Journal articles: 'MAB PURIFICATION'

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Shinagawa, Kunihiro, Katsuhiko Omoe, Naonori Matsusaka, and Shunji Sugii. "Immunological studies on staphylococcal enterotoxin D: production of murine monoclonal antibodies and immunopurification." Canadian Journal of Microbiology 37, no. 8 (August 1, 1991): 586–89. http://dx.doi.org/10.1139/m91-099.

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Eight murine monoclonal antibodies (MAbs) against staphylococcal enterotoxin D (SED) were obtained by fusion of myeloma cells with mouse spleen cells immunized with SED only or a combination of SED and either enterotoxin A (SEA) or enterotoxin E (SEE). When only SED was used as an immunogen, six MAbs were specific for SED only, whereas one MAb was reactive with both SED and SEE when both SEs were used as immunogens. One MAb reacted with SEA, SED, and SEE when both SEA and SED were used as immunogens. A MAb with the highest reactivity to SED was used to prepare an immunosorbent for purification of SED by immunoaffinity chromatography. Approximately 70% of the partially purified SED was recovered in the eluate. The purified SED was electrophoretically and antigenically pure. Immunoaffinity chromatography proved useful in the purification of SED in terms of ease of purification, percent enterotoxin, and enterotoxin purity. Key words: enterotoxin D, monoclonal antibodies, Staphylococcus aureus.

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Shi, Wenshu, Haiyang Hao, Mengran Li, Jianqin Niu, Yaning Hu, Xingbo Zhao, and Qiuyan Li. "Expression and Purification of a PEDV-Neutralizing Antibody and Its Functional Verification." Viruses 13, no. 3 (March 12, 2021): 472. http://dx.doi.org/10.3390/v13030472.

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Porcine epidemic diarrhea virus (PEDV) is a highly infectious and pathogenic virus causing high morbidity and mortality, especially in newborn piglets. There remain problems with contemporary PEDV vaccines, in part because of the rapid variation of PEDV, poor conferred immunity, and numerous side effects. The ability to produce PEDV-neutralizing antibodies suggests that we may be able to increase the success rate of PEDV prevention in piglets using these antibodies. In this study, we produced an anti-PEDV S protein monoclonal antibody (anti-PEDV mAb-2) that neutralized PEDV-CV777 (a G1 strain), PEDV-SDSX16 and PEDV-Aj1102 (two G2 strains). In vivo challenge experiments demonstrated that anti-PEDV mAb-2 inhibited the PEDV infection in piglets. We also produced three HEK293 cell lines that expressed anti-PEDV mAb-2. Overall, our study showed that anti-PEDV mAb-2 produced from hybridoma supernatants effectively inhibited PEDV infection in piglets, and the recombinant HEK293 cell lines expressed anti-PEDV mAb-2 genes.

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Deplazes, P., and B. Gottstein. "A monoclonal antibody against Echinococcus multilocularis Em2 antigen." Parasitology 103, no. 1 (August 1991): 41–49. http://dx.doi.org/10.1017/s0031182000059278.

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A monoclonal antibody (MAb G11) species-specific to the Em2 antigen of Echinococcus multilocularis was generated for (i) further biological characterization of the Em2 antigen, (ii) easy affinity-purification of Em2 antigen for immunodiagnostic and immunological investigations and (iii) development of a sandwich-ELISA for the detection of Em2 antigen in diagnostic samples and thus species-specific identification of E. multilocularis metacestode material. The MAb G11 was used in an antibody sandwich-ELISA to detect soluble Em2 antigen with a methodical sensitivity of 80 ng E. multilocularis antigen/ml of solution. MAb G11 specifically detected Em2 antigen in all of 15 E. multilocularis-isolates originating from various geographical areas and in none of other helminth isolates (e.g. Echinococcus granulosus, E. vogeli, and others). Further biological analysis by FITC-labelled MAb G11 demonstrated unique binding activity to the laminated layer of the metacestode. Also, oncospheres were binding FITC-labelled MAb G11 on an outer layer synthesized during cultivation in vitro for 13 days after hatching. Application of the MAb G11 antibody sandwich-ELISA for investigation of solubilized oncospheres confirmed the in vitro synthesis of Em2 antigen by oncospheres on day 13 p.i. Adult stages (somatic antigens) and freshly hatched oncospheres were always MAb G11 negative. Solid-phase MAb G11 was used for purification of the corresponding Em2 antigen by affinity chromatography. A preliminary serological evaluation of the Em2(G11) antigen by ELISA revealed identical immunodiagnostic characteristics, compared to Em2 obtained by classical means, thus suggesting the presented method for future isolation of large-scale Em2 antigen.

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Dasch, J. R., D. R. Pace, W. Waegell, D. Inenaga, and L. Ellingsworth. "Monoclonal antibodies recognizing transforming growth factor-beta. Bioactivity neutralization and transforming growth factor beta 2 affinity purification." Journal of Immunology 142, no. 5 (March 1, 1989): 1536–41. http://dx.doi.org/10.4049/jimmunol.142.5.1536.

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Abstract Four mAb able to recognize transforming growth factor-beta 2 (TGF-beta)2 were obtained. One of these mAb, 1D11.16, was able to neutralize the biological activity of both TGF-beta 1 and beta 2 in vitro. This was demonstrated in an Il-1, PHA-dependent thymocyte mitogenic assay that is inhibitable by TGF-beta in a dose-dependent manner. All four mAb recognized the dimeric form of TGF-beta 2 in Western blots. The mAb were also found to immunoprecipitate [125I]-TGF-beta 2. mAb 3C7.14 coupled to Sepharose could efficiently immunoaffinity purify TGF-beta 2 from a complex mixture of proteins. Affinity constants were determined for the four mAb and they ranged from 3.4 x 10(8) to 1.6 x 10(7) L/mol.

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Ziltener, H. J., I. Clark-Lewis, B. Fazekas de St Groth, P. C. Orban, L. E. Hood, S. B. Kent, and J. W. Schrader. "Monoclonal antipeptide antibodies recognize IL-3 and neutralize its bioactivity in vivo." Journal of Immunology 140, no. 4 (February 15, 1988): 1182–87. http://dx.doi.org/10.4049/jimmunol.140.4.1182.

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Abstract Mixtures of synthetic peptides corresponding to segments of murine IL-3 or synthetic IL-3 were used to raise murine mAb. Several mAb able to recognize synthetic IL-3 were obtained, two of which exhibited significant cross-reactivity with native IL-3 as shown by precipitation of biosynthetically 35S-labeled IL-3 and their effectiveness as affinity reagents for the purification of IL-3 from conditioned medium. The amino acid sequence recognized by the two mAb was determined by using synthetic peptide segments of IL-3. In both cases binding of the mAb to synthetic IL-3 was inhibited best with a hexapeptide corresponding to the amino acid residues 130-135 of IL-3, although the mAb differed in other characteristics. Neither mAb neutralized IL-3 bioactivity in vitro. However, we observed that in vivo administration of one mAb abrogated the increase in splenic mast cells and their precursors that normally occurred in mice bearing a s.c. IL-3-producing tumor, WEHI-3B.

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Harada, R., N. Okada, T. Fujita, and H. Okada. "Purification of 1F5 antigen that prevents complement attack on homologous cell membranes." Journal of Immunology 144, no. 5 (March 1, 1990): 1823–28. http://dx.doi.org/10.4049/jimmunol.144.5.1823.

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Abstract A mAb, 1F5, has the ability to cause hemolysis by human serum of human E treated with neuraminidase via the alternative C pathway. By Western blotting, this mAb reacts with a glycoprotein having a molecular mass of 20 kDa (1F5Ag). 1F5Ag was isolated from human E by affinity chromatography with mAb-coupled Sepharose. Purified 1F5Ag was then adsorbed to guinea pig E rendering them resistant to human C attack by both the classical and the alternative pathways. Furthermore, experiments with isolated C components revealed that 1F5Ag interferes with both homologous human C8 and C9 in the terminal stage of the C reaction, whereas it has little effect on hemolysis by rabbit C8 and C9. Therefore, 1F5Ag can be called HRF20, which stands for homologous restriction factor of 20 kDa.

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Kruse, Thomas, Axel Schmidt, Markus Kampmann, and Jochen Strube. "Integrated Clarification and Purification of Monoclonal Antibodies by Membrane Based Separation of Aqueous Two-Phase Systems." Antibodies 8, no. 3 (July 2, 2019): 40. http://dx.doi.org/10.3390/antib8030040.

