibody 6H8; two oxyamine-based linear monoazide linkers of similar structure were used as controls. The azide-labeled antibodies were subsequently conjugated with fluorescent dyes via SPAAC, a copper-free click reaction. Compared to their counterparts made with linear linkers, the branched conjugates possessed a higher degree of labeling. The utility of the methodology was demonstrated in the detection of the PRAME protein on the surface of the cell by flow cytometry.
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Alonso, G., and P. Siaud. "Combined use of immunoperoxidase and radioimmunocytochemistry for double immunocytochemical labeling of neurons at light and electron microscopic level." Journal of Histochemistry & Cytochemistry 37, no. 12 (December 1989): 1799–809. http://dx.doi.org/10.1177/37.12.2573630.
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Complexes formed by binding 125I- or 3H-labeled neuropeptides to one of the two binding sites of their specific antibodies allowed specific and sensitive labeling of various peptidergic neurons, which could be detected by classical autoradiographic methods. To visualize two neuronal antigens on the same material at both light and electron microscopic level, we used a new technique of double immunocytochemical labeling, combining immunoperoxidase and radioimmunocytochemistry. The main steps of the process included: (a) indirect labeling of the first antigen by its specific antibody and by a peroxidase-labeled Fab immunoglobulin fragment directed against the primary antibody; (b) direct labeling of the second antigen by a radiolabeled peptide-antibody complex; (c) revealing of the first label in the presence of peroxidase substrate; and (d) revealing of the second label by autoradiographic treatment of tissue sections. Compared with other known techniques of double immunostaining, this technique offers major advantages for combined visualization of two neuronal antigens at the electron microscopic level: (a) two neuron types can be labeled by a pre-embedding approach, allowing highly sensitive detection of neuronal antigens throughout the 50-microns thickness of vibratome sections; (b) two primary antibodies obtained in the same species can be used to label the two antigens without any risk of crossreactions between the two successive labelings; and (c) the two labels can easily be differentiated, even when they are co-localized within the same neuron structures. Application of this double immunostaining technique is illustrated by data obtained in rat hypothalamus concerning the relationships among a variety of identified neurons and the co-localization of different neuropeptides within the same neuron system.
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Bendayan, M., and S. Garzon. "Protein G-gold complex: comparative evaluation with protein A-gold for high-resolution immunocytochemistry." Journal of Histochemistry & Cytochemistry 36, no. 6 (June 1988): 597–607. http://dx.doi.org/10.1177/36.6.2452843.
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We combined the protein G-gold complex with several polyclonal and monoclonal antibodies for localization of various antigenic sites. The labelings were compared with those obtained using the protein A-gold complex. The results from either the immunodot experiment or immunoelectron microscopy have demonstrated that, for rabbit and guinea pig antibodies, both protein G-gold and protein A-gold complexes label several different specific antibodies with similar efficiency. However, with antibodies raised in goats or in mice, and particularly with mouse monoclonal antibodies, protein G-gold yielded intense and specific labeling, whereas protein A-gold yielded intense and specific labeling, whereas protein A-gold was very variable; it either gave weaker signals or failed to reveal any specific site or, as with one monoclonal, both protein G and protein A gave similar results. The higher affinity and versatility of protein G over protein A, established by the immunochemical approach, was confirmed by immunocytochemistry. Because of its enhanced reactivity with monoclonal antibodies and its broader affinity for polyclonal antibodies, protein G-gold complex appears to be a better and more versatile probe for high-resolution immunocytochemistry.
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MARTÍN, ROSARIO, JUAN I. AZCONA, CARMEN CASAS, PABLO E. HERNÁNDEZ, and BERNABÉ SANZ. "Sandwich ELISA for Detection of Pig Meat in Raw Beef Using Antisera to Muscle Soluble Proteins." Journal of Food Protection 51, no. 10 (October 1, 1988): 790–98. http://dx.doi.org/10.4315/0362-028x-51.10.790.
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A double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) has been successfully developed for the detection of defined amounts of pig meat (1–50%) in raw beef. Antibodies against pig sarcoplasmic extracts were produced in rabbits. Pig-specific antibodies were affinity purified by removing antibodies which crossreacted with horse, chicken or beef extracts followed by immunoadsorption and elution from a pig-extract column. The ELISA involved capturing antigens in sarcoplasmic extracts with pig specific antibodies immobilized on 96-well plates, detecting bound antigen with pig specific, horseradish peroxidase-labeling antibody, and measuring peroxidase activity by the conversion of a clear substrate to a colored product.
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Foa, C., P. Bongrand, J. R. Galindo, and P. Golstein. "Unexpected cell surface labeling in conjugates between cytotoxic T lymphocytes and target cells." Journal of Histochemistry & Cytochemistry 33, no. 7 (July 1985): 647–54. http://dx.doi.org/10.1177/33.7.2861226.
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Specific binding of target cells by cytotoxic T lymphocytes (CTL) is an example of tight interaction between two different cell types. The molecular events that occur at the cell membranes during these interactions are largely unknown. In the present report, we describe an electron microscopic immunostaining study made on CTL-target cell conjugates. Various membrane structures were labeled with monoclonal antibodies specific for structures possibly relevant to cytolysis (Lyt-2, LFA-1, and target cell class I major histocompatibility antigens) or probably unrelated to the cytolytic process (effector cell class I major histocompatibility antigens). Antibodies against Thy-1 were also used. Staining was achieved with immunoperoxidase or immunoferritin. With both techniques nonconjugated cells were either stained or not, depending on whether they bore the antigen corresponding to the antibody used. However, when conjugated to an antigen-bearing cell, a "non-antigen bearing" cell was labeled near the cell interaction area. No increased Fc receptor activity could be detected on bound cells near the interaction area. These data are consistent with the occurrence of limited exchange of membrane macromolecules between bound CTL and target cell.
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Pesando, JM, P. Hoffman, N. Martin, and T. Conrad. "Anti-CALLA antibodies identify unique antigens on lymphoid cells and granulocytes." Blood 67, no. 3 (March 1, 1986): 588–91. http://dx.doi.org/10.1182/blood.v67.3.588.588.
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Abstract The common acute lymphoblastic leukemia antigen (CALLA) is a 100-kd surface glycoprotein that is present on normal and malignant lymphoid cells. It is a useful marker for distinguishing between clinically important types of acute leukemia. Anti-CALLA monoclonal antibodies (MoAb) also react with mature myeloid cells (granulocytes), where they identify an antigen having a similar molecular weight (mol wt). We now report that the antigens detected by anti-CALLA MoAb on human lymphoid and myeloid cells differ in their behavior and chemistry. Surface- labeling studies indicate that the antigen on lymphoid cells has a mol wt of approximately 100 kd v 110 kd for that on granulocytes. When cells are metabolically labeled with 35S-methionine, differences in the mol wt of these antigens are again observed. Unlike the lymphoid antigen, expression of that on purified granulocytes is not modulated by incubation with specific antibody. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis analysis of proteolytic digests of the two antigens fails to clarify their chemical relationship. Thus the antigens detected on these two cell types may share an epitope(s) but be chemically distinct, or CALLA may exist in distinct forms and behave differently on lymphoid cells and granulocytes.
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Pesando, JM, P. Hoffman, N. Martin, and T. Conrad. "Anti-CALLA antibodies identify unique antigens on lymphoid cells and granulocytes." Blood 67, no. 3 (March 1, 1986): 588–91. http://dx.doi.org/10.1182/blood.v67.3.588.bloodjournal673588.
Full textAbstract:
The common acute lymphoblastic leukemia antigen (CALLA) is a 100-kd surface glycoprotein that is present on normal and malignant lymphoid cells. It is a useful marker for distinguishing between clinically important types of acute leukemia. Anti-CALLA monoclonal antibodies (MoAb) also react with mature myeloid cells (granulocytes), where they identify an antigen having a similar molecular weight (mol wt). We now report that the antigens detected by anti-CALLA MoAb on human lymphoid and myeloid cells differ in their behavior and chemistry. Surface- labeling studies indicate that the antigen on lymphoid cells has a mol wt of approximately 100 kd v 110 kd for that on granulocytes. When cells are metabolically labeled with 35S-methionine, differences in the mol wt of these antigens are again observed. Unlike the lymphoid antigen, expression of that on purified granulocytes is not modulated by incubation with specific antibody. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis analysis of proteolytic digests of the two antigens fails to clarify their chemical relationship. Thus the antigens detected on these two cell types may share an epitope(s) but be chemically distinct, or CALLA may exist in distinct forms and behave differently on lymphoid cells and granulocytes.
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Haftek, M., M. J. Staquet, J. Viac, D. Schmitt, and J. Thivolet. "Immunogold labeling of keratin filaments in normal human epidermal cells with two anti-keratin monoclonal antibodies." Journal of Histochemistry & Cytochemistry 34, no. 5 (May 1986): 613–18. http://dx.doi.org/10.1177/34.5.2422248.
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We report on application of the highly sensitive and specific immunogold labeling method for ultrastructural investigation of keratin intermediate filament antigens in human epidermal cell suspensions. Triton X-100 pretreated cells proved accessible to the colloidal gold conjugate, thus enabling keratin filament bundles to be labeled. Anti-keratin KL1 and KL2 monoclonal antibodies were raised in mice after immunization with either human stratum corneum-isolated keratins or keratins extracted from human epidermal cells suspensions, respectively. Immunoelectron microscopy confirmed immunofluorescence and immunoperoxidase results of epidermal keratinocyte staining, and revealed two different antibody reactivity patterns: KL2 reacted with keratin filaments in keratinocytes of all epidermal layers, whereas antigen to KL1 was detected only on keratin of the suprabasal layers, not on the basal keratinocyte tonofilaments. The monoclonal antibody-recognized epitopes were specific for the keratin filaments. Vimentin-rich cells (melanocytes) were not stained in the same epidermal cell suspensions. Additionally, two distinct ultrastructural patterns of keratin filament epitope labeling were observed. KL1 and KL2 monoclonal antibodies react with two different antigenic determinants, depending on the stage of keratinocyte differentiation, and may therefore be used for immunohistochemical studies of various keratin-containing cells in normal and pathologic conditions.
