Academic literature on the topic 'Adsorbed antigen'
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Journal articles on the topic "Adsorbed antigen"
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Zhang, Zhigang, Tianying Zhang, Lu Cao, Xin Wang, Jiali Cao, Xiaofen Huang, Yashuang Cai, et al. "Simultaneous in situ visualization and quantitation of dual antigens adsorbed on adjuvants using high content analysis." Nanomedicine 14, no. 19 (October 2019): 2535–48. http://dx.doi.org/10.2217/nnm-2019-0016.
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Aim: Traditional antigenicity assay requires antigen recovery from the particulate adjuvants prior to analysis. An in situ method was developed for interrogating vaccine antigens with monoclonal antibodies while being adsorbed on adjuvants. Materials & methods: The fluorescence imaging-based high content analysis was used to visualize the antigen distribution on adjuvant agglomerates and to analyze the antigenicity for adsorbed antigens. Results: Simultaneous visualization and quantitation were achieved for dual antigens in a bivalent human papillomavirus vaccine with uniquely labeled antibodies. Good agreement was observed between the in situ multiplexed assays with well-established sandwich enzyme-linked immunosorbent assays. Conclusion: The streamlined procedures and the amenability for multiplexing make the in situ antigenicity analysis a favorable choice for in vitro functional assessment of bionanoparticles as vaccine antigens.
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Laera, Donatello, Camilla Scarpellini, Simona Tavarini, Barbara Baudner, Agnese Marcelli, Carlo Pergola, Malte Meppen, and Derek T. O’Hagan. "Maturation of Aluminium Adsorbed Antigens Contributes to the Creation of Homogeneous Vaccine Formulations." Vaccines 11, no. 1 (January 11, 2023): 155. http://dx.doi.org/10.3390/vaccines11010155.
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Although aluminium-based vaccines have been used for almost over a century, their mechanism of action remains unclear. It is established that antigen adsorption to the adjuvant facilitates delivery of the antigen to immune cells at the injection site. To further increase our understanding of aluminium-based vaccines, it is important to gain additional insights on the interactions between the aluminium and antigens, including antigen distribution over the adjuvant particles. Immuno-assays can further help in this regard. In this paper, we evaluated how established formulation strategies (i.e., sequential, competitive, and separate antigen addition) applied to four different antigens and aluminium oxyhydroxide, lead to formulation changes over time. Results showed that all formulation samples were stable, and that no significant changes were observed in terms of physical-chemical properties. Antigen distribution across the bulk aluminium population, however, did show a maturation effect, with some initial dependence on the formulation approach and the antigen adsorption strength. Sequential and competitive approaches displayed similar results in terms of the homogeneity of antigen distribution across aluminium particles, while separately adsorbed antigens were initially more highly poly-dispersed. Nevertheless, the formulation sample prepared via separate adsorption also reached homogeneity according to each antigen adsorption strength. This study indicated that antigen distribution across aluminium particles is a dynamic feature that evolves over time, which is initially influenced by the formulation approach and the specific adsorption strength, but ultimately leads to homogeneous formulations.
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Oksanich, A. S., A. G. Krasko, T. G. Samartseva, E. L. Gasich, and G. M. Ignatyev. "The use of quantitative enzyme-linked immunosorbent assay for the determination of S-antigen concentration in whole-virion inactivated adsorbed coronavirus vaccines." Biological Products. Prevention, Diagnosis, Treatment 22, no. 4 (November 25, 2022): 405–13. http://dx.doi.org/10.30895/2221-996x-2022-22-4-405-413.
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The severe consequences and high mortality of COVID-19 prompted the development of a wide range of preventive vaccines. The first vaccines to be tested were developed in China and formulated as inactivated SARS-CoV-2 adsorbed on aluminium hydroxide. One of the quality indicators for inactivated adsorbed vaccines is the degree of adsorption, which can be used to control the content not only of non-adsorbed antigen, but also of specific antigen in one dose of a vaccine.The aim of the study was to investigate the possibility of desorbing SARS-CoV-2 antigen from formulated adsorbed vaccines and the possibility of measuring its concentration using the BioScan-SARS-CoV-2 (S) ELISA kit for SARS-CoV-2 S-protein content determination.Materials and methods: the study used four batches of BBIBP-CorV by CNBG, Sinopharm (China) and three batches of CoronaVac by Sinovac Biotech (China). The authors desorbed SARS-CoV-2 S antigen in accordance with monograph FS.3.3.1.0029.15 of the State Pharmacopoeia of the Russian Federation (Ph. Rus.), edition XIV, and quantified it using the BioScan-SARS-CoV-2 (S) ELISA kit by Bioservice Biotechnology Co. Ltd. (Russia).Results: mean S-antigen concentrations in the desorbed samples ranged from 61 to 129 ng/mL for BBIBP-CorV and from 461 to 533 ng/mL for CoronaVac.Conclusions: the study demonstrated the possibility of specific SARS-CoV-2 antigen desorption from the surface of aluminium hydroxide using the Ph. Rus. method, as well as the possibility of S-antigen quantification in desorbed medicinal products and supernatants using the BioScan-SARS-CoV-2 (S) ELISA kit. The authors observed 3.6- to 8.7-fold difference between the S-antigen concentrations of the desorbed preparations by the two manufacturers.