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Therapeutic monoclonal antibodies (mAb) are used for the treatment of numerous serious diseases, which have led to an increasing demand over the last decades. Increased cell density and mAb titer of the cultivation broth lead to great challenges for the subsequent clarification and capture operations in the downstream process. As an alternative approach to the conventional downstream process, a selective mAb extraction via an aqueous two-phase system (ATPS) directly from the cultivation broth of a mAb producing industrial relevant chinese hamster ovary (CHO) cell line was investigated. An efficient purification of the mAb was accomplished by the ATPS composition. The phase separation was realized by a newly developed membrane based phase separator. Moreover, a complete cell removal was integrated into this process by the used membrane. A selectivity between both phases was achieved by membrane modification. Yields up to 93% in the light phase and removal of process related impurities were obtained after aqueous two-phase extraction (ATPE). Phase separation performance as well as contact angles on the membrane were characterized for different ATPS. ATPE directly from the cultivation broth in combination with the new membrane based phase separation led to a mAb yield of 78% with a simultaneous reduction of deoxyribonucleic acid (DNA) and host cell protein (HCP) load.

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Wei, Yuping, Jiandong Xu, Liang Zhang, Yankai Fu, and Xia Xu. "Development of novel small peptide ligands for antibody purification." RSC Advances 5, no. 82 (2015): 67093–101. http://dx.doi.org/10.1039/c5ra07829f.

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Small peptide ligands which were designed based on the interactions with human immunoglobulin G (IgG) using the molecular simulations, can offer a potential alternative for mAb purification with elution condition at pH 9 and pH 3.

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Santos, Carlos F., Andrew S. Greene, Maria Cristina O. Salgado, and Eduardo B. Oliveira. "Conversion of renin substrate tetradecapeptide to angiotensin II by rat MAB elastase-2." Canadian Journal of Physiology and Pharmacology 82, no. 11 (November 1, 2004): 1000–1005. http://dx.doi.org/10.1139/y04-102.

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A new approach for the purification of rat mesenteric arterial bed (MAB) elastase-2 has been developed using the chromogenic substrates N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide to monitor the enzymatic activity during various stages of purification. The purified enzyme was evaluated in the presence of various inhibitors and confirmed to have angiotensin (Ang) II-forming ability. The active site-directed inhibitor acetyl-Ala-Ala-Pro-Leu-chloromethylketone (100 µmol·L-1), described for human pancreatic elastase-2, abolished the enzymatic activity, confirming that the enzyme is an elastase-2. Chymostatin (100 µmol·L-1), an inhibitor regarded as selective for chymases, also showed a remarkable inhibitory effect (94%), whereas captopril (100 µmol·L-1) had no effect at all on the Ang II-forming activity. The Ang II precursor renin substrate tetradecapeptide (RS-14P) was converted into Ang II by the rat MAB elastase-2 with the following kinetic constants: Km = 124 ± 21 µmol·L-1; Kcat = 629 min-1; catalytic efficiency (Kcat /Km) = 5.1 min-1 µ(mol/L)-1. In conclusion, the strategy for the purification of rat MAB elastase-2 with the chromogenic substrates proved to be simple, rapid, accurate, and highly reproducible; therefore, it can be reliably and conveniently used to routinely purify this enzyme. The kinetic parameters for the formation of Ang II from RS-14P by rat MAB elastase-2 emphasize differences in substrate specificity between this and other Ang II-forming enzymes.Key words: N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide, elastase-2, angiotensin II, renin substrate tetradecapeptide.

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Valdés, Rodolfo, Tatiana Álvarez, Andrés Tamayo, Eutimio G. Fernandez, José Montero, Déborah Geada, William Ferro, et al. "New Mab CB.Hep-1 Purification Process Eliminates the Need for Pre-Chromatographic Purification. Stability Demonstrated Over 100 Purification Cycles." Chromatographia 67, no. 11-12 (April 18, 2008): 923–27. http://dx.doi.org/10.1365/s10337-008-0607-5.

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Leyton, Lisette, Alfredo De Ioannes, Horacio B. Croxatto, Elise J. Graham, and John S. Elce. "Two satisfactory methods for purification of human acrosin." Biochemistry and Cell Biology 64, no. 10 (October 1, 1986): 1020–24. http://dx.doi.org/10.1139/o86-135.

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Acrosin has been purified from human sperm cells by two alternative procedures which give purer products and in higher yields than could be achieved previously. The products were characterized by their molecular weight, catalytic action, sensitivity to inhibitors, and reaction with a polyclonal anti-acrosin antibody. After acid extraction of the cells, one method involves removal of acrosin inhibitors by vacuum dialysis, followed by affinity chromatography on a soybean trypsin inhibitor (SBTI) column, and therefore requires that the acrosin be in an active form capable of binding to the inhibitor. The other method involves affinity chromatography on a column of a monoclonal anti-acrosin antibody (MAb) and can be used to provide either active or proenzyme forms of acrosin, by choice of extraction conditions and inclusion of appropriate inhibitors. The yield of human acrosin from the SBTI method was 104% and from the MAb column was 75%. It is hoped that these procedures will make the very scarce human acrosin more readily available for further study.

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Jugler, Collin, Jussi Joensuu, and Qiang Chen. "Hydrophobin-Protein A Fusion Protein Produced in Plants Efficiently Purified an Anti-West Nile Virus Monoclonal Antibody from Plant Extracts via Aqueous Two-Phase Separation." International Journal of Molecular Sciences 21, no. 6 (March 20, 2020): 2140. http://dx.doi.org/10.3390/ijms21062140.

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The development of monoclonal antibodies (mAbs) has provided vast opportunities to treat a wide range of diseases from cancer to viral infections. While plant-based production of mAbs has effectively lowered the upstream cost of mAb production compared to mammalian cell cultures, further optimization of downstream processing, especially in extending the longevity of Protein A resin by an effective bulk separation step, will further reduce the overall prohibitive cost of mAb production. In this study, we explored the feasibility of using aqueous two-phase separation (ATPS) in capturing and separating plant-made mAbs from host proteins. Our results demonstrated that an anti-West Nile virus mAb (E16) was efficiently separated from most plant host proteins by a single ATPS step, comprising the mixing of plant extracts containing Hydrophobin-Protein A fusion protein (HPA) and E16 and the subsequent incubation with an inexpensive detergent. This simple ATPS step yielded a highly enriched E16 mAb preparation with a recovery rate comparable to that of Protein A chromatography. The ATPS-enriched E16 retained its structural integrity and was fully functional in binding its target antigen. Notably, HPA-based ATPS was also effective in enriching E16 from plant host proteins when both HPA and E16 were produced in the same leaves, supporting the potential of further streamlining the downstream purification process. Thus, ATPS based on plant-produced HPA in unpurified extract is a cost-effective yet efficient initial capture step for purifying plant-made mAbs, which may significantly impact the approach of mAb purification.

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Brenan, M., and M. Puklavec. "The MRC OX-62 antigen: a useful marker in the purification of rat veiled cells with the biochemical properties of an integrin." Journal of Experimental Medicine 175, no. 6 (June 1, 1992): 1457–65. http://dx.doi.org/10.1084/jem.175.6.1457.

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A mouse immunoglobulin G1 monoclonal antibody (mAb), MRC OX-62 (OX-62), was raised against density gradient-enriched rat veiled (dendritic) cells obtained from lymph. In suspensions of lymphoid cells, the OX-62 mAb only labeled cells with the characteristics of veiled cells. The OX-62 mAb was used with a magnetic cell sorter to enrich or deplete veiled cells, and the enriched veiled cells were potent stimulators in the primary allogeneic mixed leukocyte reaction. Immunohistochemical staining of tissue sections showed that the OX-62 mAb did not label all classical dendritic cells and was not restricted to this cell type. In lymphoid tissues, the labeling correlated with dendritic cells, but in skin, major histocompatibility complex class II+ cells were OX-62-, while another CD3+ cell with dendritic morphology was strongly OX-62+. It seems that the OX-62 mAb may be restricted to dendritic cells and probably to gamma/delta T cells. The OX-62 mAb will be of use in delineating minor subsets of cells with dendritic morphology in various tissues. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of veiled cell-enriched populations immunoprecipitated with the OX-62 mAb gave bands with the biochemical characteristics of an integrin. The OX-62 mAb recognized the alpha-like subunit.

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Chassagne, J., P. Verrelle, C. Dionet, F. Clavel, F. Barre-Sinoussi, J. C. Chermann, L. Montagnier, J. C. Gluckman, and D. Klatzmann. "A monoclonal antibody against LAV gag precursor: use for viral protein analysis and antigenic expression in infected cells." Journal of Immunology 136, no. 4 (February 15, 1986): 1442–45. http://dx.doi.org/10.4049/jimmunol.136.4.1442.