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De Waele, M., W. Renmans, E. Segers, K. Jochmans, and B. Van Camp. "Sensitive detection of immunogold-silver staining with darkfield and epi-polarization microscopy." Journal of Histochemistry & Cytochemistry 36, no. 6 (June 1988): 679–83. http://dx.doi.org/10.1177/36.6.3259250.
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We evaluated the contribution of darkfield and epi-polarization microscopy to the detection of leukocyte cell surface antigens with immunogold-silver staining (IGSS). Lymphocyte cell surface differentiation antigens were labeled with monoclonal antibodies and IGSS as described for brightfield microscopy. In darkfield and epi-polarization microscopy the labeling appeared as bright spots on a dark background. The sensitivity of detection was much higher than that of brightfield microscopy. Sixteenfold higher dilutions of the monoclonal antibody could be used to detect all cells expressing the antigen in the cell suspension. However, non-specific staining was also better visualized. The latter could be reduced to a level comparable to that of brightfield microscopy only by use of weaker labeling conditions. A 25% reduction of the silver enhancement time was necessary for this purpose. However, these weaker labeling conditions also reduced the intensity of the specific staining. Therefore, the efficiency of IGSS, as detected with darkfield and epi-polarization microscopy, was only fourfold greater than that found with brightfield microscopy or that of an immunofluorescence procedure. Especially in combination with transmitted light, to improve cell identification, epi-polarization microscopy is a reliable and sensitive method for detection of immunogold-silver-labeled cell surface antigens for diagnostic and research purposes.
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Reed, S. G., R. Badaro, and R. M. Lloyd. "Identification of specific and cross-reactive antigens of Leishmania donovani chagasi by human infection sera." Journal of Immunology 138, no. 5 (March 1, 1987): 1596–601. http://dx.doi.org/10.4049/jimmunol.138.5.1596.
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Abstract Cloned Leishmania donovani chagasi (Ldc) promastigotes were analyzed by SDS-PAGE separation and immunoblotting with human infection sera. The patterns of antigen reactivity were compared by using sera from individuals with Ldc, Leishmania mexicana amazonensis (Lma), Trypanosoma cruzi, Mycobacterium tuberculosis, or Mycobacterium leprae infections. Sera from individuals with these infections recognized Ldc antigens in several m.w. ranges. Reactivity was due to recognition of Ldc molecules and not to Ldc culture medium components, as shown by comparing Ldc promastigotes grown in the presence or absence of fetal bovine serum (FBS), by immunoblotting of FBS, and by [35S]methionine labeling. The major findings of the study were as follows. Immunoblots with Ldc promastigotes could be used to distinguish individuals with Ldc infections from those with Lma infections. Persons with Ldc infections had antibodies to a Ldc antigen of approximately 32 to 35 kd not recognized by persons with Lma infections. Individuals cured of acute Ldc infection did not develop antibodies that differed in specificity to those present during their acute phase of infection. Ldc antigens in the 62 to 66 kd region were recognized by all individuals with Ldc or Lma infections but were not recognized by individuals in the other disease groups or by control sera. This region was found to contain at least four distinct bands, one of which appeared to be glycosylated as indicated by periodic acid-Schiff staining and concanavalin A labeling; an apparently nonglycosylated protein of 62 to 63 kd was eluted from SDS-PAGE gels and was used to diagnose Ldc infection by the ELISA. Whereas crude Ldc antigen gave false positive results with T. cruzi and mycobacteria infection sera, the eluted 62 to 63 kd protein was 100% specific and sensitive in the diagnosis of Ldc infection.
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D'Andrea, M., M. Forte, C. Chen, and J. Gu. "Quantitative correlation of immunoelectron microscopy and radioimmunoassay." Proceedings, annual meeting, Electron Microscopy Society of America 49 (August 1991): 312–13. http://dx.doi.org/10.1017/s0424820100085861.
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Indirect Immunogold electron microscopy has been widely used in biomedical research. It uses uniformly sized gold particles to label antibodies which can recognize specific tissue antigens on ultra thin sections. The method is superior to other immunoelectron microscopic procedures in that the number of good particles on antigenic sites can be counted and the results assessed comparatively. This method is not absolutely quantitative because the amounts of antigen molecules each labeling gold particle represents are not known. We developed an approach to bring a novel quantitative concept into immunoelectron microscopy (IEM) by correlating the results obtained by IEM and by radioimmunoassay (RIA). A peptide hormone, atrial natriuretic peptide (ANP) in the right atrium of the rat heart was chosen as the model because in this region this antigen is abundant and has a defined distribution, being exclusively in the specific cytoplasmic granules.
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Kurdekar, Aditya Dileep, L. A. Avinash Chunduri, C. Sai Manohar, Mohan Kumar Haleyurgirisetty, Indira K. Hewlett, and Kamisetti Venkataramaniah. "Streptavidin-conjugated gold nanoclusters as ultrasensitive fluorescent sensors for early diagnosis of HIV infection." Science Advances 4, no. 11 (November 2018): eaar6280. http://dx.doi.org/10.1126/sciadv.aar6280.
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We have engineered streptavidin-labeled fluorescent gold nanoclusters to develop a gold nanocluster immunoassay (GNCIA) for the early and sensitive detection of HIV infection. We performed computational simulations on the mechanism of interaction between the nanoclusters and the streptavidin protein via in silico studies and showed that gold nanoclusters enhance the binding to the protein, by enhancing interaction between the Au atoms and the specific active site residues, compared to other metal nanoclusters. We also evaluated the role of glutathione conjugation in binding to gold nanoclusters with streptavidin. As proof of concept, GNCIA achieved a sensitivity limit of detection of HIV-1 p24 antigen in clinical specimens of 5 pg/ml, with a detection range up to1000 pg/ml in a linear dose-dependent manner. GNCIA demonstrated a threefold higher sensitivity and specificity compared to enzyme-linked immunosorbent assay for the detection of HIV p24 antigen. The specificity of the immunoassay was 100% when tested with plasma samples negative for HIV-1 p24 antigen and positive for viruses such as hepatitis B virus, hepatitis C virus, and dengue. GNCIA could be developed into a universal labeling technology using the relevant capture and detector antibodies for the specific detection of antigens of various pathogens in the future.
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Lomueller, Jason Jakob, Adam Butchy, Yaniv Tivon, Michael Kvorjak, Natasa Miskov-Zivanov, Alexander Deiters, and Olivera J. Finn. "Covalent adaptor synNotch and chimeric antigen receptors (CARs) for programmable antigen targeting." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 71.18. http://dx.doi.org/10.4049/jimmunol.202.supp.71.18.
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Abstract Chimeric antigen receptors (CARs) are artificial T cell receptors that re-target patients’ T cells to specifically bind and kill tumor cells. Adoptive cell therapy with CAR T cells targeting CD19 has revolutionized treatment of refractory B cell acute lymphoblastic leukemia, and there is great interest in generating CAR T cells treating other cancers by targeting additional tumor antigens. Another promising class of engineered receptors are synthetic Notch (synNotch) receptors that can sense an antigen of interest on a neighboring cell and turn on expression of any transgene(s) of interest. To expand the targeting capabilities of these receptors, we have developed “universal” CAR and SynNotch receptors whose antigen-specificity can be re-directed by co-administered tumor-specific antibodies. Instead of directly targeting a tumor antigen, our universal receptors contain the SNAPtag self-labeling enzyme, which reacts with antibodies conjugated to benzylguanine (BG) to post-translationally assemble complete covalently associated antigen receptors. We demonstrate that the activation of SNAP CAR and SNAP-SynNotch receptors can be successfully re-targeted by several clinically relevant antibodies including: Rituximab, FMC63, Herceptin, and Cetuximab. SNAP-SynNotch cells demonstrate potent transgene activation, and SNAP-CAR T cells are capable of performing effector functions in a BG-antibody-directed antigen-specific manner. Additionally, the receptor response is titratable by BG-antibody dose. Finally, a continuous mathematical model was constructed to describe and optimize system activity. SNAP synNotch and SNAP CAR T cells provide a powerful new strategy to retarget engineered cells to multiple antigens.
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Carson, J. L., A. M. Collier, S. C. Hu, and J. B. McLachlan. "Variability in distribution and populations of gap junctions in ferret trachea during postnatal development." American Journal of Physiology-Lung Cellular and Molecular Physiology 268, no. 4 (April 1, 1995): L576—L583. http://dx.doi.org/10.1152/ajplung.1995.268.4.l576.
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Immunocytochemical probes have been used to characterize gap junction distribution in the postnatal ferret trachea by epifluorescence and by laser scanning confocal and electron microscopy. A battery of antibodies directed against fragments of different connexins localized beta 1- and beta 2-gap junction antigens (connexins 32 and 26, respectively) at the intercellular borders of the superficial epithelium while alpha 1-gap junction antigen (connexin 43) was localized to the loose connective tissues. Gap junction labeling in the superficial epithelium declined in the first weeks of life but persisted in the developing submucosal glands to the weanling stage. Localization of the alpha- and beta-antigens was specific for connective tissues and epithelial layers, respectively. These observations suggest that communication competence is an important component of early development in the mammalian airways.
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Fakan, S., G. Leser, and T. E. Martin. "Immunoelectron microscope visualization of nuclear ribonucleoprotein antigens within spread transcription complexes." Journal of Cell Biology 103, no. 4 (October 1, 1986): 1153–57. http://dx.doi.org/10.1083/jcb.103.4.1153.
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The ultrastructural distribution of nuclear ribonucleoproteins (RNP) within spread active chromatin has been investigated using specific anti-RNP antibodies. Monoclonal antibodies directed against the core proteins of heterogeneous nuclear (hn)RNP or against small nuclear (sn)RNP have been incubated directly with lysed mouse or Drosophila tissue culture cells and the bound antibodies visualized by means of a protein A-colloidal gold complex. The hnRNP core proteins have been localized on growing RNP fibrils within non-nucleolar transcription complexes. Anti-snRNP antibodies, directed either against the Sm-antigen (common for nucleoplasmic snRNP species containing U1, U2, U4, U5, and U6 RNAs) or against U1-snRNP, were bound by two morphological types of RNP structures. Within areas of chromatin that do not completely disperse, labeling was observed on RNP-fibril gradient type structures or on groups of fibrogranular material. In the well dispersed regions containing individual nonribosomal transcription complexes, snRNP antigens were associated with growing RNP fibrils. Our results provide direct evidence for association of some U-snRNP species (including U1-snRNP) with extranucleolar RNA as early as during transcription elongation. In addition, the presence of core hnRNP proteins on the same type of nascent RNA transcripts has been confirmed.