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TRUJILLO, Mary, Luz M. SALAZAR, and Jesús VALENCIA. "VACCINE FORMULATION: ADSORPTION OF <I>Plasmodium falciparum</I> MSP-1 PEPTIDE 1585 ON ALUMINIUM HYDROXIDE." Vitae 18, no. 2 (September 2, 2011): 183–91. http://dx.doi.org/10.17533/udea.vitae.10070.
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The Plasmodium falciparum merozoite surface protein 1 has been studied due to its potential to become a vaccine; likewise, the peptide 1585 which is located in the 42-kDa amino-terminal fragment induces protective immunity in primates. Despite the importance of antigen adsorption in the formulation and production of vaccines containing aluminium adjuvant, the protein fragment adsorption on aluminium hydroxide has not been thoroughly studied. Electrostatic attraction, hydrophobic interaction and ligand exchange have been identified as the major mechanisms involved in antigen retention on the adsorbent surface. Peptide 1585 was synthesized, and its solubility, adsorption on aluminium hydroxide, as well as its molecule release have been studied here. Results allowed us to raise a model for the adsorption and release of this peptide, which are important parameters to establish optimal conditions for peptideadsorbent interaction and, therefore, their response as a vaccine. Results also established the reversibility of such process due to the phosphate ion effect.
Thus, this work provides a starting point for research works, leading to further development of vaccine formulations containing highly purified synthetic antigens adsorbed on aluminium adjuvant.
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Miletich, JP, and GJ Jr Broze. "Human plasma protein Z antigen: range in normal subjects and effect of warfarin therapy." Blood 69, no. 6 (June 1, 1987): 1580–86. http://dx.doi.org/10.1182/blood.v69.6.1580.bloodjournal6961580.
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In contrast to the other well-studied vitamin K-dependent proteins that circulate in plasma, protein Z antigen is much more variable. The concentration in plasmas collected in EDTA from 455 normal, healthy donors is normally distributed with a mean of 2.9 micrograms/mL (46 nmol/L) and a SD of 1.0 microgram/mL (95% interval of 32% to 168% of the mean). No significant correlation to age or sex could be detected. In comparison, the concentration of protein C antigen measured with the same type of assay on the same 455 samples has a log normal distribution with a mean of 4.0 micrograms/mL (65 nmol/L) and a 95% interval of 70% to 138% of the mean. Also in marked contrast to other plasma vitamin K-dependent proteins, the total protein Z antigen level is extremely low in patients on stable warfarin therapy (range 1% to 16% of normal). Moreover, even though greater than 95% of the antigen in normal plasmas adsorbs to barium citrate (a crude reflection of the presence of gamma-carboxyglutamic acid (Gla) residues), in the patients taking warfarin almost all of the small amount of the antigen failed to adsorb, suggesting that virtually no protein Z had its full complement of Gla residues. Total protein C antigen in the same 25 patients averaged 53% of normal (34% to 72%) and 54% (average) of the total remaining antigen still adsorbed to barium citrate. The concentration of protein Z antigen in the plasma of a normal individual given a loading dose of warfarin fell at an initial rate of approximately 20% a day, indicating a plasma half-life (t1/2) of 2 to 3 days.
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Dunstan, RA, MB Simpson, RW Knowles, and WF Rosse. "The origin of ABH antigens on human platelets." Blood 65, no. 3 (March 1, 1985): 615–19. http://dx.doi.org/10.1182/blood.v65.3.615.615.
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Abstract ABH antigens are present on platelets from individuals of the corresponding red cell phenotype, but the extent to which these antigens are intrinsic or adsorbed remains undefined. To evaluate platelets for intrinsic H substance, an IgM mouse monoclonal antibody against type 2H chain (the intrinsic H structure found on erythrocytes) was labeled with 125I and incubated with platelets from donors of different ABO type. The antibody showed dose-response saturation curves, and binding to platelets paralleled that of the red cell ABO type, with O greater than B greater than A1 greater than A1B greater Oh cells, giving a single factor variance F of 190 (P less than .0005). Passive adsorption of A antigens by platelets has been previously reported. To verify this phenomenon for A and B antigens and to quantitate the elution of A and B antigens from platelets, the following assay system was used. Platelets from group A1 and B donors were incubated in plasma from group O donors, and platelets from group O donors were incubated in plasma from different ABO, Lewis, and presumed secretor-type donors. Human IgG anti-A or anti-B was added to the platelets. The amount of antibody bound was determined with a 125I- labeled mouse monoclonal anti-human IgG. When incubated for 96 hours in group O plasma, group A1 platelets showed a 45% to 50% decrease in binding of anti-A. There was no significant change in the level of type 2H antigen on these platelets during the same incubation period. Group O platelets incubated in A or B plasmas rapidly acquired the antigens, but if returned to their original plasma, 95% of this passively adsorbed antigen eluted off within 18 hours. The maximum uptake of A and B substances was influenced by the Lewis and secretor type of donor plasma. Our present study demonstrates that ABH antigens on platelets consist of type 2H chains, which are presumably intrinsic as when found on red cells, and of passively adsorbed ABH structures, which are presumably type 1H chains.