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Abstract A murine monoclonal antibody (MAb), named C.V.K., was produced after immunization with highly purified and sonicated lymphadenopathy-associated virus (LAV). No monoclonal antibody was observed with intact virus used as immunogen. C.V.K. MAb recognizes an epitope present on the precursor gag protein of 55 kilodaltons. Western blot analysis and pulse-chase experiments support the interpretation that after p55 cleavage into p25, p18, and p13, only p18 expresses this epitope. C.V.K. MAb selectively stained only LAV-infected lymphocytes. This intracytoplasmic staining appears 3 days after the infection and is correlated with reverse transcriptase activity. Neither membrane immunofluorescence of infected lymphocytes nor neutralizing activity was observed with C.V.K. MAb. These facts suggest that p55 and p18 are not expressed at the cell membrane or on the viral envelope. C.V.K. MAb should prove useful not only in the purification of core proteins but also for detecting infected cells producing the virus in suspension or on histologic sections.

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Jaso-Friedmann, L., D. T. Harris, A. St John, H. S. Koren, and D. L. Evans. "A monoclonal antibody-purified soluble target cell antigen inhibits nonspecific cytotoxic cell activity." Journal of Immunology 144, no. 6 (March 15, 1990): 2413–18. http://dx.doi.org/10.4049/jimmunol.144.6.2413.

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Abstract We have previously reported the characterization of mAb derived against NC-37 target cells. mAb 18C2 and 1E7 inhibit fish cytotoxicity by binding to target cells and thus preventing the formation of conjugates with fish nonspecific cytotoxic cells (NCC). It was therefore presumed that these inhibitory mAb were specific for the target cell Ag necessary for effector cell recognition. mAb 1D4 and 7C6 bind to NC-37 cells but do not inhibit fish cytotoxic activity. We now report the isolation and purification of the Ag recognized by mAb 18C2 (inhibitor) and 1D4 (noninhibitor) by affinity chromatography of solubilized NC-37 target cell extracts. The 18C2-purified soluble target Ag (STAg) caused inhibition of cytotoxicity when preincubated with fish NCC. This inhibitory activity was reversible and dose-dependent ranging from 20 to 70% inhibition with 25 to 100 micrograms 18C2 purified STAg/10(6) NCC. STAg purified by 1D4 affinity chromatography had no effect on fish cytotoxicity. mAb 18C2 and 1E7 preabsorbed with 18C2 STAg lost their inhibitory activity when tested in the fish NCC cytotoxicity assay. Preabsorption of the same mAb with 1D4 STAg had no effect on their activity.

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Barnard, Gavin C., Maria D. Hougland, and Yashas Rajendra. "High-throughput mAb expression and purification platform based on transient CHO." Biotechnology Progress 31, no. 1 (November 28, 2014): 239–47. http://dx.doi.org/10.1002/btpr.2012.

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Rucker-Pezzini, Joanna, Lindsay Arnold, Kevin Hill-Byrne, Tom Sharp, Maksim Avazhanskiy, and Chris Forespring. "Single pass diafiltration integrated into a fully continuous mAb purification process." Biotechnology and Bioengineering 115, no. 8 (April 27, 2018): 1949–57. http://dx.doi.org/10.1002/bit.26708.

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Shinagawa, Kunihiro, Teimi Takechi, Naonori Matsusaka, and Shunji Sugii. "Purification of an enterotoxin produced by Bacillus cereus by immunoaffinity chromatography using a monoclonal antibody." Canadian Journal of Microbiology 38, no. 2 (February 1, 1992): 153–56. http://dx.doi.org/10.1139/m92-025.

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A murine monoclonal antibody (MAb) with high reactivity to an enterotoxin produced by Bacillus cereus was used to prepare an immunoadsorbent for purification of the enterotoxin. By immunoaffinity chromatography using the immunoadsorbent, approximately 25% of crude enterotoxin applied was recovered in the eluate. The purified enterotoxin was found to be electrophoretically and antigenically homogeneous. It also showed vascular permeability activity and mouse lethality, and caused fluid accumulation in mouse ligated intestinal loops, whereas it did not show any haemolytic and lecithinase activities. Thus, immunoaffinity chromatography proved useful in the purification of enterotoxin produced by B. cereus in terms of recovery, purity, and relative ease of performing the purification. Key words: purification, immunoaffinity chromatography, enterotoxin, monoclonal antibody, Bacillus cereus.

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Pan, A. A., and D. McMahon-Pratt. "Amastigote and epimastigote stage-specific components of Trypanosoma cruzi characterized by using monoclonal antibodies. Purification and molecular characterization of an 83-kilodalton amastigote protein." Journal of Immunology 143, no. 3 (August 1, 1989): 1001–8. http://dx.doi.org/10.4049/jimmunol.143.3.1001.

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Abstract Stage-specific mAb have been produced to amastigotes and epimastigotes of Trypanosoma cruzi (Brazil strain). mAb C-1 through C-6 reacted specifically with T. cruzi strains; no cross-reactions were found with membranes of promastigotes or amastigotes of Leishmania species. One mAb produced against the epimastigote membranes (C-5) was found to be specific against this stage by radioimmune binding assay, immunofluorescence, and radioimmunoprecipitation. mAb C-5 recognized a novel epimastigote protein at Mr (greater than 200,000) on immunoprecipitation with radiolabeled epimastigotes. Three amastigote stage-specific monoclonal antibodies were produced against membrane-enriched preparations of T. cruzi (Brazil strain) amastigotes grown in axenic culture (C-1 through C-3). By indirect immunofluorescence assay, monoclonal antibody C-2 bound only to T. cruzi amastigotes; no reaction with either tissue culture-derived trypomastigotes or epimastigotes was observed. mAb C-1 and C-2 each specifically immunoprecipitated a single protein molecule with Mr 83,000 from [35S]-methionine-labeled amastigotes. mAb C-2 was also used to affinity purify an 83-kDa Ag that was recognized by human Chagasic sera from patients of endemic countries of Latin America in an enzyme immunoassay. Amino acid composition and preliminary sequence data of the 83-kDa protein are presented. These mAb and/or purified Ag may be useful in studying stage differentiation, monitoring transformation, and for further taxonomic, epidemiologic, and immunologic studies of Chagas' disease.

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Primakoff, P., H. Hyatt, and J. Tredick-Kline. "Identification and purification of a sperm surface protein with a potential role in sperm-egg membrane fusion." Journal of Cell Biology 104, no. 1 (January 1, 1987): 141–49. http://dx.doi.org/10.1083/jcb.104.1.141.

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Sperm-egg plasma membrane fusion during fertilization was studied using guinea pig gametes and mAbs to sperm surface antigens. The mAb, PH-30, strongly inhibited sperm-egg fusion in a concentration-dependent fashion. When zona-free eggs were inseminated with acrosome-reacted sperm preincubated in saturating (140 micrograms/ml) PH-30 mAb, the percent of eggs showing fusion was reduced 75%. The average number of sperm fused per egg was also reduced by 75%. In contrast a control mAb, PH-1, preincubated with sperm at 400 micrograms/ml, caused no inhibition. The PH-30 and PH-1 mAbs apparently recognize the same antigen but bind to two different determinants. Both mAbs immunoprecipitated the same two 125I-labeled polypeptides with Mr 60,000 (60 kD) and Mr 44,000 (44 kD). Boiling a detergent extract of sperm severely reduced the binding of PH-30 but had essentially no effect on the binding of PH-1, indicating that the two mAbs recognize different epitopes. Immunoelectron microscopy revealed that PH-30 mAb binding was restricted to the sperm posterior head surface and was absent from the equatorial region. The PH-30 and PH-1 mAbs did not bind to sperm from the testis, the caput, or the corpus epididymis. PH-30 mAb binding was first detectable on sperm from the proximal cauda epididymis, i.e., sperm at the developmental stage where fertilization competence appears. After purification by mAb affinity chromatography, the PH-30 protein retained antigenic activity, binding both the PH-30 and PH-1 mAbs. The purified protein showed two polypeptide bands of 60 and 44 kD on reducing SDS PAGE. The two polypeptides migrated further (to approximately 49 kD and approximately 33 kD) on nonreducing SDS PAGE, showing that they do not contain interchain disulfide bonds, but probably have intrachain disulfides. 44 kD appears not to be a proteolytic fragment of 60 kD because V8 protease digestion patterns did not reveal related peptide patterns from the 44- and 60-kD bands. In the absence of detergent, the purified protein precipitates, suggesting that either 60 or 44 kD could be an integral membrane polypeptide.

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Santarelli, Xavier, and Charlotte Cabanne. "Mixed Mode Chromatography: A Novel Way Toward New Selectivity." Current Protein & Peptide Science 20, no. 1 (November 9, 2018): 14–21. http://dx.doi.org/10.2174/1389203718666171024121137.