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Schmauder-Chock, E. A., and S. P. Chock. "Localization of cyclo-oxygenase and prostaglandin E2 in the secretory granule of the mast cell." Journal of Histochemistry & Cytochemistry 37, no. 9 (September 1989): 1319–28. http://dx.doi.org/10.1177/37.9.2504812.
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The application of anti-cyclo-oxygenase and anti-prostaglandin E2 immunoglobulins to A23187-stimulated rat connective tissue mast cells has permitted the localization of cyclooxygenase activity (prostaglandin H2 synthetase) and the site of prostaglandin E2 (PGE2) formation in the secretory granules. Because binding was carried out after stimulation but before dehydration and embedding, we have limited the loss of these antigens due to normal degradation and to aqueous and solvent washes. As this method permits labeling of exposed cell surfaces, only granules that have been exteriorized can be labeled. Contrary to what might have been expected, no labeling was associated with plasma membranes or with any portion of damaged cells. Antibodies to PGE2 were bound evenly over the surface of the granule matrix, whereas antibodies to cyclo-oxygenase appeared to be bound to strands of proteo-heparin projecting from the surface of the granule matrix. Where granule matrix had become unraveled and dispersed, label appeared to adhere throughout the ribbon-like proteo-heparin strands. These results support our previous conclusion that the secretory granule is the site of the arachidonic acid cascade during exocytosis.
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Pringle, G. A., and C. M. Dodd. "Immunoelectron microscopic localization of the core protein of decorin near the d and e bands of tendon collagen fibrils by use of monoclonal antibodies." Journal of Histochemistry & Cytochemistry 38, no. 10 (October 1990): 1405–11. http://dx.doi.org/10.1177/38.10.1698203.
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Two monoclonal antibodies, 6D6 and 7B1, previously shown to recognize different epitopes on different regions of the protein core of decorin were used to localize the protein core in relation to the positively stained bands in the D period of bovine tendon collagen fibrils. Peroxidase-antiperoxidase staining revealed that the antigen is associated with the surface of all fibrils and suggested that the axial distance between antigens is D-periodic. Immunoferritin labeling with each antibody produced a distribution of ferritin particles that showed that both epitopes of the protein core are localized near the d and e bands in the D period. The data indicate that the decorin protein core binding site(s) on tendon collagen fibrils is/are located near these bands, axially, within the D period.
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Howe, Daniel K., Amy C. Crawford, David Lindsay, and L. David Sibley. "The p29 and p35 Immunodominant Antigens ofNeospora caninum Tachyzoites Are Homologous to the Family of Surface Antigens of Toxoplasma gondii." Infection and Immunity 66, no. 11 (November 1, 1998): 5322–28. http://dx.doi.org/10.1128/iai.66.11.5322-5328.1998.
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ABSTRACT Neospora caninum is an apicomplexan parasite that is closely related to Toxoplasma gondii and has been found to be associated with neurological disorders in dogs and congenital infections and abortions in cattle. We have identified two surface proteins of 29 and 35 kDa (designated Ncp29 and Ncp35, respectively) from N. caninum tachyzoites that are the predominant antigens recognized by antisera from Neospora-infected animals. Monoclonal antibodies against Ncp29 and Ncp35 were used to analyze several independent and diverse N. caninumisolates; both antigens were recognized in all isolates, suggesting that they are well conserved. Localization studies and surface labeling with biotin demonstrated that Ncp29 and Ncp35 are membrane associated and displayed on the surface of the parasite. After treatment with phosphatidylinositol-specific phospholipase C, parasite lysates were analyzed with antibodies against the cross-reacting determinant of glycosylphosphatidylinositol anchors. Approximately six glycolipid-anchored surface proteins were identified, with the two most prominent corresponding to Ncp29 and Ncp35. Sequence comparisons of Ncp29 and Ncp35 with GenBank indicated that they are most similar to the T. gondii surface antigen 1 (SAG1) and surface antigen 1-related sequence 2 (SRS2), respectively. Consequently, Ncp29 has been designated NcSAG1 and Ncp35 has been designated NcSRS2. Both NcSAG1 and NcSRS2 contain a tandemly duplicated motif and 12 absolutely conserved cysteines which are also found in all of the SAG and SRS proteins ofT. gondii. Maintenance of these motifs and the 12 cysteine residues suggests that these surface antigens share a similar secondary and tertiary structure that is presumably important for a conserved function that these antigens serve during infection.
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Moreira, J. E., L. A. Tabak, G. S. Bedi, D. J. Culp, and A. R. Hand. "Light and electron microscopic immunolocalization of rat submandibular gland mucin glycoprotein and glutamine/glutamic acid-rich proteins." Journal of Histochemistry & Cytochemistry 37, no. 4 (April 1989): 515–28. http://dx.doi.org/10.1177/37.4.2926128.
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We studied the subcellular localization of two major secretory products of adult rat submandibular gland (RSMG), blood group A-reactive mucin glycoprotein and glutamine/glutamic acid-rich protein (GRP), by light and electron microscopic immunocytochemistry. The structure of the major neutral oligosaccharide of the mucin was shown to be: GalNAc alpha 1,3(Fuc alpha 1,2)Gal beta 1,3GalNAc. A mouse monoclonal antibody (1F9) with specificity for blood group A determinants was prepared against the mucin. The antibody recognized a single band of approximately 114 KD on Western blots of RSMG extract. A previously characterized monoclonal antibody (59) against GRP (Mirels et al.: J Biol Chem 262: 7289, 1987) reacted with a doublet of 45-50 KD on Western blots of extraparotid saliva. Immunofluorescence and immunoperoxidase staining of cryostat sections of RSMG with anti-mucin antibodies and anti-GRP antibodies revealed reactivity in acinar cells of the gland. No specific labeling was seen in duct cells of RSMG or in mucous acinar cells of the adjacent sublingual gland. Post-embedding immunogold labeling of thin sections of glutaraldehyde-fixed RSMG with anti-mucin showed strong labeling of the Golgi apparatus and secretory granules of acinar cells. Gold particles were seen mainly over electron-lucent areas of the granules. No labeling occurred over the endoplasmic reticulum. The labeling pattern with the anti-GRP antibodies was similar, except that both electron-dense and -lucent areas of the granules were labeled, and the endoplasmic reticulum was reactive. Double labeling with two different sizes of gold particles showed that both mucin and GRP co-localized in the same granules. Pre-absorption of the antibodies with their respective antigens eliminated immunolabeling of the acinar cells. These antibodies will be useful in studies of cell differentiation in RSMG and of synthesis, processing, and packaging of RSMG secretory products.
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Franz, Bettina, Kenneth F. May, Glenn Dranoff, and Kai Wucherpfennig. "Ex vivo characterization and isolation of rare memory B cells with antigen tetramers." Blood 118, no. 2 (July 14, 2011): 348–57. http://dx.doi.org/10.1182/blood-2011-03-341917.
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Abstract Studying human antigen-specific memory B cells has been challenging because of low frequencies in peripheral blood, slow proliferation, and lack of antibody secretion. Therefore, most studies have relied on conversion of memory B cells into antibody-secreting cells by in vitro culture. To facilitate direct ex vivo isolation, we generated fluorescent antigen tetramers for characterization of memory B cells by using tetanus toxoid as a model antigen. Brightly labeled memory B cells were identified even 4 years after last immunization, despite low frequencies ranging from 0.01% to 0.11% of class-switched memory B cells. A direct comparison of monomeric to tetrameric antigen labeling demonstrated that a substantial fraction of the B-cell repertoire can be missed when monomeric antigens are used. The specificity of the method was confirmed by antibody reconstruction from single-cell sorted tetramer+ B cells with single-cell RT-PCR of the B-cell receptor. All antibodies bound to tetanus antigen with high affinity, ranging from 0.23 to 2.2 nM. Furthermore, sequence analysis identified related memory B cell and plasmablast clones isolated more than a year apart. Therefore, antigen tetramers enable specific and sensitive ex vivo characterization of rare memory B cells as well as the production of fully human antibodies.
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Jiang, Li, Zhangbin Yu, Zuming Tang, Tao Jiang, Chunxiu Zhang, and Zuhong Lu. "Protein Arrays Based on Biotin-Streptavidin System for the Simultaneous Detection of TORCH Infections." Journal of Nanoscience and Nanotechnology 8, no. 5 (May 1, 2008): 2286–92. http://dx.doi.org/10.1166/jnn.2008.276.
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In this paper, we developed a protein array based on biotin-streptavidin system (PABS) used in the identification of IgM antibodies against TORCH antigens, including toxoplasma gondii (TOX), rubella virus (RuV), cytomegalovirus (CMV) and herpes simplex virus types II (HSV-2) antigens. The detection signal intensities and sensitivities between the PABS and the direct labeling array system (DLAS) were compared. The linear ranges of detectable IgM antibodies in PABS were 0.485–1000 μg/mL, which was more sensitive than DLAS. Quantitatively, the lowest detectable amount for IgM antibodies on each spot of the PABS was 0.25 pg. Furthermore, sixty serum samples from patients were tested with the PABS in TORCH detection. All the results were correspondingly confirmed with ELISA assay. No significant differences in identifying TORCH specific IgM antibodies were found between the PABS and ELISA assay. There was a good concordance between PABS and ELISA in the classification of sera. The results suggested that the PABS was more sensitive, sample-saving and suitable for multi-pathogens parallel clinical detection.
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Gillitzer, R., R. Berger, and H. Moll. "A reliable method for simultaneous demonstration of two antigens using a novel combination of immunogold-silver staining and immunoenzymatic labeling." Journal of Histochemistry & Cytochemistry 38, no. 3 (March 1990): 307–13. http://dx.doi.org/10.1177/38.3.1689333.