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Dunstan, RA, MB Simpson, RW Knowles, and WF Rosse. "The origin of ABH antigens on human platelets." Blood 65, no. 3 (March 1, 1985): 615–19. http://dx.doi.org/10.1182/blood.v65.3.615.bloodjournal653615.
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ABH antigens are present on platelets from individuals of the corresponding red cell phenotype, but the extent to which these antigens are intrinsic or adsorbed remains undefined. To evaluate platelets for intrinsic H substance, an IgM mouse monoclonal antibody against type 2H chain (the intrinsic H structure found on erythrocytes) was labeled with 125I and incubated with platelets from donors of different ABO type. The antibody showed dose-response saturation curves, and binding to platelets paralleled that of the red cell ABO type, with O greater than B greater than A1 greater than A1B greater Oh cells, giving a single factor variance F of 190 (P less than .0005). Passive adsorption of A antigens by platelets has been previously reported. To verify this phenomenon for A and B antigens and to quantitate the elution of A and B antigens from platelets, the following assay system was used. Platelets from group A1 and B donors were incubated in plasma from group O donors, and platelets from group O donors were incubated in plasma from different ABO, Lewis, and presumed secretor-type donors. Human IgG anti-A or anti-B was added to the platelets. The amount of antibody bound was determined with a 125I- labeled mouse monoclonal anti-human IgG. When incubated for 96 hours in group O plasma, group A1 platelets showed a 45% to 50% decrease in binding of anti-A. There was no significant change in the level of type 2H antigen on these platelets during the same incubation period. Group O platelets incubated in A or B plasmas rapidly acquired the antigens, but if returned to their original plasma, 95% of this passively adsorbed antigen eluted off within 18 hours. The maximum uptake of A and B substances was influenced by the Lewis and secretor type of donor plasma. Our present study demonstrates that ABH antigens on platelets consist of type 2H chains, which are presumably intrinsic as when found on red cells, and of passively adsorbed ABH structures, which are presumably type 1H chains.
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Werthén, Maria, and Håkan Nygren. "Cooperativity in the antibody binding to surface-adsorbed antigen." Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 1162, no. 3 (March 1993): 326–32. http://dx.doi.org/10.1016/0167-4838(93)90298-6.
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Miletich, JP, and GJ Jr Broze. "Human plasma protein Z antigen: range in normal subjects and effect of warfarin therapy." Blood 69, no. 6 (June 1, 1987): 1580–86. http://dx.doi.org/10.1182/blood.v69.6.1580.1580.
Full textAbstract:
Abstract In contrast to the other well-studied vitamin K-dependent proteins that circulate in plasma, protein Z antigen is much more variable. The concentration in plasmas collected in EDTA from 455 normal, healthy donors is normally distributed with a mean of 2.9 micrograms/mL (46 nmol/L) and a SD of 1.0 microgram/mL (95% interval of 32% to 168% of the mean). No significant correlation to age or sex could be detected. In comparison, the concentration of protein C antigen measured with the same type of assay on the same 455 samples has a log normal distribution with a mean of 4.0 micrograms/mL (65 nmol/L) and a 95% interval of 70% to 138% of the mean. Also in marked contrast to other plasma vitamin K-dependent proteins, the total protein Z antigen level is extremely low in patients on stable warfarin therapy (range 1% to 16% of normal). Moreover, even though greater than 95% of the antigen in normal plasmas adsorbs to barium citrate (a crude reflection of the presence of gamma-carboxyglutamic acid (Gla) residues), in the patients taking warfarin almost all of the small amount of the antigen failed to adsorb, suggesting that virtually no protein Z had its full complement of Gla residues. Total protein C antigen in the same 25 patients averaged 53% of normal (34% to 72%) and 54% (average) of the total remaining antigen still adsorbed to barium citrate. The concentration of protein Z antigen in the plasma of a normal individual given a loading dose of warfarin fell at an initial rate of approximately 20% a day, indicating a plasma half-life (t1/2) of 2 to 3 days.
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Zhao, Xiubo, Fang Pan, Luis Garcia-Gancedo, Andrew J. Flewitt, Gregory M. Ashley, Jikui Luo, and Jian R. Lu. "Interfacial recognition of human prostate-specific antigen by immobilized monoclonal antibody: effects of solution conditions and surface chemistry." Journal of The Royal Society Interface 9, no. 75 (May 2, 2012): 2457–67. http://dx.doi.org/10.1098/rsif.2012.0148.