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Mixed mode chromatography offers a diversity of ligands, each providing a new selectivity. This allows the design of novel purification processes with reduced column steps. Structure of ligands is based on both hydrophobic and ionic groups. Thanks to its salt tolerance, crude extracts or post-IEX samples can be loaded directly without conditioning. The selectivity could be enhanced by modulating elution parameters or by using additives. More importantly, mixed mode chromatography could be as effective as affinity chromatography for mAb purification processes. Mixed mode chromatography opens the way to short and economical processes.

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Chavez-Olortegui, C., M. Resende, and C. A. P. Tavares. "Purification and characterization of a 47 kDa protease from Schistosoma mansoni cercarial secretion." Parasitology 105, no. 2 (October 1992): 211–18. http://dx.doi.org/10.1017/s0031182000074138.

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SUMMARYFractionation of Schistosoma mansoni cercariae gland secretion on a Sephadex G-150 column followed by a Superose-12 column in an FPLC system, isolated a 47 kDa protease which migrated as a single band on SDS–PAGE gels. A monoclonal antibody (MAb) was produced which recognizes only the 47 kDa protease, and an immuno-affinity column with the MAb was used to isolate the protease. The 47 kDa protease showed activity on several macromolecules such as elastin and collagen type VI besides gelatin and casein. This suggests that this enzyme can be one of the enzymes that might facilitate invasion of the cercariae through host skin. The optimal pH of the protease against the synthetic substrate, Ac-Phe-Arg-Nan, in Tris–HCI buffer was 10. Experiments with protease inhibitors indicate that the purified enzyme is a serine protease.

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Fujii, Yuki, Mika K. Kaneko, and Yukinari Kato. "MAP Tag: A Novel Tagging System for Protein Purification and Detection." Monoclonal Antibodies in Immunodiagnosis and Immunotherapy 35, no. 6 (December 2016): 293–99. http://dx.doi.org/10.1089/mab.2016.0039.

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Luisa Nolli, Maria, Angelo Corti, Adolfo Soffientini, and Giovanni Cassani. "A Monoclonal Antibody that Recognizes the Receptor Binding Region of Human Urokinase Plasminogen Activator." Thrombosis and Haemostasis 56, no. 02 (1986): 214–18. http://dx.doi.org/10.1055/s-0038-1661643.

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SummaryAn anti-urokinase monoclonal antibody 5B4 (MAB 5B4) was obtained by fusing the murine myeloma cell line X63-Ag8.653 with the spleen cells from a female BALB/c mouse immunized with high-molecular-weight urokinase (HMW-uPA).MAB 5B4 is an IgGl that binds selectively to the single-chain form of uPA (sc-uPA), to HMW-uPA and to the 17,000 Mr aminoterminal fragment of the A-chain (ATF) but not to the low-molecular-weight urokinase (LMW-uPA) nor to the reduced form of HMW-uPA. This strongly suggests that MAB 5B4 recognizes a conformational determinant on the A-chain.The antibody has an affinity constant for uPA-Sepharose of 1.42 × 107 M−1, calculated from equilibrium binding data, and can be used for one step purification of HMW-uPA by immunoaffinity chromatography.MAB 5B4 and the previously obtained antibody 105IF10 (16) recognize the A-chain: the epitopes, however, are distinct as shown by double-antibody-sandwich enzyme immunoassay.Finally MAB 5B4 inhibits the binding of ATF to the uPA receptor of different human cells, whereas 105IF10 does not. Thus this antibody represents a potentially, useful tool for the study of uPA receptor physiology.

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Yang, Yun, Na Chen, Zhe Li, Xian-Jiang Wang, Sheng-Yu Wang, Tingwu, Fang-Hong Luo, and Jiang-Hua Yan. "Preparation, Purification, and Identification of a Monoclonal Antibody Against NRP2 b1b2 Domain." Monoclonal Antibodies in Immunodiagnosis and Immunotherapy 34, no. 5 (October 2015): 354–59. http://dx.doi.org/10.1089/mab.2015.0025.

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Gallo, J. M., D. Escalier, P. Grellier, E. Précigout, M. Albert, G. David, and J. Schrével. "Characterization of a monoclonal antibody to human proacrosin and its use in acrosomal status evaluation." Journal of Histochemistry & Cytochemistry 39, no. 3 (March 1991): 273–82. http://dx.doi.org/10.1177/39.3.1704391.

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Among the monoclonal antibodies (MAb) selected after immunization of mice with a detergent-insoluble fraction from human spermatozoa, MAb 4D4 was found to stain in immunofluorescence the principal part of the acrosome of human spermatozoa. Acrosome reaction induced decreased and spotty 4D4 immunofluorescence staining. Immunoelectron microscopy before or after embedding revealed that the epitope defined by MAb 4D4 was sequestered in the anterior acrosomal matrix and, after the acrosome reaction, remained partly bound on matrix elements attached to the inner acrosomal membrane. Western blot analysis of sperm extracts showed that the epitope defined by MAb 4D4 was located on a 55 KD polypeptide in whole cells and on 55 and 50 KD polypeptides in non-ionic detergent fractions. Human proacrosin-enriched fraction obtained by FPLC purification exhibited several proteolytic activities against gelatin in gel enzymography: a 50 KD major band and two minor bands in the 20-30 KD area; the 50 KD polypeptide reacted with MAb 4D4 in Western blots. Furthermore, the 4D4-immunoprecipitated polypeptide from sperm extract showed that the 50 KD band exhibited proteolytic activity with an optimal pH at 8.0 that was strongly inhibited by soybean trypsin inhibitor and ZnCl2. MAb 4D4 also reacted with the acrosome of the monkey Macaca fascicularis but not with the acrosome of any of the other non-primate mammalian species examined so far. Various shape defects of the acrosomal principal region were revealed by 4D4 labeling of spermatozoa with head anomalies from infertile patients. MAb 4D4 also recognized proacrosin in paraffin-embedded human testis sections. These data make the monoclonal antiproacrosin antibody 4D4 an efficient tool for evaluation of the acrosomal status of human spermatozoa and spermatids.

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Muller, W. A., C. M. Ratti, S. L. McDonnell, and Z. A. Cohn. "A human endothelial cell-restricted, externally disposed plasmalemmal protein enriched in intercellular junctions." Journal of Experimental Medicine 170, no. 2 (August 1, 1989): 399–414. http://dx.doi.org/10.1084/jem.170.2.399.

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We have raised an mAb to a previously undescribed 135-kD externally disposed integral membrane protein that is enriched in the intercellular junctional domain of cultured human umbilical vein endothelial cells. This protein localizes at the appositional surfaces of cells as they become confluent and is stably expressed in the junctional zones of confluent monolayers. This protein is expressed in situ on continuous endothelia of all blood vessels in all human tissues examined. Moreover, this protein, as determined by mAb immunocytochemistry, is not expressed by any other cell type. This protein may mediate endothelial-specific functions restricted to the intercellular domain. It may also serve as a unique cell surface marker for the identification and purification of human endothelial cells.

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Kopyrulina, M. E., E. N. Zakharova, T. N. Zabotina, D. Yu Blokhin, and P. K. Ivanov. "Тhe technology of creation and quality testing of immunofluorescent probes with dye alexa-488 for analysis of celular populations by flow cytometry." Russian Journal of Biotherapy 17, no. 1 (November 27, 2018): 70–75. http://dx.doi.org/10.17650/1726-9784-2018-17-1-70-75.

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Fluorescent probes based on monoclonal antibodies (MAb) are widely used in scientific and clinical research in the field of oncology, hematology, immunology, epidemiology. Objective: to create of fluorescent probes based on the MAb and the fluorescent dye Alexa-488 for the analysis of cellular populations by flow cytometry. Materials and methods. MAb to B lymphocyte antigen (clone ICO-180), fluorescent dye Alexa-488 were used in the work. MAb was isolated from ascitic fluid by combined purification of the immunoglobulin fraction with caprylic acid and salting out with ammonium sulfate. Gel filtration on a PD-10 column was used to purify the conjugates (immunofluorescent probes, IFP), the concentration and labeling density of the IFP were determined spectrophotometrically. The determination of the working titer of the IFP was performed using the antibody titration method proposed by C.C. Stewart. Results. The optimal time of incubation of MAb with a fluorophore was experimentally determined. The optimal conditions for labeling MAb of the IСO series with the dye are: a carbonate buffer with pH 8,3, the concentration of antibodies in the reaction mixture is 1 mg/ml, molar ratio of active dye – 10–100 mmol per 1 mmol of protein, the incubation time is 90 minutes, the temperature is 18–25 °C. We obtained a panel of conjugates of MAb with Alexa-488, differing in their different labeling densities. Evaluation of the biological activity of the resulting conjugates was carried out on peripheral blood cells of donors in the concentration range of MAb 0,5–100 μg/ml. Conclusion. The optimal conditions for labeling MAb of the IСO series with the dye are: a carbonate buffer with pH 8,3, the concentration of antibodies in the reaction mixture is 1 mg/ml, the incubation time is 90 minutes, the temperature is 18–25 °C. The optimum density of labeling is in the range 5–13,5 M:M, the optimal concentration of antibodies is in the range of 5–25 μg/ml.