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We have developed a reliable and sensitive immunohistochemical staining technique which allows the simultaneous demonstration of two different antigens expressed in or on the same cell (referred to as mixed labeling), together with the evaluation of the general histopathological appearance of the tissue. The staining procedure combines a three-step (streptavidin-biotin) immunogold-silver staining (IGSS) with a three-step immunoenzymatic labeling. For this purpose, we investigated the compatibility of IGSS with various substrates of peroxidase or alkaline phosphatase (AP). Highly reliable and discernible mixed labeling was achieved only after initial labeling with IGSS followed by AP labeling using the substrates naphthol AS-MX phosphate/Fast Blue or naphthol AS-BI phosphate/New Fuchsin, respectively. To ensure utmost specificity, we applied FITC-conjugated mouse monoclonal antibodies and rabbit anti-FITC immunoglobulins visualized by AP-labeled immunoglobulins and the respective substrate in a final step. This novel approach provides an excellent means for demonstration of immunocompetent cells and unequivocal determination of the percentage of specific cell subsets in infiltrated tissue. The advantages of this method, as compared with double immunofluorescence or double immunoenzymatic labeling, were investigated and are discussed.
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Fok, A. K., M. S. Aihara, M. Ishida, K. V. Nolta, T. L. Steck, and R. D. Allen. "The pegs on the decorated tubules of the contractile vacuole complex of Paramecium are proton pumps." Journal of Cell Science 108, no. 10 (October 1, 1995): 3163–70. http://dx.doi.org/10.1242/jcs.108.10.3163.
Full textAbstract:
Our previous study has shown that the decorated tubules (collectively known as the decorated spongiome) of the contractile vacuole complex (CVC) in Paramecium are the site of fluid segregation, as the binding of microinjected monoclonal antibody (mAb) DS-1 to the tubules reduced the CVC's fluid output. In this study, we showed by immunogold labeling on cryosections that the antigenic sites for mAb DS-1 were located on the 15 nm ‘pegs’ protruding from the cytosolic surface of the decorated tubules. In immunofluorescence studies, both polyclonal antibodies against the subunits of the V-ATPase of Dictyostelium discoideum and against the 57 kDa B-subunit of the V-ATPase of chromaffin granules gave identical labeling patterns to that produced by mAb DS-1. On cryosections, all three antigens were located most consistently near or on the pegs of the decorated tubules. These data support the notion that the pegs on the membrane of the decorated tubules represent the V1 complex of a proton pump. Concanamycin B, a potent inhibitor of V-ATPase activity and of acidification of lysosomes and endosomes, strongly and reversibly inhibited fluid output from the CVC but had minimal effect on the integrity of the decorated spongiome as observed by immunofluorescence. Such inhibition suggests that a V-ATPase is intimately involved in fluid segregation. Exposing Paramecium to 12 degrees C or 1 degrees C for 30 minutes resulted in the dissociation of the decorated tubules from the smooth spongiome that borders the collecting canals; thus the DS-1-reactive A4 antigen, the 75 kDa and 66 kDa antigens were all found dispersed in the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)
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Manjunath, R., R. F. Graziano, and W. R. Green. "The specificity of H-2-restricted cytotoxic T lymphocytes directed to AKR/Gross leukemia virus-induced tumors. III. Coordinate alterations in viral gp70 antigen expression and restoration of CTL-susceptibility to insusceptible variant tumors." Journal of Immunology 136, no. 6 (March 15, 1986): 2271–79. http://dx.doi.org/10.4049/jimmunol.136.6.2271.
Full textAbstract:
Abstract Two variant subclones, called cl.18-5 and cl.18-12, were derived from the AKR.H-2bSL1 tumor cell line that were, in contrast to the parental cells, selectively insusceptible to H-2-restricted anti-AKR/Gross virus cytotoxic T lymphocytes (CTL). Cell surface expression of viral envelope (env) and group-specific antigens (gag) on these CTL-resistant variants were analyzed and compared with the expression of these antigens on AKR.H-2bSL1 and two other CTL-susceptible clones, cl.1 and cl.5, also derived from AKR.H-2bSL1. Although normal levels of gag-encoded and H-2 antigens were displayed on the CTL-resistant variants, the expression of five distinct determinants of viral gp70 antigen as defined by monoclonal antibodies was significantly decreased on these CTL-resistant variants relative to their expression on the CTL-susceptible cell lines. However, similar dramatic changes in cell surface gp70 antigen expression were undetectable as defined by anti-gp70-specific antiserum. Immunoprecipitation and gel electrophoretic analysis revealed that gp70 molecules from cl.18-5 cells had a lower m.w. than those of AKR.H-2bSL1, but there were no differences in the m.w. of gp70 antigens from AKR.H-2bSL1, cl.5, and cl.18-12 cells. Expression of the five gp70 antigenic determinants mentioned above was completely restored by exposure of cl.18-5 and cl.18-12 cells to the halogenated pyrimidine, iododeoxyuridine (IudR). Treatment of cl.18-5 and cl.18-12 cells with IudR simultaneously restored CTL susceptibility of these cells to anti-AKR/Gross virus CTL without affecting gag and H-2 antigen expression. Viral gp70 antigen immunoprecipitated from IudR-treated cl.18-5 cells had a mobility slightly lower, but different from that of untreated cl.18-5 cells. Pulse-labeling with [35S]-methionine showed that IudR treatment of cl.18-5 cells caused the expression of an additional high m.w. gp70 precursor protein originally absent in untreated cl.18-5 cells but present on parental AKR.H-2bSL1 cells. Collectively, these results pointed to the involvement of viral gp70 antigenic determinants in the recognition of AKR/Gross virus-induced tumor targets by anti-AKR/Gross virus CTL.
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Pelchen-Matthews, Annegret, Beatrice Kramer, and Mark Marsh. "Infectious HIV-1 assembles in late endosomes in primary macrophages." Journal of Cell Biology 162, no. 3 (July 28, 2003): 443–55. http://dx.doi.org/10.1083/jcb.200304008.
Full textAbstract:
Although human immunodeficiency virus type 1 (HIV-1) is generally thought to assemble at the plasma membrane of infected cells, virions have been observed in intracellular compartments in macrophages. Here, we investigated virus assembly in HIV-1–infected primary human monocyte-derived macrophages (MDM). Electron microscopy of cryosections showed virus particles, identified by their morphology and positive labeling with antibodies to the viral p17, p24, and envelope proteins, in intracellular vacuoles. Immunolabeling demonstrated that these compartments contained the late endosomal marker CD63, which was enriched on vesicles within these structures and incorporated into the envelope of budding virions. The virus-containing vacuoles were also labeled with antibodies against LAMP-1, CD81, and CD82, which were also incorporated into the viral envelope. To assess the cellular source of infectious viruses derived from MDM, virus-containing media from infected cells were precipitated with specific antibodies. Only antibodies against antigens found in late endosomes precipitated infectious virus, whereas antibodies against proteins located primarily on the cell surface did not. Our data indicate that most of the infectious HIV produced by primary macrophages is assembled on late endocytic membranes and acquires antigens characteristic of this compartment. This notion has significant implications for understanding the biology of HIV and its cell–cell transmission.
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Contegiacomo, A., R. Mariani Costantini, R. Muraro, P. Battista, C. Valli, L. Frati, R. Calderopoli, et al. "Cell Kinetics and Tumor-Associated Antigen Expression in Human Mammary Carcinomas." International Journal of Biological Markers 6, no. 3 (July 1991): 159–66. http://dx.doi.org/10.1177/172460089100600304.
Full textAbstract:
Twenty-six primary breast carcinomas were studied to evaluate cell proliferation as assessed by thymidine labeling index (TLI), and antigenic phenotype, as defined by immunohistochemistry using eight monoclonal antibodies (MAbs) to tumor-associated antigens (TAAs). The majority of tumors had low TLI values. Reactivity to MAbs B72.3, CC49, CC83 (anti TAG 72), COL-12 (anti CEA) and MOv2 (against a tumor-associated mucoprotein) was restricted to < 50% of the tumors studied, while MAbs B1.1 (anti CEA), MBrl and MBr8 (to tumor-associated carbohydrates) reacted with > 50% of the cases. Correlations between expression of TAAs and proliferative activity showed that the tumors could be divided into three groups, two characterized by either high proliferative activity and absence of antigenic expression or low proliferative activity and strong antigenic expression, and the third showing no relation between these two biological features. We defined two antigenic phenotypes associated with specific cellular kinetics: one characterized by negative immunoreaction with MAbs, CC49, CC83 and COL-12 and high proliferative activity; the other characterized by intense immunoreactivity with these antibodies and low proliferative activity. The data suggest that cell proliferation and antigenic phenotype may define biologic subsets of breast carcinomas
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Morrissette, N. S., E. S. Gold, J. Guo, J. A. Hamerman, A. Ozinsky, V. Bedian, and A. A. Aderem. "Isolation and characterization of monoclonal antibodies directed against novel components of macrophage phagosomes." Journal of Cell Science 112, no. 24 (December 15, 1999): 4705–13. http://dx.doi.org/10.1242/jcs.112.24.4705.
Full textAbstract:
In order to identify novel proteins associated with various stages of macrophage phagocytosis, we have generated monoclonal antibodies that recognize phagosomes. Purified Fc receptor-mediated phagosomes, isolated by feeding IgG-conjugated magnetic beads to LPS-primed murine peritoneal macrophages, were used as the immunogen. An immunofluorescence screen was used to isolate and single-cell clone approximately 150 monoclonal antibodies that recognize mouse macrophage phagosomes as well as labeling other cellular components in patterns which are frequently distinct from those observed with previously characterized phagosome-associated proteins. Predominant morphological categories (in addition to phagosome labeling) include staining of one or more of the following: cytoskeletal patterns, vesicular patterns and plasma membrane localization. In this paper, we describe the antibody screen, preliminary characterization of the antibodies and our identification of the antigens for three representative monoclonal antibodies. These antibodies identify a plasma membrane associated receptor (Mac-1, a subunit of the complement receptor), an actin binding protein (coronin-2) and a vesicular protein (amphiphysin II). Some of the antibodies recognize many cell types, whereas other antibodies are apparently macrophage specific as assessed by flow cytometry and histology. Remarkably, several of the antibodies cross-react with the phagocytic slime mold, Dictyostelium discoideum, recognizing phagosomes and other cellular elements as assessed by immunofluorescence and immunoblots. These results indicate that macrophage phagocytosis has both conserved ancestral features and unique specialized aspects associated with the role of these phagocytes in immunity.