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The specific recognition between monoclonal antibody (anti-human prostate-specific antigen, anti-hPSA) and its antigen (human prostate-specific antigen, hPSA) has promising applications in prostate cancer diagnostics and other biosensor applications. However, because of steric constraints associated with interfacial packing and molecular orientations, the binding efficiency is often very low. In this study, spectroscopic ellipsometry and neutron reflection have been used to investigate how solution pH, salt concentration and surface chemistry affect antibody adsorption and subsequent antigen binding. The adsorbed amount of antibody was found to vary with pH and the maximum adsorption occurred between pH 5 and 6, close to the isoelectric point of the antibody. By contrast, the highest antigen binding efficiency occurred close to the neutral pH. Increasing the ionic strength reduced antibody adsorbed amount at the silica–water interface but had little effect on antigen binding. Further studies of antibody adsorption on hydrophobic C8 (octyltrimethoxysilane) surface and chemical attachment of antibody on (3-mercaptopropyl)trimethoxysilane/4-maleimidobutyric acid N -hydroxysuccinimide ester-modified surface have also been undertaken. It was found that on all surfaces studied, the antibody predominantly adopted the ‘flat on’ orientation, and antigen-binding capabilities were comparable. The results indicate that antibody immobilization via appropriate physical adsorption can replace elaborate interfacial molecular engineering involving complex covalent attachments.
Dissertations / Theses on the topic "Adsorbed antigen"
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Hogan, Victoria. "Characterization of HLA Class I antigens on platelets as integral or adsorbed membrane proteins." Thesis, University of Ottawa (Canada), 1991. http://hdl.handle.net/10393/7708.
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To determine whether HLA-A,B antigens on platelets are integral membrane constituents or simply represent adsorbed plasma proteins, the degree to which they are adsorbed, and the relative ease with which they elute off platelet membranes was studied using various treatments known to elute passively adsorbed membrane proteins. In addition, this question was investigated at the RNA level using phytohemagglutinin stimulation to determine whether platelets have the capacity to respond with de-novo synthesis of HLA antigens and by enzymatic amplification of platelet derived mRNA to attempt to demonstrate the presence of nascent message encoding for these antigens. HLA antigen present on platelet membranes did not elute when platelets were incubated in autologous plasma or in plasma from homologous, antigen negative donors. When HLA-A2 negative platelets were incubated in HLA-A2 positive plasma a small amount of HLA-A2 antigen was detectable indicating that platelets possess the ability, to a limited extent, to absorb HLA antigen from the plasma, in vitro. The results indicate that the majority of HLA antigen present on platelet membranes can be selectively eluted without a concomitant loss of known integral membrane proteins such as GPIIIa. These findings argue in favor of the existent hypothesis that HLA antigens are absorbed platelet membrane proteins. (Abstract shortened by UMI.)
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HUETZ, PHILIPPE, and Pierre Schaaf. "Etude de la reactivite d'anticorps vis-a-vis de leurs antigenes adsorbes a une interface solide/solution aspects dynamiques de processus interfaciaux." Université Louis Pasteur (Strasbourg) (1971-2008), 1994. http://www.theses.fr/1994STR13223.
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Le but de cette these est d'etudier les phenomenes de reactivite et d'echange entre des proteines a une interface solide/liquide. Elle comprend une description des differents aspects lies aux proteines utilisees (caracteristiques physicochimiques du fibrinogene et des immunoglobulines g, structure, fonction) et a la structure de l'adsorbant (silice). Elle resume certaines proprietes importantes concernant l'adsorption, la desorption et l'echange de proteines a l'interface, et presente la liste de toutes les techniques et methodes experimentales utilisees. Les trois derniers chapitres concernent (1) l'etude de la reactivite d'anticorps vis-a-vis de leurs antigenes, lorsque ces derniers sont adsorbes directement sur une surface solide, (2) l'etude de la reactivite des memes systemes antigene/anticorps, lorsque l'antigene est adsorbe par un processus d'echange sur une couche proteique preadsorbee, et (3) une analyse dynamique et quantitative du processus d'echange heterogene entre des immunoglobulines g adsorbees et du fibrinogene en solution
Book chapters on the topic "Adsorbed antigen"
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Scheicher, Christoph, Maria Mehlig, Hans-Peter Dienes, and Konrad Reske. "Uptake of Bead-Adsorbed Versus Soluble Antigen by Bone Marrow Derived Dendritic Cells Triggers Their Activation and Increases Their Antigen Presentation Capacity." In Advances in Experimental Medicine and Biology, 253–55. Boston, MA: Springer US, 1995. http://dx.doi.org/10.1007/978-1-4615-1971-3_56.
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Huetz, P., P. Schaaf, J. C. Voegel, E. K. Mann, B. Miras, V. Ball, M. Freund, and J. P. Cazenave. "Reactivity of Antibodies on Antigens Adsorbed on Solid Surfaces." In ACS Symposium Series, 334–49. Washington, DC: American Chemical Society, 1995. http://dx.doi.org/10.1021/bk-1995-0602.ch024.
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Şenel, Behiye, and Gülay Büyükköroğlu. "Nanocarriers for Vaccine and Gene Delivery Application." In Multifunctional Nanocarriers for Contemporary Healthcare Applications, 381–414. IGI Global, 2018. http://dx.doi.org/10.4018/978-1-5225-4781-5.ch014.