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Fujii, Yuki, Mika K. Kaneko, Satoshi Ogasawara, Shinji Yamada, Miyuki Yanaka, Takuro Nakamura, Noriko Saidoh, Kanae Yoshida, Ryusuke Honma, and Yukinari Kato. "Development of RAP Tag, a Novel Tagging System for Protein Detection and Purification." Monoclonal Antibodies in Immunodiagnosis and Immunotherapy 36, no. 2 (April 2017): 68–71. http://dx.doi.org/10.1089/mab.2016.0052.

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30

Rattanapisit, Kaewta, Anchalee Srijangwad, Taksina Chuanasa, Suchada Sukrong, Angkana Tantituvanont, Hugh Mason, Dachrit Nilubol, and Waranyoo Phoolcharoen. "Rapid Transient Production of a Monoclonal Antibody Neutralizing the Porcine Epidemic Diarrhea Virus (PEDV) in Nicotiana benthamiana and Lactuca sativa." Planta Medica 83, no. 18 (June 2, 2017): 1412–19. http://dx.doi.org/10.1055/s-0043-112344.

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AbstractPorcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration, weight loss, and high mortality rate in neonatal piglets. Porcine epidemic diarrhea (PED) has been reported in Europe, America, and Asia including Thailand. The disease causes substantial losses to the swine industry in many countries. Presently, there is no effective PEDV vaccine available. In this study, we developed a plant-produced monoclonal antibody (mAb) 2C10 as a prophylactic candidate to prevent the PEDV infection. Recently, plant expression systems have gained interest as an alternative for the production of antibodies because of many advantages, such as low production cost, lack of human and animal pathogen, large scalability, etc. The 2C10 mAb was transiently expressed in Nicotiana benthamiana and lettuce using geminiviral vector. After purification by protein A affinity chromatography, the antibody was tested for the binding and neutralizing activity against PEDV. Our result showed that the plant produced 2C10 mAb can bind to the virus and also inhibit PEDV infection in vitro. These results show excellent potential for a plant-expressed 2C10 as a PEDV prophylaxis and a diagnostic for PEDV infection.

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Belitsos, P. C., J. E. Hildreth, and J. T. August. "Homotypic cell aggregation induced by anti-CD44(Pgp-1) monoclonal antibodies and related to CD44(Pgp-1) expression." Journal of Immunology 144, no. 5 (March 1, 1990): 1661–70. http://dx.doi.org/10.4049/jimmunol.144.5.1661.

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Abstract The present study shows that a mAb (H4C4) developed against human peripheral blood adherent cells has the unusual property of inducing in vitro homotypic aggregation of several types of hemopoietic cells and cell lines. The Ag recognized by mAb H4C4 is a 85-kDa glycoprotein that corresponds to the human Ag CD44 (equivalent to murine Pgp-1), as determined by protein purification, immunologic cross-reactivity studies, and tryptic fragment sequencing. In addition to H4C4, other mAb directed against some, but not all, epitopes of CD44(Pgp-1) were capable of inducing cell aggregation. This process was temperature sensitive and was almost totally abrogated by cytochalasin B but was unaffected by sodium azide, colchicine, EGTA, trifluoperazine, or staurosporin. A role for CD44 (Pgp-1) in cell-to-cell adhesion was further indicated by an inverse relationship observed between spontaneous aggregation of some hemopoietic cell lines and cell-surface expression of CD44(Pgp-1). These observations provide evidence for a fundamental role of CD44(Pgp-1) in cellular aggregation phenomena with an involvement of the cytoskeleton.

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Song, Ilchan, Yang Joo Kang, Su-Lim Choi, Dalmuri Han, Deuk-Su Kim, Hae Kyung Lee, Joon-Chul Lee, Jeanho Park, Do-Sun Kim, and Kisung Ko. "Purification of plant-derived anti-virus mAb through optimized pH conditions for coupling between protein A and epoxy-activated beads." PeerJ 7 (May 21, 2019): e6828. http://dx.doi.org/10.7717/peerj.6828.

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The main goal of this research was to determine optimum pH conditions for coupling between protein A and epoxy-activated Sepharose beads for purification of monoclonal antibodies (mAbs) expressed in plants. To confirm the effect of pH conditions on purification efficacy, epoxy-activated agarose beads were coupled to protein A under the pH conditions of 8.5, 9.5, 10.5, and 11.5 (8.5R, 9.5R, 10.5R, and 11.5R, respectively). A total of 300 g of fresh leaf tissue of transgenic Arabidopsis expressing human anti-rabies mAb (mAbP) SO57 were harvested to isolate the total soluble protein (TSP). An equal amount of TSP solution was applied to five resin groups including commercial protein A resin (GR) as a positive control. The modified 8.5R, 9.5R, 10.5R, and 11.5R showed delayed elution timing compared to the GR control resin. Nano-drop analysis showed that the total amount of purified mAbPSO57 mAbs from 60 g of fresh leaf mass were not significantly different among 8.5R (400 μg), 9.5R (360 μg), 10.5R (380 μg), and GR (350 μg). The 11.5R (25 μg) had the least mAbPSO57. SDS–PAGE analysis showed that the purity of mAbPSO57 was not significantly different among the five groups. Rapid fluorescent focus inhibition tests revealed that virus-neutralizing efficacies of purified mAbPSO57 from all the five different resins including the positive control resin were similar. Taken together, both pH 8.5 and 10.5 coupling conditions with high recovery rate should be optimized for purification of mAbPSO57 from transgenic Arabidopsis plant, which will eventually reduce down-stream cost required for mAb production using the plant system.

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Chen, Serene W., and Wei Zhang. "Current trends and challenges in the downstream purification of bispecific antibodies." Antibody Therapeutics 4, no. 2 (April 1, 2021): 73–88. http://dx.doi.org/10.1093/abt/tbab007.

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ABSTRACT Bispecific antibodies (bsAbs) represent a highly promising class of biotherapeutic modality. The downstream processing of this class of antibodies is therefore of crucial importance in ensuring that these products can be obtained with high purity and yield. Due to the various fundamental structural similarities between bsAbs and monoclonal antibodies (mAbs), many of the current bsAb downstream purification methodologies are based on the established purification processes of mAbs, where affinity, charge, size, hydrophobicity and mixed-mode-based purification are frequently employed. Nevertheless, the downstream processing of bsAbs presents a unique set of challenges due to the presence of bsAb-specific byproducts, such as mispaired products, undesired fragments and higher levels of aggregates, that are otherwise absent or present in lower levels in mAb cell culture supernatants, thus often requiring the design of additional purification strategies in order to obtain products of high purity. Here, we outline the current major purification methods of bsAbs, highlighting the corresponding solutions that have been proposed to circumvent the unique challenges presented by this class of antibodies, including differential affinity chromatography, sequential affinity chromatography and the use of salt additives and pH gradients or multistep elutions in various modes of purification. Finally, a perspective towards future process development is offered.

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34

Mejan, Odile, Vincent Fert, Maryléne Delezay, Michel Delaage, Rose Cheballah, and Alain Bourgois. "Immunopurification of Human Factor VIII/vWF Complex from Plasma." Thrombosis and Haemostasis 59, no. 03 (1988): 364–71. http://dx.doi.org/10.1055/s-0038-1647497.

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SummaryIn this study we describe a process for immunopurification of FVIII/vWF complex directly from plasma. A mAb against vWF has been selected that is able to bind, under physiologic conditions, the FVIII/vWF complex and to release it in slightly alkaline conditions while preserving its activity.After investigating the influence of solid supports and of coupling methods on the recovery of active FVIII we produced an immunoadsorbent by immobilisation of the selected mAb onto a Sephacryl S-1000 support using a benzoquinone coupling method. With this immunoadsorbent we developed a purification process directly from plasma with an excellent recovery (50%) of both FVIII and vWF activities. The product obtained is very enriched (the FVIII: C specific activity is 20 IU/mg of protein) and is stable after lyophilization.

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Morrison, Christopher J., Pete Gagnon, and Steven M. Cramer. "Purification of monomeric mAb from associated aggregates using selective desorption chromatography in hydroxyapatite systems." Biotechnology and Bioengineering 108, no. 4 (December 22, 2010): 813–21. http://dx.doi.org/10.1002/bit.22971.

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36

Brichory, F., B. Collet, C. Pineau, B. Desrues, L. Toujas, J. P. Le Pennec, and L. Dazord. "Purification of a Tumoral Marker Recognized by Monoclonal Antibody Po66 and Associated with Human Lung Squamous Cell Carcinoma." International Journal of Biological Markers 11, no. 3 (July 1996): 148–52. http://dx.doi.org/10.1177/172460089601100302.