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Kanbour, A., H. N. Ho, D. N. Misra, T. A. MacPherson, H. W. Kunz, and T. J. Gill. "Differential expression of MHC class I antigens on the placenta of the rat. A mechanism for the survival of the fetal allograft." Journal of Experimental Medicine 166, no. 6 (December 1, 1987): 1861–82. http://dx.doi.org/10.1084/jem.166.6.1861.
Full textAbstract:
In some mating combinations in rats, there is a maternal antibody response to the maternal antigenic components of the placenta without any previous immunization of the mother. The highest response occurs in the WF (u) female mated to the DA (a) male, and it is against a unique MHC-encoded class I antigen, the Pa antigen, and not against the major allele-specific transplantation antigen of the DA strain, RT1.Aa. The development of mAbs to the Pa and Aa antigens allowed us to localize these antigens on the placenta and to explore the reason for the differential antibody response to them using immunohistochemical and biochemical techniques. Both antibodies reacted with the WF X DA placenta and stained the endovascular and interstitial trophoblast of the decidua, the basal trophoblast, Reichert's membrane, and the yolk sac epithelium, but they did not stain the labyrinthine trophoblast. Blocking studies showed that each antibody reacted with a separate molecule in the placenta. Anti-class II mAbs reactive with the a or u haplotype did not stain the WF X DA, DA X DA, or WF X WF placenta; hence, there are no class II antigens in the placenta. Electron microscopic studies of the semiallogeneic WF X DA placenta using the immunogold technique with both single- and double-labeling showed that only the Pa antigen was expressed on the surface of the basal trophoblast, but that both the Pa and Aa antigens were in the cytoplasm of these cells; neither antigen was found in the labyrinthine trophoblast. By contrast, the placenta from the syngeneic DA X DA mating expressed both the Pa and Aa antigens on the surface of the basal trophoblast as well as in the cytoplasm; neither antigen was found in the labyrinthine trophoblast. These observations were quantified morphometrically using electron photomicrographs of single-labeled tissues. Both the Pa and Aa antigens isolated from the plasma membrane of lymphocytes have heavy chains of 46 kD, but those antigens isolated from the plasma membrane of basal trophoblast cells have heavy chains of 43 kD. Based on densitometric measurements of autoradiographs, the Pa/Aa ratio in the basal trophoblast membrane is 23.5, whereas it is 0.46 in lymphocyte membranes. These studies show that there is differential regulation of the expression of class I antigens on basal trophoblast cells in semiallogeneic pregnancies, but not in syngeneic pregnancies, such that the major allele-specific transplantation antigen is scarcely expressed on the surface of the basal trophoblast.(ABSTRACT TRUNCATED AT 400 WORDS)
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Hainfeld, James F., and Frederic R. Furuya. "Aldehyde gold clusters for molecular labeling." Proceedings, annual meeting, Electron Microscopy Society of America 53 (August 13, 1995): 858–59. http://dx.doi.org/10.1017/s042482010014066x.
Full textAbstract:
Glutaraldehyde is a useful tissue and molecular fixing reagents. The aldehyde moiety reacts mainly with primary amino groups to form a Schiff's base, which is reversible but reasonably stable at pH 7; a stable covalent bond may be formed by reduction with, e.g., sodium cyanoborohydride (Fig. 1). The bifunctional glutaraldehyde, (CHO-(CH2)3-CHO), successfully stabilizes protein molecules due to generally plentiful amines on their surface; bovine serum albumin has 60; 59 lysines + 1 α-amino. With some enzymes, catalytic activity after fixing is preserved; with respect to antigens, glutaraldehyde treatment can compromise their recognition by antibodies in some cases. Complicating the chemistry somewhat are the reported side reactions, where glutaraldehyde reacts with other amino acid side chains, cysteine, histidine, and tyrosine. It has also been reported that glutaraldehyde can polymerize in aqueous solution. Newer crosslinkers have been found that are more specific for the amino group, such as the N-hydroxysuccinimide esters, and are commonly preferred for forming conjugates. However, most of these linkers hydrolyze in solution, so that the activity is lost over several hours, whereas the aldehyde group is stable in solution, and may have an advantage of overall efficiency.
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Yang, Yexin, Rebecca S. Treger, Juan Hernandez-Bird, and Akiko Iwasaki. "Glycan-reactive natural antibodies mediate blockade of endogenous retroviruses emergence." Journal of Immunology 206, no. 1_Supplement (May 1, 2021): 114.09. http://dx.doi.org/10.4049/jimmunol.206.supp.114.09.
Full textAbstract:
Abstract Endogenous retroviruses (ERVs), comprising a substantial portion of the vertebrate genome, are remnants of ancient genetic invaders. Although multiple layers of cell-intrinsic control are utilized to prevent retroviral reactivation, ERVs with intact coding potential are able to reactivate in immunodeficient mice. While previous studies have indicated that B cells are indispensable for preventing ERV reactivation, it is not yet clear which B cell population mediates the blockade of ERV emergence to prevent subsequent damage in the host. Here, we employed direct labeling of B cells reactive with emerged ERV particles to characterize the B cell population and clonal repertoire responsible for recognition of ERV, and to study the mechanism by which B cells provide protection against ERV emergence. We found that ERV-reactive B cells are enriched in innate-like B-1 cell compartment that predominantly reside in peritoneal and pleural cavities. B-1 cells specificity is biased toward conserved epitopes shared by bacterial- and self-antigens due to distinct developmental pathways in fetal haematopoiesis. We identified ERV-reactive antibodies in unimmunized mice, the level of which further increases upon innate sensor stimulation. B cell receptor repertoire profiling of ERV-reactive B-1 cells revealed increased usage of Igh VH genes that give rise to glycan-specific antibodies targeting glycan structures exhibited by bacterial and viral antigens. We demonstrated that these glycan-specific natural antibodies engage complement pathway to facilitate clearance of reactivated ERV particles. In conclusion, we elucidated the role of glycan-specific B-1 cells and secreted natural antibodies in mediating blockade of ERV emergence.
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Otsuki, Y., L. E. Maxwell, S. Magari, and H. Kubo. "Immunogold-silver staining method for light and electron microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies." Journal of Histochemistry & Cytochemistry 38, no. 8 (August 1990): 1215–21. http://dx.doi.org/10.1177/38.8.2365991.
Full textAbstract:
We used the immunogold-silver staining method (IGSS) for detection of lymphocyte cell surface antigens with monoclonal antibodies in light and electron microscopy and compared this procedure with the immunogold staining method. Two different sizes of colloidal gold particles (5 nm and 15 nm) were used in this study. Immunolabeling on cell surfaces was visualized as fine granules only by IGSS in light microscopy. The labeling density (silver-gold complexes/cell) and diameters of silver-enhanced gold particles on cell surfaces were examined by electron microscopy. Labeling density was influenced not by the enhancement time of the physical developer but by the size of the gold particles. However, the development of shells of silver-enhanced gold particles correlated with the enhancement time of the physical developer rather than the size of the colloidal gold particles. Five-nm gold particles enhanced with the physical developer for 3 min were considered optimal for this IGSS method because of reduced background staining and high specific staining in the cell suspensions in sheep lymph. Moreover, this method may make it possible to show the ultrastructure of identical positive cells detected in 1-micron sections counterstained with toluidine blue by electron microscopy, in addition to the percentage of positive cells by light microscopy.
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Eckhard, Andreas H., Jennifer T. O’Malley, Joseph B. Nadol, and Joe C. Adams. "Mechanical Compression of Coverslipped Tissue Sections During Heat-induced Antigen Retrieval Prevents Section Detachment and Preserves Tissue Morphology." Journal of Histochemistry & Cytochemistry 67, no. 6 (January 29, 2019): 441–52. http://dx.doi.org/10.1369/0022155419826940.
Full textAbstract:
Heat-induced antigen retrieval (HIAR) is routinely employed on aldehyde-fixed tissue sections to enhance the reactivity of antibodies that exhibit weak or no specific interactions with tissue antigens when applied in conventional immunohistochemical protocols. A major drawback of HIAR protocols is, however, the heat-induced detachment of sections from the microscope slide with resultant impaired tissue morphology or loss of the section. We developed a method in which tissue sections mounted on glass slides are temporally coverslipped, and a clamp is used to compress the sections on the microscope slide during HIAR treatment. This “pressurized coverslipping” during HIAR was tested on various formalin-fixed tissues (murine kidneys and temporal bones, human tonsils and temporal bones) that were embedded in paraffin or celloidin. The method reliably kept the sections adherent to the slide, preserved the tissue morphology, and effectively retrieved tissue antigens for improved results in immunohistochemical labeling, even for exceptionally delicate, large, and poorly adhering sections, that is, decalcified human temporal bone sections. In summary, we present a simple method for improved slide adherence and morphological preservation of tissue sections during HIAR treatment that can be combined with all HIAR protocols and that requires only basic lab equipment.
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Sapozhnikova, Ksenia A., Vsevolod A. Misyurin, Dmitry Y. Ryazantsev, Egor A. Kokin, Yulia P. Finashutina, Anastasiya V. Alexeeva, Igor A. Ivanov, et al. "Sensitive Immunofluorescent Detection of the PRAME Antigen Using a Practical Antibody Conjugation Approach." International Journal of Molecular Sciences 22, no. 23 (November 27, 2021): 12845. http://dx.doi.org/10.3390/ijms222312845.
Full textAbstract:
Bioconjugation of antibodies with various payloads has diverse applications across various fields, including drug delivery and targeted imaging techniques. Fluorescent immunoconjugates provide a promising tool for cancer diagnostics due to their high brightness, specificity, stability and target affinity. Fluorescent antibodies are widely used in flow cytometry for fast and sensitive identification and collection of cells expressing the target surface antigen. Nonetheless, current approaches to fluorescent labeling of antibodies most often use random modification, along with a few rather sophisticated site-specific techniques. The aim of our work was to develop a procedure for fluorescent labeling of immunoglobulin G via periodate oxidation of antibody glycans, followed by oxime ligation with fluorescent oxyamines. Here, we report a novel technique based on an in situ oxime ligation of ethoxyethylidene-protected aminooxy compounds with oxidized antibody glycans. The approach is suitable for easy modification of any immunoglobulin G, while ensuring that antigen-binding domains remain intact, thus revealing various possibilities for fluorescent probe design. The technique was used to label an antibody to PRAME, a cancer-testis protein overexpressed in a number of cancers. A 6H8 monoclonal antibody to the PRAME protein was directly modified with protected-oxyamine derivatives of fluorescein-type dyes (FAM, Alexa488, BDP-FL); the stoichiometry of the resulting conjugates was characterized spectroscopically. The immunofluorescent conjugates obtained were applied to the analysis of bone marrow samples from patients with oncohematological diseases and demonstrated high efficiency in flow cytometry quantification. The approach can be applied for the development of various immunofluorescent probes for detection of diagnostic and prognostic markers, which can be useful in anticancer therapy.