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Nanocarriers with various compositions and biological properties are frequently used systems for in-vitro/in-vivo vaccination and gene transfer. In recent years, developments in nanotechnology have focused on the design and synthesis of nanocarriers that have new properties and can be modified for gene and vaccine delivery. In the favorable results obtained from in-vivo studies performed, they increase interest in these developments and pave the way for their therapeutic use. Nanocarriers have become increasingly important because they can stabilize vaccine antigens and serve as adjuvants, with the advantage of easily transporting genetic material to the target site. In nanocarriers, the molecules involved are adsorbed to the surface or encapsulated in particulates. At the same time, surface modification of nanoparticles allows these systems to carry cargo molecules easily to target site. Among the most studied nanocarriers, lipidic and polymeric systems dendrimers, inorganic nanoparticles, cyclodextrins, cell penetration peptides, and ISCOMs are attracting attention.
Conference papers on the topic "Adsorbed antigen"
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Kieffer, N., M. Titeux, A. Henri, J. Breton-Gorius, and W. Vainchenker. "MEGAKARYOCYTIC ORIGIN OF PLATELET HLA CLASS I ANTIGEN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643546.
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The existence of HLA class I antigens on human platelets is well established. However, several authors have suggested that platelet HLA antigens are not integral membrane components but are acquired from soluble plasma sources and adsorbed to the platelet surface.In the present study, we used the monoclonal antibody W6/32, directed against a monomorphic epitope of the HLA class I antigen for the immunochemical characterization of platelet HLA. Immunoprecipitation experiments, performed after in vitro metabolic radiolabeling of human platelets revealed a band of molecular weight 44,000 identical to that precipitated from metabolic labeled U937 or HEL cells. When the same antibody was tested by indirected immunofluorescence in a double labeling technique on in vitro cultures of human megakaryocytes, performed in the absence of human serum in the culture medium, megakaryocytes identified by an anti-vWF MoAb revealed a membrane staining with W6/32 identical to that observed on other bone marrow cells, e.g. macrophages. Our results provide evidence that platelet HLA has a megaka-ryocytic origin and that residual biosynthesis of HLA antigen does still occur in circulating platelets. However, our results do not exclude the ability of human platelets to adsord circulating HLA class I antigen from plasma.
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Gao, Yali, Philip M. Sherman, Yu Sun, and Dongqing Li. "Multiplexed High-Throughput Electrokinetically-Controlled Immunoassay on a Chip for the Detection of Specific Bacterial Antibodies in Human Serum." In ASME 2007 International Mechanical Engineering Congress and Exposition. ASMEDC, 2007. http://dx.doi.org/10.1115/imece2007-42512.
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This work presents a multiplexed electrokinetically-controlled heterogeneous immunoassay that can process ten samples in parallel. The immunoassay microchip was soft-lithographically fabricated using poly(dimethylsiloxane) and glass. Controlling parameters of the electrokinetically-driven flow in the microfluidic network was determined by numerically simulating transport processes. Multiple passively adsorbed antigens captured antibodies present in samples, which then bound with TRITC-labeled detection antibodies to generate fluorescent signals. Antibodies against Escherichia coli O157:H7 and Helicobacter pylori were studied as model analytes. After conditions for antigen-coating were optimized, a 24-minute assay detected E. coli O157:H7 antibody in the concentration range of 0.02–10 μg/mL, and H. pylori antibody in the range of 0.1–50 μg/mL. In testing human serum samples, non-specific binding of serum components was effectively suppressed by using 10% (w/v) bovine serum albumin. An accuracy of 100% was achieved in detecting either E. coli O157:H7 antibody or H. pylori antibody from human serum samples. Simultaneous screening of both antibodies was also successfully demonstrated. The immunoassay chip shows an excellent potential for efficiently detecting multiple pathogenic infections in clinical environments.
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Bilyi, Olexander I., Yevgen M. Kiselyov, and Volodymyr P. Novikov. "Creation of aggregate latex complexes with protein adsorbed on their surface for the study of antigen-antibody reactions with light-scattering method." In BiOS 2000 The International Symposium on Biomedical Optics, edited by Tuan Vo-Dinh, Warren S. Grundfest, and David A. Benaron. SPIE, 2000. http://dx.doi.org/10.1117/12.384926.
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Mannhalter, C., and E. Deutsch. "IMMUN0BL0TTING STUDIES OF THE INTERACTION OF PLASMA-FACT0R XI WITH KAOLIN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644806.
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An immunoblotting technique with a commercially available, polyclonal Factor XI antibody was developed for Factor XI in plasma samples.Aliquots containing equivalents of 6 ul plasma were treated with appropriate amounts of SDS, electrophoresed on 7.5 % SDS-PAGE and immunoblotted onto nitrocellulose membranes. The blots were quenched with low fat milk and incubated with 2 % Factor Xl-antiserum, which was preadsorbed with Factor XI deficient plasma. The bound Factor XI antibody was identified by incubation with 125I-labelled Factor XI followed by autoradiography. Using this method, the interaction of Factor XI with kaolin was studied in plasma. Normal plasma was incubated with kaolin for various time periods. Aliquots were removed, centrifuged and the plasma supernatant and the kaolin pellets were examined after treatment with SDS on 7.5 % SDS-PAGE followed by immunoblotting.Factor XI was adsorbed onto kaolin and, after five minutes no Factor XI antigen was present in the plasma supernatant.However, after twenty minutes a band corresponding to Factor XI became again visible in the plasma, indicating a release of Factor XI from the kaolin surface. The electrophoretic results obtained with the SDS-eluates of the kaolin pellets confirm these observations. After thirty seconds a significant amount of Factor XI was present on the kaolin, and it exhibited the same electrophoretic mobility as Factor XI in the starting material. After ten minutes incubation, the protein concentration adsorbed onto the kaolin had increased. However, after twenty minutes clearly less Factor XI was present on the kaolin than after ten minutes.Thus, our results indicate that Factor XI is quickly adsorbed from plasma onto kaolin. However, Factor XI or XIa do not remain surface-bound but are released into solution.