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Monoclonal antibody (MAb) Po66, a murine IgG1, was raised by immunization against human lung squamous cell carcinoma. When injected intravenously, Po66 showed prolonged retention in the tumor. It recognized an intracellular antigen. The human lung squamous carcinoma cell line SK-MES-1 expresses the antigen recognized by MAb Po66 and was used as a source of biological material for its purification. The SK-MES-1 cell line was labeled in culture with [35S]methionine and its lysate was immunoprecipitated with Po66 immobilized on Protein G-Sepharose. The precipitate contained three proteins (47, 50 and 69 kDa) absent in the controls. The 69 kDa polypeptide was further purified by anion exchange and immunoaffinity chromatographies. To date, no other tumor marker expressed in non-small cell lung cancer with these characteristics has been described and as such this marker is interesting for future use in immunotherapy and in diagnosis.

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37

Lee, Song Hee, Tae-Kyun Oh, Sung Oh, Seongdae Kim, Han Byul Noh, Nagarajan Vinod, Ji Yoon Lee, Eun Sun Moon, and Chang Won Choi. "Development of a Kit for Rapid Immunochromatographic Detection of Sacbrood Virus Infecting Apis cerana (AcSBV) Based on Polyclonal and Monoclonal Antibodies Raised against Recombinant VP1 and VP2 Expressed in Escherichia coli." Viruses 13, no. 12 (December 4, 2021): 2439. http://dx.doi.org/10.3390/v13122439.

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A Korean isolate of the sacbrood virus infecting Apis cerana (AcSBV-Kor) is the most destructive honeybee virus, causing serious economic damage losses in Korean apiculture. To address this, here, we attempted to develop an assay for the rapid detection of AcSBV-Kor based on immunochromatographic detection of constituent viral proteins. Genes encoding VP1 and VP2 proteins of AcSBV-Kor were cloned into an expression vector (pET-28a) and expressed in Escherichia coli BL21(DE3). During purification, recombinant VP1 (rVP1) and VP2 (rVP2) proteins were found in the insoluble fraction, with a molecular size of 26.7 and 24.9 kDa, respectively. BALB/c mice immunized with the purified rVP1 and rVP2 produced polyclonal antibodies (pAbs) such as pAb-rVP1 and pAb-rVP2. Western blot analysis showed that pAb-rVP1 strongly reacted with the homologous rVP1 but weakly reacted with heterologous rVP2. However, pAb-rVP2 strongly reacted not only with the homologous rVP2 but also with the heterologous rVP1. Spleen cells of the immunized mice fused with SP2/0-Ag14 myeloma cells produced monoclonal antibodies (mAbs) such as mAb-rVP1-1 and mAb-rVP2-13. Western blot analysis indicated that pAb-rVP1, pAb-rVP2, mAb-rVP1-1, and mAb-rVP2-13 reacted with AcSBV-infected honeybees and larvae as well as the corresponding recombinant proteins. These antibodies were then used in the development of a rapid immunochromatography (IC) strip assay kit with colloidal gold coupled to pAb-rVP1 and pAb-rVP2 at the conjugate pad and mAb-rVP1-1 and mAb-rVP2-13 at the test line. One antibody pair, pAb-rVP1/mAb-VP1-1, showed positive reactivity as low as 1.38 × 103 copies, while the other pair, pAb-rVP2/mAb-VP2-13, showed positive reactivity as low as 1.38 × 104 copies. Therefore, the antibody pair pAb-rVP1/mAb-VP1-1 was selected as a final candidate for validation. To validate the detection of AcSBV, the IC strip tests were conducted with 50 positive and 50 negative samples and compared with real-time PCR tests. The results confirm that the developed IC assay is a sufficiently sensitive and specific detection method for user-friendly and rapid detection of AcSBV.

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Verkerke, Hans P., James A. Williams, Miklos Guttman, Cassandra A. Simonich, Yu Liang, Modestas Filipavicius, Shiu-Lok Hu, Julie Overbaugh, and Kelly K. Lee. "Epitope-Independent Purification of Native-Like Envelope Trimers from Diverse HIV-1 Isolates." Journal of Virology 90, no. 20 (August 10, 2016): 9471–82. http://dx.doi.org/10.1128/jvi.01351-16.

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ABSTRACTSoluble forms of trimeric HIV-1 envelope glycoprotein (Env) have long been sought as immunogens and as reagents for analysis of Env structure and function. Isolation of trimers that mimic native Env, derived from diverse viruses, however, represents a major challenge. Thus far, the most promising native-like (NL) structures have been obtained by engineering trimer-stabilizing mutations, termed SOSIP, into truncated Env sequences. However, the abundances of NL trimeric conformers vary among Envs, necessitating purification by monoclonal antibodies (MAbs) like PGT145, which target specific epitopes. To surmount this inherent limitation, we developed an approach that uses lectin affinity chromatography, ion-exchange chromatography, hydrophobic-interaction chromatography (HIC), and size exclusion chromatography (SEC) to isolate NL trimers from nonnative Env species. We validated this method with SOSIP trimers from HIV-1 clades A and B. Analyses by SEC, blue native PAGE, SDS-PAGE, and dynamic light scattering indicated that the resulting material was homogeneous (>95% pure), fully cleaved, and of the appropriate molecular weight and size for SOSIP trimers. Negative-stain electron microscopy further demonstrated that our preparations were composed of NL trimeric structures. By hydrogen/deuterium-exchange mass spectrometry, these HIC-pure trimers exhibited structural organization consistent with NL trimers and inconsistent with profiles seen in nonnative Envs. Screened for antigenicity, some Envs, like BS208.b1 and KNH1144 T162A, did not present the glycan/quaternary structure-dependent epitope for PGT145 binding, suggesting that these SOSIPs would be challenging to isolate by existing MAb affinity methods. By selecting based on biochemical rather than antigenic properties, our method offers an epitope-independent alternative to MAbs for isolation of NL Env trimers.IMPORTANCEThe production and purification of diverse soluble Env trimers that maintain native-like (NL) structure present technical challenges that must be overcome in order to advance vaccine development and provide reagents for HIV research. Low levels of NL trimer expression amid heterogeneous Env conformers, even with the addition of stabilizing mutations, have presented a major challenge. In addition, it has been difficult to separate the NL trimers from these heterogeneous mixtures. While MAbs with specificity for quaternary NL trimer epitopes have provided one approach to purifying the desirable species, such methods are dependent on the Env displaying the proper epitope. In addition, MAb affinity chromatography can be expensive, the necessary MAb may be in limited supply, and large-scale purification may not be feasible. Our method based on biochemical separation techniques offers an epitope-independent approach to purification of NL trimers with general application to diverse Envs.

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Chapman, M. D., R. C. Aalberse, M. J. Brown, and T. A. Platts-Mills. "Monoclonal antibodies to the major feline allergen Fel d I. II. Single step affinity purification of Fel d I, N-terminal sequence analysis, and development of a sensitive two-site immunoassay to assess Fel d I exposure." Journal of Immunology 140, no. 3 (February 1, 1988): 812–18. http://dx.doi.org/10.4049/jimmunol.140.3.812.

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Abstract Two mAb were used to develop new techniques for the purification and quantitation of the major feline salivary allergen, Felis domesticus allergen I (Fel d I). The allergen was purified from aqueous house dust extract with a high Fel d I content by affinity chromatography over a monoclonal immunosorbent and elution with 4 mM HCl, pH 2.5. This single step procedure gave 40 to 50% recovery of 90% pure allergen which, following final purification by size exclusion HPLC, showed a single line on immunodiffusion and crossed immunoelectrophoresis against monospecific anti-Fel d I and polyclonal anti-cat dander antibodies. The m.w. of native Fel d I was 39,000 on size exclusion HPLC, and 17,000 under nonreducing conditions on gel electrophoresis. The N-terminal amino acid sequence (33 residues) showed no homology with other known protein sequences. The combination of the SDS-PAGE and N-terminal sequence data suggests that Fel d I is a non-covalently linked homodimer. A two-site RIA was developed using mAb directed against different epitopes on Fel d I. This assay was species-specific, highly sensitive (0.0004 U/ml), and showed an excellent correlation with a polyclonal inhibition RIA (n = 27, r = 0.93, p less than 0.001). Cat allergen extracts used for immediate skin tests showed marked differences in Fel d I content (from 0.1 to 30 U/ml). Consistently high Fel d I levels were found at monthly intervals in six dust samples from four houses with cats (10 to 100 U/g of dust). Comparisons of Fel d I and mite and pollen allergen levels showed that house dust can contain greater than 100 micrograms/g of either of these allergens and is a potent source of foreign environmental antigens. Monoclonal affinity chromatography provides a major breakthrough in the purification of Fel d I, from a source material that would otherwise have been considered impossible (house dust). The mAb assay for Fel d I is both more sensitive and more easily standardized than existing techniques. These techniques will allow full structural and antigenic analysis of Fel d I and more detailed studies on the relationship between cat antigen exposure and the development of asthma.