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Janckila, Anthony J., Wen-Kuang Yang, Ruey-Jen Lin, Chia-Jen Tseng, Hsin-Yu Chang, Jia-Ming Chang, and Lung T. Yam. "Flow Cytoenzymology of Intracellular Tartrate-resistant Acid Phosphatase." Journal of Histochemistry & Cytochemistry 51, no. 9 (September 2003): 1131–37. http://dx.doi.org/10.1177/002215540305100903.
Full textAbstract:
Tartrate-resistant acid phosphatase (TRACP) is a cytochemical marker for hairy cell leukemia, macrophages, dendritic cells, and osteoclasts. Our purpose was to develop multicolor cytofluorometric methods to evaluate intracellular TRACP enzymic activity using a fluorogenic cytochemical reaction in combination with immunochemical stains for distinct surface membrane antigens. Monocyte-derived dendritic cells (DCs) were the model TRACP-expressing cells studied. Intracellular TRACP activity was disclosed using naphthol-ASBI phosphate as substrate with fast red-violet LB salt as coupler for the reaction product. Before the TRACP enzymic reaction, surface antigens, CD86 and CD11c of DCs, were bound with specific fluorescent antibodies to test compatibility of surface labeling and intracellular staining. TRACP activity varied in DCs from donor to donor but was reproducible on repeated examinations of each sample. Samples could be stained for simultaneous analysis of surface antigens and intracellular TRACP activity, provided certain technical details were observed. The TRACP reaction time should not exceed 9 min and the cell number should not exceed 2 × 105/100 μl test. Fluorescent surface labels did not affect the intensity of the TRACP stain, but the intensity of some surface labels may be diminished by elution of low-affinity antibodies during the TRACP reaction. Readjustment of the threshold settings in triple-labeled cells is needed to compensate for this phenomenon. Intracellular TRACP activity can be quantitated in subpopulations of cells within mixed cell populations by flow cytofluorometry using simple cytochemical methods in combination with fluorescent antibodies to cell-surface and other differentiation antigens. The cytochemical method should be useful for basic investigations of differentiation, maturation, and function of macrophages, DCs, and osteoclasts, and for diagnosis and management of hairy cell leukemia.
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Chakraborty, Biswanath, Usha Chakraborty, Kuldip Rai, Kiran Sumar, and Pannalal Dey. "Serological and molecular detection of Macrophomina phaseolina, causing root rot of Citrus reticulata." NBU Journal of Plant Sciences 6, no. 1 (2012): 77–86. http://dx.doi.org/10.55734/nbujps.2012.v06i01.012.
Full textAbstract:
Polyclonal antibodies (PAbs) were raised against mycelial antigens of Macrophomina phaseolina a causal organism of root rot disease of mandarin plants. IgG was purified and further packaged into immunological formats such as immuno diffusion, Plate trapped antigen (PTA)-ELISA, dot immunobinding assay, Western blot analysis and indirect immunofluorescence for quick and accurate detection of pathogen from soil. Indirect staining of mycelia and sclerotia of M. phaseolina with homologous PAb and labeling with goat antirabbit IgG conjugated with FITC developed strong fluorescence in young hyphal tips and sclerotia of M. phaseolina. Genomic DNA prepared from mycelia of M. phaseolina was purified and PCR amplification of 18S rDNA was done using ITS region specific primer pair. The amplified DNA was sequenced and aligned against ex-type strain sequences from NCBI GenBank using BLAST and phylogenetic analysis was obtained using MEGA4 software. Amplification of ITSI region of the rDNA can be considered as a rapid technique for identifying pathogens successfully in all cases.
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Shindler, K. S., and K. A. Roth. "Double immunofluorescent staining using two unconjugated primary antisera raised in the same species." Journal of Histochemistry & Cytochemistry 44, no. 11 (November 1996): 1331–35. http://dx.doi.org/10.1177/44.11.8918908.
Full textAbstract:
Monoclonal antibodies (MAbs) capable of recognizing developmental stage-specific neuronal epitopes are becoming increasingly available. Because most of these MAbs are raised in a single species (mouse), simultaneous immunofluorescent detection of multiple epitopes has been difficult. We have taken advantage of the high sensitivity of tyramide signal amplification to develop a protocol that permits simultaneous detection of two antibodies raised in the same species. One primary antibody was applied at a concentration below the detection limit of fluorescently labeled secondary antibodies, yet sufficient for detection with the tyramide system. This first primary antibody was then effectively neglected during application of a second primary antibody that was detected by conventional fluorescently labeled secondary antibodies. Specifically, dual labeling for nestin and MAP2 was used to distinguish neuronal stem cells and precursor cells from immature postmitotic neurons, and synapsin I and GAP43 immunostaining was used to distinguish neurons with established synaptic connections from developing neurons. We have used this technique for staining both tissue sections and cultured cells from the embryonic mouse brain. This technique should be widely applicable and offers a simple procedure for simultaneously detecting two antigens when antibodies from only a single species are available.
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GAUNITZ, CHRISTINE, JÖRG GABERT, ERNST LÜCKER, JOHANNES SEEGER, and TOBIAS STAHL. "Suitability of Antigens PGP 9.5 and Neurofilament Light as Marker Proteins for Detection of Neuronal Tissue in Processed Meat Products." Journal of Food Protection 72, no. 5 (May 1, 2009): 1070–77. http://dx.doi.org/10.4315/0362-028x-72.5.1070.
Full textAbstract:
The enforcement of rules for food labeling and quantitative ingredient declaration presupposes appropriate test systems. Additionally, central nervous system (CNS) tissue of ruminants is classified as specified risk material for the transmission of prion diseases, and its detection is needed to support the specified risk material ban. Existing antibody-based test systems are hampered by relatively high limits of detection and susceptibility to food processing conditions. For that reason we tested a broad panel of commercially available monoclonal antibodies to identify marker antigens appropriate for the development of a sensitive test system. Western blot analysis using organ-specific samples from cow, pig, and chicken and differently processed meat products containing defined amounts of CNS tissue revealed neurofilament light (NF-L) and protein gene product 9.5 (PGP 9.5) as suitable antigens for the organ-specific and sensitive detection of porcine and bovine CNS tissue. None of the tested PGP 9.5 antibodies displayed cross-reactivity to chicken tissues. Both ntigens could be detected in moderately (F10121.1 equals; 0.84) and strongly (F10121.1 = 4.01) heated processed meat products containing 5% (NF-L) or 0.2% (PGP 9.5) CNS tissue, respectively. Further, two monoclonal antibodies (clones 13C4 and 31A3) directed against PGP 9.5 were used for the development of a sandwich enzyme-linked immunosorbent assay. The limits of detection of the enzyme-linked immunosorbent assay were ∼2% added CNS tissue in fresh processed meat products and ∼0.5% for strongly heated processed meat products (F10121.1 = 4.01). In conclusion this test system constitutes a valuable supplementation to existing procedures, which could improve enforcement of food safety regulations.
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Yang, Yexin, Rebecca Treger, Juan Hernandez-Bird, and Akiko Iwasaki. "Glycan-specific B-1 cells mediate blockade of endogenous retroviruses emergence through recognition of conserved glycan epitopes." Journal of Immunology 208, no. 1_Supplement (May 1, 2022): 126.31. http://dx.doi.org/10.4049/jimmunol.208.supp.126.31.
Full textAbstract:
Abstract Endogenous retroviruses (ERVs), comprising a substantial portion of the vertebrate genome, are remnants of ancient genetic invaders. Although multiple layers of cell-intrinsic control are utilized to prevent retroviral reactivation, ERVs with intact coding potential were found to reactivate in immunodeficient mice. While previous studies have indicated that B cells are indispensable for preventing ERV reactivation, it is not yet clear which B cell population mediates the blockade of ERV emergence to prevent subsequent damage in the host. Here, we employed direct labeling of B cells reactive with emerged ERV particles to characterize the B cell population and clonal repertoire responsible for recognition of ERV, and to study the mechanism by which B cells provide protection against ERV emergence. We found that ERV-reactive B cells are enriched in innate-like B-1 cell compartment that predominantly reside in peritoneal and pleural cavities. We identified ERV-reactive antibodies in unimmunized mice, the level of which further increases upon innate sensor stimulation. B cell receptor repertoire profiling of ERV-reactive B-1 cells revealed increased usage of Igh VH genes that give rise to glycan-specific antibodies, which were further determined to target terminal N-Acetylglucosamine moieties exhibited by endogenous and exogenous viral antigens. We demonstrated that these glycan-specific natural antibodies engage complement pathway to facilitate clearance of reactivated ERV particles. In conclusion, we elucidated the role of glycan-specific B-1 cells and secreted natural antibodies in mediating blockade of ERV emergence through recognition of conserved glycan epitopes. Supported by grants from Howard Hughes Medical Institute
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Cramer, EM, G. Berger, and MC Berndt. "Platelet alpha-granule and plasma membrane share two new components: CD9 and PECAM-1." Blood 84, no. 6 (September 15, 1994): 1722–30. http://dx.doi.org/10.1182/blood.v84.6.1722.1722.