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Eckhardt, Th, S. Haas, B. Lange, and H. Pfeiffer. "MEASUREMENT OF ELASTASE-INDUCED FIBRINOGEN-DERIVED PEPTIDES IN VITRO." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643165.
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Leukocyte elastase (EL) cleaves peptides from the N-terminal fibrinogen (fbg) Aα and B6 chains. The Aα peptide (Aα 1-21) and the as yet unidentified BIB peptide contain thrombin (TB)cleavage sites. As we were interested in fbg proteolysis in sepsis and leukemia, we have tried to measure these peptides (generated by incubating a crude granulocyte extract with fbg) using available fibrinopeptide A (FPA) - und Bß 15-42-antigen-RIA techniques. As the indirect measurement of Aα 1-21 in terms of FPA releasable by TB treatment in vitro (TB-inducible FPA, TIFPA) requires the use of a highly specific anti-FPA-antiserum to avoid cross reaction of Aα 1-21 we tested the specificity of several antisera. Only R2, provided by Dr. J. Owen, proved suitable whereas two commercial antisera measured 6-15% of Aα 1-21 as apparent FPA. Simultaneous recovery of FPA and Aα 1-21 from plasma using R2 was between 80-100% after precipitation of cross-reacting fbg by ethanol, whereas bentonite adsorbed Aα 1-21 and the EL induced BIB peptide. TIFPA was fully recovered after incubation with plasma (37°C, 2 hr) whereas cross-reactivity of Aα 1-21 in the FPA-assay did not increase. EL-induced proteolysis in plasma in terms of TIFPA generation did not occur unless the normal granulocyte-plasma ratio was increased about 300-400 fold. The Bß 15-42 antigen RIA allowed quantitative measurement of the as yet undefined EL-induced Bß peptide, since the "Bß 15-42" concentration measured was equal to the amount of FPB (measured as Bß 1-13) releasable from the EL-induced BIB-peptide by TB. The immunoreactivity of this peptide is stable in plasma and completely recovered after ethanol precipitation. This finding suggests that the EL-induced BIB peptide is longer than Bß 1-4 2 and not susceptible to C-terminal degradation of the Bß41/42 region which is crucial for recognition of the peptide by the antiserum (supported by Deutsche Forschungs-gemeinschaft).
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Hubbard, A. R., and C. A. Jennings. "TISSUE FACTOR-FACTOR VII INHIBITION REQUIRES FACTOR Xa AND PLASMA LIPOPROTEINS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643291.
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Tissue factor rapidly loses procoagulant activity when incubated with defibrmated normal plasma and calcium ions. Inhibition is apparently directed against the tissue factor-Factor VII complex (TF-EVII) and requires Factor Xa and a component(s) found in A1 (OH)3-adsorbed plasma (AP). We have developed a two stage assay for the inhibitor which involves first, the incubation of a TF-FVII complex with test material in the presence of Factor Xa, followed by the amidolytic assay of residual TF-EVII activity.Our studies have indicated that the component of AP responsible for this effect is lipoprotein. Incubation of AP with antiserum to apo-lipoprotein B (apo B) reduced the inhibitory activity by 73%, whereas antisera to antithrombin III and a2-macroglobulin had no effect. Inhibition by AP does not appear to be caused by an artefact of adsorption, since the inhibitory-capacity of AP was 59% of normal, defibrinated plasma. This correlated well with the apo B antigen in AP, which was 64% of normal. Moreover, the dose/response lines of AP and normal plasma were parallel, suggesting that the inhibitor assay is not affected by the presence of normal levels of coagulation factors.Purified lipoprotein-rich fractions prepared from AP using density gradient ultracentrifugation all contained inhibitory activity. Incubation of these fractions with anti-apo B greatly reduced the inhibition by the very low density and low density lipoprotein-rich fractions (VLDL and LDL) but had essentially no effect on the high density lipoprotein-rich fractions (HDL). Incubation of LDL with Factor Xa produced an inhibitory component which eluted together with the apo B antigen during gel filtration. Inhibition appears to require the interaction of Factor Xa with plasma lipoproteins, particularly LDL. The product of this interaction is then able to bind and inhibit the TF EVII complex. The requirement of Factor Xa in order for inhibition to be expressed is indicative of a feedback anticoagulant response which may have physiological significance.
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Mahmoud, M., F. Hammerschmidt, H. Scheuerlein, and P. J. Gaffney. "IMMUNOASSAYS FOR SINGLE CHAIN URINARY-TYPE PLASMINOGEN ACTIVATOR (SCUPA) IN PLASMA AND IN CELL CULTURE SUPERNATANTS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643604.