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King, C. H., A. Hull, P. J. Kleinhenz, N. F. Phillips, and J. A. Marino. "Structural and functional analysis of the human neutrophil 1-15 antigen, an Mr 65,000 to 70,000 activation-associated membrane proteinase." Journal of Immunology 146, no. 9 (May 1, 1991): 3115–23. http://dx.doi.org/10.4049/jimmunol.146.9.3115.

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Abstract Previous studies with the anti-neutrophil/antichymotrypsin mAb 1-15 have identified an activation-associated, chymotrypsin-like activity within the membrane fraction of isolated human neutrophils (PMN). In the present study, the molecular and biochemical characteristics of mAb 1-15 Ag/proteinase were determined. On casein/acrylamide sizing gels, PMN membrane preparations were found to contain an Mr 58,000 to 84,000 band of Ca2(+)-dependent proteinase activity. Reducing and nonreducing SDS-PAGE of mAb 1-15-affinity-purified membrane proteins demonstrated specific recovery of an enzymatically active Mr 65,000 to 70,000 chymotrypsin-like Ag. The presence of a distinct membrane serine esterase of isoelectric point 6.3/Mr 65,000 to 70,000 was confirmed in active site-labeling experiments with the serine proteinase inhibitor [3H]diisopropylfluorophosphate (DFP). Substrate-affinity chromatography with phe-Sepharose or FMLP-Sepharose provided partial purification of enzyme activity among Mr 65,000 to 70,000 FMLP- or phe-binding proteins. Enzyme inhibition was obtained by incubation with mAb 1-15, DFP, N-carbobenzoxyl-phe-chlormethyl ketone, or PMSF, but not tosyl-amide-phenylethylchlormethyl ketone, bestatin, aprotinin, or phosphoramidon. In HPLC analysis, [3H]DFP labeled proteinase was found to comigrate with one of three FMLP-affinity-labeled membrane peaks, but unlike the FMLP surface receptor the DFP-labeling membrane proteinase was not modified by endoglycosidase F. We conclude that the mAb 1-15 Ag, which appears to play a role in PMN activation, is a distinct, active, Mr 65,000 to 70,000 serine proteinase with affinity for substrate sites containing aromatic amino acids.

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Yoshimura, T., M. Takeya, K. Takahashi, J. Kuratsu, and E. J. Leonard. "Production and characterization of mouse monoclonal antibodies against human monocyte chemoattractant protein-1." Journal of Immunology 147, no. 7 (October 1, 1991): 2229–33. http://dx.doi.org/10.4049/jimmunol.147.7.2229.

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Abstract We developed five different hybridoma cell lines that produced mAb against human monocyte chemoattractant protein-1 (MCP-1). The subclass of all five antibodies was IgG1. All five mAb formed complexes with metabolically labeled MCP-1 that could be demonstrated by immunoprecipitation. The antibodies were specific for MCP-1. They did not cross-react by immunoprecipitation with structurally related host defense cytokines present in metabolically labeled PHA- or LPS-stimulated mononuclear cell culture fluids, nor did they cross-react in a direct ELISA with neutrophil attractant/activation protein-1, with crude platelet lysate proteins, or with pure platelet proteins that have amino acids sequences similar to that of MCP-1. The mAb also reacted with rMCP-1 expressed in Escherichia coli, suggesting that they recognize protein structure rather than the glycosylated portion of human MCP-1. When the mAb were mixed with MCP-1, the monocyte chemotactic response to MCP-1 was inhibited. A sandwich ELISA was developed to detect MCP-1 in biologic fluids containing relatively high concentrations of other proteins. The sensitivity was 300 pg/ml, or 30 pg/ELISA well. An anti-MCP-1 mAb column was used in an improved method of MCP-1 purification. Approximately 240 micrograms of MCP-1 were purified from 5 liters of FCS-containing U-105MG cell culture supernatant. The yield was at least 60%. In addition to two forms of MCP-1 reported previously by us, two more forms of MCP-1 were found in a mixture of culture supernatants of PHA- and LPS-stimulated human PBMC.

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Barnard, R., P. G. Bundesen, D. B. Rylatt, and M. J. Waters. "Evidence from the use of monoclonal antibody probes for structural heterogeneity of the growth hormone receptor." Biochemical Journal 231, no. 2 (October 15, 1985): 459–68. http://dx.doi.org/10.1042/bj2310459.

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We describe the use of four monoclonal antibodies (MAbs) to the rabbit liver growth hormone (GH) receptor and one raised against purified rat liver GH receptor to characterize liver receptor subtypes which differ in their hormone-binding regions. The anti-(rat liver GH receptor) MAb both inhibited and precipitated rat and rabbit GH receptors, but only one-half of 125I-oGH (ovine GH) binding to liver microsomes could be inhibited by excess antibody. Conversely, only one-half of 125I-anti-(rat GH receptor) MAb binding was inhibited by excess oGH and Scatchard plots for this MAb exhibited two components. Although only 50% of 125I-oGH binding to membranes was inhibited by this MAb, all solubilized receptor could be immunoprecipitated. We postulate two epitopes for the anti-(rat GH receptor) MAb, one located at the hormone-binding site (inhibitory site) and one elsewhere (immunoprecipitating site). A second, rabbit-specific antibody (MAb 7) inhibited 85% of hormone binding but only 30% of 125I-anti-(rat GH receptor) MAb binding to rabbit liver microsomes. A combination of this MAb with the anti-(rat GH receptor) MAb totally inhibited 125I-oGH binding. MAb 7 alone totally inhibited 125I-rat GH binding to rabbit liver microsomes, as it did with 125I-oGH binding to purified receptor. On the basis of these results and others we postulate three types of GH receptor in rabbit liver membranes and ascribe approximate extents of 125I-oGH binding to each. A cytosolic ‘GH receptor’ which is not poly(ethylene glycol)-precipitable is shown to share five epitopes with ‘type 2’ microsomal receptors. Purified plasma membrane and endoplasmic reticulum fractions derived from a rabbit liver microsomal preparation have identical antigenic characteristics with respect to the GH-binding region, indicating that the heterogeneity we describe is not related to receptor processing. Of the three types of GH receptor in the plasma membrane of the rabbit (and possibly rat) we postulate that one (type 1) corresponds to the GH receptor involved in stimulating growth and possesses all of the epitopes studied here. A second (type 2) appears to be identical with the cytosolic ‘GH receptor’ and lacks the epitope for the anti-(rat GH receptor) MAb in the hormone binding site region. A third (type 3) does not possess the epitope for the inhibitory anti-(rabbit GH receptor) MAb, appears not to bind rat GH and is lost during purification. The availability of type-specific MAbs will facilitate assignment of specific functions to liver receptor subtypes which mediate the multiple functions of GH.

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Hatami Giklou Jajan, Leila, Seyed Nezamedin Hosseini, Mohsen Abolhassani, and Masoud Ghorbani. "Progress in affinity ligand-functionalized bacterial magnetosome nanoparticles for bio-immunomagnetic separation of HBsAg protein." PLOS ONE 17, no. 7 (July 25, 2022): e0267206. http://dx.doi.org/10.1371/journal.pone.0267206.

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Efficient Bio-immunomagnetic separation (BIMS) of recombinant hepatitis B surface antigen (rHBsAg) with high binding capacity was studied using affinity ligand immobilized bacterial magnetosome nanoparticles (Magnetospirillum gryphiswaldense strain MSR-1 bacteria) as an immunomagnetic sorbent. Our results showed immunomagnetic adsorption, acted by affinity interactions with the immobilized monoclonal antibody, offered higher antigen adsorption and desorption capacities as compared with the commercially available immunoaffinity sorbents. Four different ligand densities of the Hep-1 monoclonal antibody were examined during covalent immobilization on Pyridyl Disulfide-functionalized magnetosome nanoparticles for HBsAg immunomagnetic separation. The average of adsorption capacity was measured as 3 mg/ml in optimized immunomagnetic sorbent (1.056 mg rHBsAg/ml immunomagneticsorbent/5.5 mg of total purified protein) and 5mg/ml in immunoaffinity sorbent (0.876 mg rHBsAg/ml immunosorbent/5.5 mg total purified protein during 8 runs. Immunomagnetic sorbent demonstrated ligand leakage levels below 3 ng Mab/Ag rHBsAg during 12 consecutive cycles of immunomagnetic separation (IMS). The results suggest that an immunomagnetic sorbent with a lower ligand density (LD = 3 mg Mab/ml matrix) could be the best substitute for the immunosorbent used in affinity purification of r-HBsAg there are significant differences in the ligand density (98.59% (p-value = 0.0182)), adsorption capacity (97.051% (p-value = 0.01834)), desorption capacity (96.06% (p-value = 0.036)) and recovery (98.97% (p-value = 0.0231)). This study indicates that the immunosorbent approach reduces the cost of purification of Hep-1 protein up to 50% as compared with 5 mg Mab/ml immunoaffinity sorbent, which is currently used in large-scale production. As well, these results demonstrate that bacterial magnetosome nanoparticles (BMs) represent a promising alternative product for the economical and efficient immobilization of proteins and the immunomagnetic separation of Biomolecules, promoting innovation in downstream processing.