Full textAbstract:
Abstract CD9 (p24) and PECAM1 (CD31) antigens are well-defined components of the platelet plasma membrane. Both are integral glycoproteins (GPs) implicated in the adhesive and aggregative properties of human platelets. In the present report, we have investigated their subcellular localization using immunoelectron microscopy. The monospecificity of the two polyclonal antibodies used was confirmed by immunoblotting. On normal resting platelets, immunolabeling for CD9 and PECAM1 was found lining the plasma membrane and the luminal face of the open canalicular system. Some labeling was also consistently found on the alpha-granule limiting membrane. This was confirmed by double labeling experiments in which fibrinogen and von Willebrand factor (vWF) were used as alpha-granule markers. CD9 and PECAM-1 were found lining the membrane of the same granules that contained fibrinogen and vWF in their matrix. CD9 and PECAM-1 thus appear to have an intracellular distribution identical to GPIIb-IIIa, a major aggregation platelet receptor. To rule out a cross-reactivity of the two polyclonal antibodies with GPIIb/IIIa, we studied PECAM1 and CD9 expression on the platelets from a patient with type I Glanzmann's thrombasthenia whose platelets are devoid of GPIIb/IIIa. The same pattern of labeling was observed for both antigens as for normal platelets. Normal platelets were further observed after stimulation by agonists that either fail to induce (ADP) or induce granule secretion (thrombin). After treatment with ADP, platelets changed shape and centralized their granules; the plasma membrane immunolabeling remained unchanged; and gold particles were still found decorating the periphery of the centralized alpha- granules. After thrombin treatment, alpha-granules fused with the platelet membrane and secretion occurred. A significant increase of labeling was then observed on the platelet surface. From these results we conclude that the alpha-granule membrane contains two additional receptors in common with the plasma membrane. This suggests that alpha- granule membrane receptors may originate from a dual mechanism: direct targeting from the Golgi complex in megakaryocytes (for alpha-granule- specific receptors such as P-selectin) or by endocytosis from the plasma membrane (for proteins distributed in the two compartments).
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Cramer, EM, G. Berger, and MC Berndt. "Platelet alpha-granule and plasma membrane share two new components: CD9 and PECAM-1." Blood 84, no. 6 (September 15, 1994): 1722–30. http://dx.doi.org/10.1182/blood.v84.6.1722.bloodjournal8461722.
Full textAbstract:
CD9 (p24) and PECAM1 (CD31) antigens are well-defined components of the platelet plasma membrane. Both are integral glycoproteins (GPs) implicated in the adhesive and aggregative properties of human platelets. In the present report, we have investigated their subcellular localization using immunoelectron microscopy. The monospecificity of the two polyclonal antibodies used was confirmed by immunoblotting. On normal resting platelets, immunolabeling for CD9 and PECAM1 was found lining the plasma membrane and the luminal face of the open canalicular system. Some labeling was also consistently found on the alpha-granule limiting membrane. This was confirmed by double labeling experiments in which fibrinogen and von Willebrand factor (vWF) were used as alpha-granule markers. CD9 and PECAM-1 were found lining the membrane of the same granules that contained fibrinogen and vWF in their matrix. CD9 and PECAM-1 thus appear to have an intracellular distribution identical to GPIIb-IIIa, a major aggregation platelet receptor. To rule out a cross-reactivity of the two polyclonal antibodies with GPIIb/IIIa, we studied PECAM1 and CD9 expression on the platelets from a patient with type I Glanzmann's thrombasthenia whose platelets are devoid of GPIIb/IIIa. The same pattern of labeling was observed for both antigens as for normal platelets. Normal platelets were further observed after stimulation by agonists that either fail to induce (ADP) or induce granule secretion (thrombin). After treatment with ADP, platelets changed shape and centralized their granules; the plasma membrane immunolabeling remained unchanged; and gold particles were still found decorating the periphery of the centralized alpha- granules. After thrombin treatment, alpha-granules fused with the platelet membrane and secretion occurred. A significant increase of labeling was then observed on the platelet surface. From these results we conclude that the alpha-granule membrane contains two additional receptors in common with the plasma membrane. This suggests that alpha- granule membrane receptors may originate from a dual mechanism: direct targeting from the Golgi complex in megakaryocytes (for alpha-granule- specific receptors such as P-selectin) or by endocytosis from the plasma membrane (for proteins distributed in the two compartments).
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Rieder, C. L., and S. S. Bowser. "Correlative immunofluorescence and electron microscopy on the same section of epon-embedded material." Journal of Histochemistry & Cytochemistry 33, no. 2 (February 1985): 165–71. http://dx.doi.org/10.1177/33.2.3881520.
Full textAbstract:
Semithick (0.25-0.50 micron) sections, cut from cells stained with fluorescein isothiocyanate (FITC)-conjugated antibodies prior to embedding in Epon, show high resolution patterns of immunofluorescence against a background void of autofluorescence. These same sections can then be viewed, after uranyl and lead staining, in the electron microscope. We clearly establish the specificity of this same-section correlative immunofluorescence-electron microscopy approach by showing that the immunofluorescent patterns observed in such sections of cells, stained prior to embedding for the indirect immunofluorescent localization of tubulin, follows the distribution of microtubules within the same sections as determined by electron microscopy. We then use this method to demonstrate for the first time that the 57 kD core protein of wound tumor virus is associated, at the ultrastructural level, with two distinct cellular inclusions in virally infected AC-20 cells. In some instances the fidelity in the correlation between the distribution of immunofluorescently labeled antigens and the ultrastructure in the same section eliminates the need to employ more complex procedures for labeling antigens for ultrastructural detection. This technique, therefore, provides a rapid and simple first approach to many problems that require the ultrastructural localization of specific antigens.
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Gebert, A. "M-cells in the rabbit tonsil exhibit distinctive glycoconjugates in their apical membranes." Journal of Histochemistry & Cytochemistry 44, no. 9 (September 1996): 1033–42. http://dx.doi.org/10.1177/44.9.8773569.
Full textAbstract:
The tonsil crypt epithelium contains membranous (M)-cells that transport antigens from the lumen to underlying lymphoid cells, thereby initiating specific immune responses. Mechanisms mediating the adhesion of antigens to the M-cell surface are important for effective and selective uptake of potential pathogens but are still poorly understood. Therefore, the carbohydrates present on crypt epithelial cells of the rabbit palatine tonsil were studied by lectin histochemistry. Ultrathin LR White sections were incubated with a panel of eight lectins conjugated to colloidal gold or biotin. The glycocalyx of the apical membrane of M-cells was selectively labeled by UEA-I, LTA, HPA, and VVA, whereas that of the remaining squamous epithelial cells preferentially bound RCA-I and PNA. WGA and ConA showed only little binding, with no discernible preference for any of the cell types. Double labeling of UEA-1 together with anti-vimentin antibodies revealed that UEA-I-positive epithelial cells also contained the rabbit M-cell marker vimentin, and vice versa. The results show that a specific composition of glycoconjugates, which differs from that on squamous epithelial cells, is found on M-cells of the rabbit tonsil. The M-cell-specific glycoproteins and glycolipids could be selectively targeted by microorganisms that adhere to M-cells and enter the host along this pathway.
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Tamura-Sakaguchi, Risako, Rie Aruga, Mika Hirose, Toru Ekimoto, Takuya Miyake, Yohei Hizukuri, Rika Oi, et al. "Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion." Acta Crystallographica Section D Structural Biology 77, no. 5 (April 19, 2021): 645–62. http://dx.doi.org/10.1107/s2059798321002527.
Full textAbstract:
Antibody labeling has been conducted extensively for structure determination using both X-ray crystallography and electron microscopy (EM). However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, an alternative method has been developed to prepare an antibody complex by inserting an exogenous epitope into the target. It has already been demonstrated that the Fab of the NZ-1 monoclonal antibody can form a stable complex with a target containing a PA12 tag as an inserted epitope. Nevertheless, it was also found that complex formation through the inserted PA12 tag inevitably caused structural changes around the insertion site on the target. Here, an attempt was made to improve the tag-insertion method, and it was consequently discovered that an alternate tag (PA14) could replace various loops on the target without inducing large structural changes. Crystallographic analysis demonstrated that the inserted PA14 tag adopts a loop-like conformation with closed ends in the antigen-binding pocket of the NZ-1 Fab. Due to proximity of the termini in the bound conformation, the more optimal PA14 tag had only a minor impact on the target structure. In fact, the PA14 tag could also be inserted into a sterically hindered loop for labeling. Molecular-dynamics simulations also showed a rigid structure for the target regardless of PA14 insertion and complex formation with the NZ-1 Fab. Using this improved labeling technique, negative-stain EM was performed on a bacterial site-2 protease, which enabled an approximation of the domain arrangement based on the docking mode of the NZ-1 Fab.
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Rieder, C. L., S. P. Alexander, and S. S. Bowser. "Same-section correlative light and electron microscopy." Proceedings, annual meeting, Electron Microscopy Society of America 47 (August 6, 1989): 896–97. http://dx.doi.org/10.1017/s0424820100156468.
Full textAbstract:
Epoxy embedded biological material, sectioned for conventional, intermediate or high-voltage electron microscopy (EM), can be visualized within the section with good contrast and detail by phase-contrast or dark-field light microscopy (LM). The contrast of such material is not substantially influenced by the type of embedding resin or section support substrate. It is, however, influenced by the type of fixation (glutaraldehyde with and without osmium postfixation), by heavy metal (uranyl and lead) staining, and by the section thickness. The ability to examine the specimen with the LM, within a section cut for EM, allows each section to be rapidly screened for content prior to examination in the EM. We have found this approach to be particularly useful for studies requiring the ultrastructural examination of a selected area or structure which is large enough to be visualized with the LM but which comprises only a small volume of the embedded material (e.g., centrosomes and nuclei within oocytes; specific regions of large protists.)Same section LM-EM can also be extended to sections cut from cells stained prior to embedding for the immunofluorescent localization of antigens using fluorescein, rhodamine or Texas red-conjugated antibodies. Under these conditions the sections show high resolution patterns of antigen-specific fluorescence against a background void of autoflouorescence. The fidelity in the correlation between the distribution of immunofluorescently labeled antigens and the ultrastructure in the same section can be extended to structures as small as a single microtubule or just a few actin filaments . This approach of same section correlative fluorescence microscopy (FLM) and EM can eliminate the need, in many instances, to employ more complex procedures such as immunoferritin or immunogold for labeling antigens for ultrastructural detection.
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Keene, D. R., L. Y. Sakai, R. E. Burgeson, and H. P. Bächinger. "Direct visualization of IgM antibodies bound to tissue antigens using a monoclonal anti-type III collagen IgM as a model system." Journal of Histochemistry & Cytochemistry 35, no. 3 (March 1987): 311–18. http://dx.doi.org/10.1177/35.3.3546481.