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Monoclonal antibodies (mabs) to SCUPA have been generated in balb/C mice by conventional means and have been demonstrated to have no crossreactivity with two chain urinary-type plasminogen activator (TCUPA). These mabs have been used to develop two types of specific assay for SCUPA. Mabs coated on polyvinyl plates in conjunction with polyclonal antibodies (pabs) to TCUPA have allowed the development of a catcher-tag ELISA using alkaline phosphatase-label led goat anti-rabbit IgG as a final step. The sensitivity range of the assay was 0.5 - 10.0 iu/ml. A second bioimmunoassay (BIA) using SCUPA mabs as catcher and inplate development with glu-plasminogen and S-2251 has yielded an assay with a sensitivity range of 0.5 - 10.0 iu/ml. The international unitage ascribed in these assays was derived by comparing the hydrolysis of S-2444 by the I.S. for TCUPA with the purified SCUPA following full activation with plasmin.Using these assays it was found that normal pooled plasmas contained about 1.0 iu of SCUPA antigen which was fully inhibited such that no activity was evident by the BIA assay for SCUPA. This suggests that urokinase in plasma is present in two forms: SCTJPA bound to inhibitor and TCUPA which is biologically active when assayed using a BIA based on immobilised pabs to TCUPA. Cell supernatants from cultured human lung fibroblasts yield a SCTJPA/TCUPA ratio of 70/30 using S-2444 chromogenic assay following a plasmin-mediated SCTJPA-TCUPA conversion step. It was also shown that, in these cell supernatants, SCTJPA was secreted with no inhibitor bound to it, since the BIA and ELISA data were quite similar. A curious feature of these assays, which is as yet unexplained, is the observation that urokinase (both plasmin activated SCTJPA and TCUPA), when immunologically adsorbed on to the PVC-immobilised specific mabs or pabs used in this study, readily activated plasminogen but showed no hydrolytic activity on the chromogenic substrate, S-2444.
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Boyer-Neumann, C., M. Wolf, J. Amiral, A. M. Guyager, D. Meyer, and M. J. Larrieu. "FAMILIAL TYPE I PROTEIN S DEFICIENCY ASSOCIATED WITH SEVERE VENOUS THROMBOSIS. A STUDY OF FIVE CASES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642943.
Full textAbstract:
Protein S deficiency has been demonstrated in 5 members from the same family with a history of severe recurrent venous thrombosis over three generations. The propositus, a 16 year old female, had a first spontaneous thrombotic episode at age 15. Phlebography revealed a total obstruction of her left ilio-femoral vein with an extension to the vena cava. She was treated with heparin followed by oral anticoagulant therapy. The four other affected members (mother, aunts and uncle of the propositus) had also presented recurrent thrombosis with onset at a young age. The grandfather, not tested, had died from massive pulmonary embolism at age 54. The immunological assay of protein S was performed in plasma by Laurell, using a monospecific antiserum to human protein S, or by an ELISA, using a kit from Diagnostica Stago (Asserachrom Protein S). In order to separate free protein S, the functionally active form, from protein S complexed with C4-binding protein, plasma was adsorbed with 3.75 % polyethyleneglycol (PEG 6000). Following PEG precipitation, the levels of free protein S antigen remaining in the supernatant were quantitated by the usual immunological methods. In addition, two-dimensional immunoelectrophoresis (DDIE) also provided information on the distribution of both forms. In plasma protein S levels were decreased (40 to 55 % of the normal range) in two untreated patients and lower levels (17 to 20 96) were observed in the three others, including the propositus, who were under dicoumarol therapy. In PEG treated-plasma, protein S was undetectable (less than 5 %) in all patients, indicating a lack of free protein S. This was confirmed by DDIE : whereas protein S migrated as two distinct peaks, corresponding to free and complexed protein S in normal plasma, only a single peak of complexed protein S was observed in all affected patients. These results clearly demonstrate a total lack of free protein S which appears to be responsible for the thromboembolic disorder in this family as there was no deficiency of the other plasma inhibitors (antithrombin III, heparin cofactor II and protein C). According to the classification recently proposed by Comp et al., this family belongs to type I protein S deficiency, with an autosomal dominant mode of inheritance.
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Church, W., T. Messier, P. Howard, J. Amiral, D. Meyer, and K. Mam. "A SHARED EPITOPE ON HUMAN PROTEIN C, FACTOR X, FACTOR VII, AND PROTTOBIN DEFINED BY A MONOCLONAL ANTIBODY." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643937.