Full textAbstract:
A mouse monoclonal IgM antibody directed against human Type III collagen was utilized to immunolocalize Type III collagen by transmission and scanning electron microscopy without the use of an electron-dense conjugate. Because bound IgM can be directly visualized, primary or secondary antibody conjugates, such as ferritin, HRP, colloidal gold, etc., are unnecessary in this method. Immunolocalization to Type III collagen in the matrix of human skin and to fibrils formed in vitro using only IgM antibody reveals uninterrupted IgM binding which exactly matches the banding period of the collagen fibrils. In contrast, colloidal gold-conjugated secondary antibody complexes directed against primary IgM binding sites reveal less precise labeling. The data suggest that direct visualization of primary monoclonal IgM antibodies may be useful in a wide variety of highly specific ultrastructural immunolocalization studies without requiring the use of electron-dense conjugates.
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Manara, G. C., G. De Panfilis, and C. Ferrari. "Ultrastructural characterization of human large granular lymphocyte subsets defined by the expression of HNK-1 (Leu-7), Leu-11, or both HNK-1 and Leu-11 antigens." Journal of Histochemistry & Cytochemistry 33, no. 11 (November 1985): 1129–33. http://dx.doi.org/10.1177/33.11.3932517.
Full textAbstract:
A peroxidase-colloidal gold double labeling system in immunoelectron microscopy was used to investigate the ultrastructural features of human large granular lymphocytes (LGL) subpopulations. Three subsets of LGL, Leu-7+-Leu-11-, Leu-7+-Leu-11+, Leu-7- -Leu-11+, were characterized using combinations of the monoclonal antibodies anti-Leu-7 and anti-Leu-11. They showed different ultrastructural patterns. In fact, Leu-7+-Leu-11- cells showed a high nuclear/cytoplasmic ratio (N/C), a round nucleus, a cytoplasm with few organelles, and a rather even surface. Moreover, most of them lacked electron-dense granules. On the other hand, Leu-11+ cells displayed a low N/C, an irregular-shaped nucleus, and a cytoplasm containing a well-developed Golgi apparatus, many mitochondria, vacuoles, vesicles, and numerous electron-dense granules. Moreover, they exhibited an irregular cell surface. Thus, Leu-7+-Leu-11- cells seemed to represent an immature form of LGL, while cells expressing the Leu-11 antigen showed a fine structure specific for functional NK cells. Our findings suggest that the expression of HNK-1 (Leu-7) and Leu-11 antigens respectively represents subsequent stages in NK cell differentiation.
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Dewulf, Jonatan, Karuna Adhikari, Christel Vangestel, Tim Van Den Wyngaert, and Filipe Elvas. "Development of Antibody Immuno-PET/SPECT Radiopharmaceuticals for Imaging of Oncological Disorders—An Update." Cancers 12, no. 7 (July 11, 2020): 1868. http://dx.doi.org/10.3390/cancers12071868.
Full textAbstract:
Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) are molecular imaging strategies that typically use radioactively labeled ligands to selectively visualize molecular targets. The nanomolar sensitivity of PET and SPECT combined with the high specificity and affinity of monoclonal antibodies have shown great potential in oncology imaging. Over the past decades a wide range of radio-isotopes have been developed into immuno-SPECT/PET imaging agents, made possible by novel conjugation strategies (e.g., site-specific labeling, click chemistry) and optimization and development of novel radiochemistry procedures. In addition, new strategies such as pretargeting and the use of antibody fragments have entered the field of immuno-PET/SPECT expanding the range of imaging applications. Non-invasive imaging techniques revealing tumor antigen biodistribution, expression and heterogeneity have the potential to contribute to disease diagnosis, therapy selection, patient stratification and therapy response prediction achieving personalized treatments for each patient and therefore assisting in clinical decision making.
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Cooper, Christopher D. O., Amanda P. Liggins, Kamel Ait-Tahar, Alison H. Banham, and Karen Pulford. "Protein Expression Profiles Confirm PASD1 as a Cancer Testis Antigen and a Potential Candidate for Lymphoma Immunotherapy." Blood 106, no. 11 (November 16, 2005): 2825. http://dx.doi.org/10.1182/blood.v106.11.2825.2825.
Full textAbstract:
Abstract Advances in gene expression profiling and immunolabeling techniques have provided evidence of clinically relevant subtypes within the heterogeneous disease entity diffuse large B-cell lymphoma (DLBCL). The development of improved treatment regimens still remains a priority as more than half of DLBCL patients are incurable using combination CHOP chemotherapy. Evidence that the immune system plays a critical role in cancer biology is rapidly accumulating with many tumor proteins being recognized by an anti-tumor immune response. Such tumor-associated antigens (TAAs) have the potential to provide new diagnostic, prognostic and therapeutic options for cancer patients. Of particular interest are the cancer testis antigens (CTAs), whose restricted normal tissue expression but widespread expression in tumors makes them attractive targets for therapy. SEREX was previously used to identify a DLBCL-associated TAA, PAS domain containing 1 (PASD1) that was immunologically recognized by multiple high-risk DLBCL patients. PASD1 maps to chromosome Xq28, in common with many other CTA genes, and encodes a novel putative transcription factor. Since PASD1 mRNA expression in normal tissues is restricted to testis with transcripts being expressed in both DLBCL cell lines and solid tumours, it therefore represents a new CTA and potential immunotherapeutic target. Two PASD1 transcripts were identified, v1 encoding a 639 aa N-terminus while v2 encodes a longer protein (773 aa) with a unique C-terminus. Both proteins were nuclear when expressed in transfected COS-1 cells. We have raised a panel of monoclonal antibodies against the PASD1 proteins, including two antibodies (2ALCC16 and 2ALCC136) recognizing both variants, and one antibody (antibody 2ALCC128) specific for the C-terminus of the longer protein, to study PASD1 protein distribution in both normal and neoplastic tissues. In normal tissues, PASD1 expression is restricted to the nuclei of cells in the basal layer of testis, a subset of primary spermatogonia (2ALCC16 also labeled the cytoplasm of spermatogonia) and a small subset of cells in the salivary gland. PASD1 protein was detected in a range of malignant cell lines with nuclear labeling being observed in OCI-Ly3 (DLBCL), KM-H2 (Hodgkin’s lymphoma), K562 (chronic myeloid leukaemia) and RPMI 8226 (myeloma) lines, whilst cytoplasmic labeling was detected in Karpas 299 (t(2;5) anaplastic large cell lymphoma), SUDHL6, SUDHL10 (both DLBCL) and Jurkat (T-acute lymphoblastic leukaemia) cell lines. Tumor tissue was available from two patients with seroreactivity to PASD1; PASD1-positive tumour cells were observed using all three antibodies in one patient, with scattered positive cells being observed with 2ALCC136 in the other. It is of note that, whereas strong cytoplasmic labeling was obtained with 2ALCC136, nuclear labeling was observed with 2ALCC128 recognizing the longer PASD1 variant. Variations in the staining patterns and western blotting data using different antibodies may reflect the presence of multiple PASD1 isoforms. Preliminary results demonstrate PASD1 expression in a subset of DLBCL, mantle cell lymphoma, follicular lymphoma, multiple myeloma and Hodgkin’s disease. Further studies of a larger series of hematological malignancies are in progress to determine the potential of this molecule, both in the identification of high-risk patients and as an immunotherapeutic target.
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Clofent-Sanchez, Gisèle, Catherine Jais, Emilse Bermejo, Lionel Leroux, Pierre Coste, Alan Nurden, and Paquita Nurden. "Delayed immunologic thrombocytopenia induced by abciximab." Thrombosis and Haemostasis 92, no. 10 (2004): 820–28. http://dx.doi.org/10.1160/th04-04-0237.
Full textAbstract:
SummaryAbciximab is an anti-GPIIb-IIIa drug widely used to prevent thrombotic complications during percutaneous coronary intervention. We now report on the immunologic origin of thrombocytopenia developing between 7 and 12 days after the onset of abciximab infusion. Antibodies directed against abciximabcoated platelets were located in 5 patients with delayed thrombocytopenia, just as they were present in a patient whose platelet count fell within a few hours after receiving the drug. Abciximab-dependent IgG antibody was revealed in serum using control platelets in the monoclonal antibody immobilization of platelet antigens assay (MAIPA) performed with SZ22, a MoAb to GPIIb. The presence of IgG antibodies specific for platelets sensitized with abciximab was confirmed by flow cytometry. They were not located in 13 patients receiving abciximab but whose platelet counts remained stable. For three patients, antibodies were transient and their presence related to the extent of the thrombocytopenia. Surprisingly, antibodycontaining plasma from three patients induced abciximabdependent activation and aggregation of normal platelets, a finding confirmed by electron microscopy. Immunogold labeling revealed that abciximab was associated with platelets in the aggregate, suggesting that its inhibitory effect was overcome by the platelet stimulation. In summary, these results show that abciximab-dependent thrombocytopenia can be delayed and potentially prothrombotic.
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Takizawa, Toshihiro, Takuma Saito, and John M. Robinson. "Freeze-fracture Cytochemistry: A New Method Combining Immunocytochemistry and Enzyme Cytochemistry on Replicas." Journal of Histochemistry & Cytochemistry 46, no. 1 (January 1998): 11–17. http://dx.doi.org/10.1177/002215549804600103.
Full textAbstract:
We describe a new freeze-fracture cytochemical technique consisting of combined immunocytochemistry and enzyme cytochemistry. This technique reveals the relationship between molecules in biological membranes by double labeling with two different cytochemical markers (i.e., immunogold probes and cerium). In this method, antigens were detected with specific primary antibodies and appropriate secondary immunoprobes. Subsequently, alkaline phosphatase activity was detected with cerium as the capture agent on the same replicas. Octyl-glucoside (OG) digestion before the cytochemical reactions was crucial to the success of this combined method. OG is an efficient detergent and OG digestion can preserve both immunocytochemical antigenicity and enzyme activity on replicas. As an initial examination, we applied this technique to the study of glycosyl-phosphatidyl-inositol-anchored proteins and adhesion molecules in human neutrophils. The method described here should serve as a unique additional approach for the study of topology and dynamics of molecules in biomembranes.