Full textAbstract:
A monoclonal antibody prepared against hunan protein C (HPC) was found to react with several other vitamin K-dependent blood proteins. Using a competitive inhibition solid-phase radioinminoassay with HPC, binding of 125I-HPC to the antibody was inhibited by purified prothrombin, Factor X, and Factor VII in addition to protein C. Other vitamin K-dependent proteins including Factor IX, protein S, and bone-GLA protein did not compete for binding of 125I-HPC to the antibody. The effect of calciun ion on the binding of antibody to 125I-HPC was examined in a solid-phase imnunoassay system with the antibody bound to rabbit anti-mouse inminoglobulin adsorbed to microtiter plates. In the presence of 5 mM calciun ion, radiolabeled protein C did not bind to the antibody; radiolabeled protein C did bind, however, in the presence of 5 nM EDTA suggesting that the epitope is expressed only after removal of calciun ion. The antibody bound to prothrombin and to decarboxylated prothrombin after adsorption of the antigens onto nitrocellulose indicating that the presence of GLA was not required for antibody binding. Iimunoblotting of proteins which were reduced, the peptides separated by SDS-PAGE, and transferred to nitrocellulose showed that the antibody reacts with a determinant found on the light chains of protein C and Factor X and with prothrombin Fragment 1. Comparison of the protein sequences of protein C light chain, Factor X light chain, Factor VII, and prothrombin Fragment 1 identified a segment of amino acid sequence that is highly conserved in all four proteins and might contain the antigenic site. The monoclonal antibody thus defines an antigenic determinant which is masked by calcium ion and is found on the surface of several related, yet different coagulation proteins. This antibody should prove useful in understanding the evolutionary relationships amongst the vitamin K-dependent proteins and also in understanding the effect of calcium ion on the structure of protein C, Factor X, prothrombin, Factor VII and possibly other related proteins. (Supported by NIH grant MHLBI HL35058)
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Fujikawa, K., T. Funakoshi, R. L. Heimark, and J. F. Tait. "HUMAN PLACENTAL ANTICOAGULANT PROTEIN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642949.
Full textAbstract:
Endothelium is important to maintain blood fluidity preventing coagulation. Glycosaminoglycan in the endothelial cell plasma membrane has been thought to prevent activation of blood coagulation. Heparin-like compound, which is a potent anticoagulant activity, has been localized on the surface of the cultured endothelial cells. Anticoagulant action associated with thrombomodulin, which is present in endothelial cells, is another mechanism to provide hemostatic nature of endothelial cells.We wondered whether any other intracellular protein(s) is involved in coagulation. We looked for such a protein(s) in cultured bovine aortic endothelial cells. We soon found an anticoagulant activity in the soluble fraction of endothelial cells and it was partially purified. This activity was adsorbed to DEAE-Sepharose and eluted from a gel filtration column in a molecular weight range of 30,000-40,000. However, limited amounts of the cells made it difficult to purify this activity. We then chose human placenta as a substitute source of this protein and have continued the purification of this anticoagulant activity.In this communication, we describe the isolation and characterization of a placental anticoagulant protein, called "PAP", which is silmilar or possible same as the endothelial anticoaguant protein. PAP was purified from the soluble fraction of human placenta by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose, Sephadex G-75, and mono S (Pharmacia). Approximately 20 mg of the protein was purified from one placenta. The purified protein gave a single band by SDS polyacrylamide gel electrophoresis with a molecular weight of 36,500. This protein inhibited both kaolin- and thromboplastin-induced partial thromboplastin times of normal human plasma. It also inhibited the clotting time of platelet-rich plasma induced by factor Xa, but did not affect the thrombin activity of fibrinogen-fibrin conversion. The purified protein completely inhibited the prothrombin activation by reconstituted prothrombinase. The protein neither inhibited the amidolytic activity of factor Xa nor bound factor Xa. This protein specifically bound to phospholipid vesicles (20% phosphatidylserine and 80% phosphatidylcholine) in the presence of calcium ions. These results indicate that PAP inhibits coagulation through the binding to phospholipid vesicles. The study on the amino acid sequence of PAP is in progress in our laboratory. Surprisingly, the sequence analysis of the cyanogen bromide fragments revealed that PAP is a new member of the lipocortin or calpactin family. The sequences of several cyanogen bromide fragments of PAP aligns with the sequences of lipocortin I and II with over 50% identity.Since PAP interacts directly with phospholipid rather than factor Xa, other activation steps in the coagulation cascade, in which phospholipid is involved, are pro^|bly affected by PAP. These reactions are the activation of factor X by a complex of factor IXa-factor VIIIa-phospholipid-Ca++ and the activations of factor X and factor IX by a tissue factor-factor VIIa-Ca++ complex.Reutelingsperger et. al,, have reported the isolation of a novel inhibitor from arteries of human umbilical cord. This protein inhibited the prothrombin activation by prothrombinase. The authors proposed that the inhibition mechanism of this inhibitor was a competition with factor Xa for binding to phospholipid. This protein is very similar to PAP as to the mode of inhibition. The molecular weight of this inhibitor is 32,000, which is slightly smaller than PAP. With the limited chemical characterization of this protein, presently it is difficult to identify this inhibitor with PAP.At the present time, the physiological role and origin of PAP is not known. PAP may originate from the endothelium of placenta, because we have detected a PAP-like anticoagulant activity in bovine aortic endothelial cells. This activity and PAP were quite alike in the purification up to the gel filtration step. If PAP antibody recognizes the antigen in the endothelial cells, it is interesting to see whether PAP localizes on the surface or inside the cells. Nevertheless, if PAP is present in the endothelial cells, it may play an important role to maintain the hemostatic nature of endothelium. PAP may bind phospholipid components at injured sites, before coagulation factors come in contact with lipid components and initiate thrombolytic events.