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Meng, Pei-Pei, Zhe Li, Sheng-Yu Wang, Wen-Wen Zhou, Malik Samiullah, Na Chen, Fang-Hong Luo, Ting Wu, and Jiang-Hua Yan. "Preparation, Purification, and Identification of a Monoclonal Antibody Against the C-Terminal Domain of Semaphorin3F." Monoclonal Antibodies in Immunodiagnosis and Immunotherapy 37, no. 1 (February 2018): 52–58. http://dx.doi.org/10.1089/mab.2017.0040.

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Palani, B., S. Shiva Ranjini, N. S. Jayaprakash, and M. A. Vijayalakshmi. "Development, Purification, and Characterization of Monoclonal Antibodies Against Recombinant Histidine-rich Protein 3 ofPlasmodium falciparum." Monoclonal Antibodies in Immunodiagnosis and Immunotherapy 32, no. 5 (October 2013): 341–48. http://dx.doi.org/10.1089/mab.2013.0018.

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Dreja, Hanna, Laurent Gros, Sylvie Villard, Estanislao Bachrach, Anna Oates, Claude Granier, Thierry Chardes, Jean-Claude Mani, Marc Piechaczyk, and Mireia Pelegrin. "Monoclonal Antibody 667 Recognizes the Variable Region A Motif of the Ecotropic Retrovirus CasBrE Envelope Glycoprotein and Inhibits Env Binding to the Viral Receptor." Journal of Virology 77, no. 20 (October 15, 2003): 10984–93. http://dx.doi.org/10.1128/jvi.77.20.10984-10993.2003.

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ABSTRACT Monoclonal antibody (MAb) 667 is a neutralizing mouse monoclonal antibody recognizing the envelope glycoprotein (Env) of the ecotropic neurotropic murine retrovirus CasBrE but not that of other murine retroviruses. Since 667 can be used for preclinical studies of antiviral gene therapy as well as for studying the early events of retroviral infection, we have cloned its cDNAs and molecularly characterized it in detail. Spot technique-based experiments showed that 667 recognizes a linear epitope of 12 amino acids located in the variable region A of the receptor binding domain. Alanine scanning experiments showed that six amino acids within the epitope are critical for MAb binding. One of them, D57, is not present in any other murine retroviral Env, which suggests a critical role for this residue in the selectivity of 667. MAb 667 heavy- and light-chain cDNAs were functionally characterized by transient transfection into Cos-7 cells. Enzyme-linked immunosorbent assays and Biacore studies showed that the specificities as well as the antigen-binding thermodynamic and kinetic properties of the recombinant 667 MAb (r667) produced by Cos-7 cells and those of the parental hybridoma-produced MAb (h667) were similar. However, h667 was shown to contain contaminating retroviral and/or retrovirus-like particles which interfere with both viral binding and neutralization experiments. These contaminants could successfully be removed by a stringent purification protocol. Importantly, this purified 667 could completely prevent retrovirus binding to target cells and was as efficient as the r667 MAb produced by transfected Cos-7 cells in neutralization assays. In conclusion, this study shows that the primary mechanism of virus neutralization by MAb 667 is the blocking of the retroviral receptor binding domain of CasBrE Env. In addition, the findings of this study constitute a warning against the direct use of hybridoma cell culture supernatants for studying the initial events of retroviral cell infection as well as for carrying out in vivo neutralization experiments and suggest that either recombinant antibodies or highly purified antibodies are preferable for these purposes.

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Akanji, Akinkunmi Ganiyu, Emiko Muramoto, José de Souza Caldeira Filho, Renata Martinussi Couto, and Elaine Bortoletti de Araújo. "Radiolabeling and biodistribution of monoclonal antibody (MAb) anti-CD20 with iodine-131." Brazilian Archives of Biology and Technology 48, spe2 (October 2005): 69–72. http://dx.doi.org/10.1590/s1516-89132005000700010.

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Radioactive isotopes of iodine, mainly iodine-131 have been broadly used in many monoclonal antibody radioiodination settings, employing different methods. In this study, using a Chloramine-T procedure, the influence of incubation time, CT mass, and Ab/activity ratio on the radiochemical yield of the anti-CD20 antibody labeled with iodine-131 was observed. Radiochemical yield was > 97 %, employing different Chloramine-T masses, independently of incubation time. Radiochemical purity was above 99 % after purification of the labeled compound. The relationship between Ab mass and radioactivity was tested and no difference was observed when 90.6 MBq (2.45mCi) of activity was incorporated in the Ab-mass range studied. Biodistribution study in normal Swiss mice showed higher uptake by the liver and intestines. Low thyroid uptake indicated a suitable in vivo stability. Slight blood uptake was considered a result of circulating radioactivity in normal organs and tissues. A favorable biological distribution of 131I-anti-CD20 suggests this radiopharmaceutical may be effectively used in the therapy of Non Hodgkin Lymphoma.

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Leng, Chunsheng, Qingwei Li, Fenfang Wu, Liyong Chen, and Peng Su. "Detection of the Single-Chain Precursor in the Production and Purification Process of Recombinant Human Insulin." Monoclonal Antibodies in Immunodiagnosis and Immunotherapy 32, no. 4 (August 2013): 255–61. http://dx.doi.org/10.1089/mab.2013.0013.

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Liu, Di, Xiaozhe Lv, Yanjun Cong, and Linfeng Li. "A Method for Screening Proteases That Can Specifically Hydrolyze the Epitope AA83-105 of αs1-Casein Allergen." Foods 11, no. 21 (October 23, 2022): 3322. http://dx.doi.org/10.3390/foods11213322.

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Milk protein hydrolysates are common in infant formula, but some of them retain allergenicity due to incomplete hydrolysis of the epitopes for milk allergens. This study explored a method for screening proteases that could specifically hydrolyze the epitope of αs1-casein allergen. Firstly, the αs1-casein epitope AA83-105 was synthesized by the solid-phase synthesis method. Then, after purification and identification, the complete antigen was prepared through coupling with bovine serum albumin (BSA) and was used to raise monoclonal antibodies (mAb) in BALB/c mice. Additionally, an indirect competitive-enzyme-linked immunosorbent assay (icELISA) was established. The mAb raised against αs1-casein protein was used as a control. The results showed that the purity of the synthetic epitope was >90%, and the coupling rate with BSA was 6.31. The mAb subtype is IgG1, with a titer of 1:320,000. The mAb reacted specifically with αs1-casein but did not cross-react with soybean protein. The linear regression equation of the competitive inhibition curve was y = −9.22x + 100.78 (R2 = 0.9891). The detection limit of icELISA method was more sensitive, and the method showed good accuracy and repeatability. The amounts of antigen residues in papain protease hydrolysates were relatively small, and the epitope fragment was detected in papain hydrolysate via mass spectrometry. This study provides ideas and methods for screening proteases that specifically hydrolyze the epitopes of milk allergens and also provides a superior foundation for the development of an advanced hypoallergenic formula.

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González, Angel, Beatriz L. Gómez, Soraya Diez, Orville Hernández, Angela Restrepo, Andrew J. Hamilton, and Luz E. Cano. "Purification and Partial Characterization of a Paracoccidioides brasiliensis Protein with Capacity To Bind to Extracellular Matrix Proteins." Infection and Immunity 73, no. 4 (April 2005): 2486–95. http://dx.doi.org/10.1128/iai.73.4.2486-2495.2005.

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ABSTRACT Microorganisms adhere to extracellular matrix proteins by means of their own surface molecules. Paracoccidioides brasiliensis conidia have been shown to be capable of interacting with extracellular matrix proteins. We aimed at determining the presence of fungal proteins that could interact with extracellular matrix protein and, if found, attempt their purification and characterization. Various extracts were prepared from P. brasiliensis mycelial and yeast cultures (total homogenates, β-mercaptoethanol, and sodium dodecyl sulfate [SDS] extracts) and analyzed by ligand affinity assays with fibronectin, fibrinogen and laminin. Two polypeptides were detected in both fungal forms. SDS extracts that interacted with all the extracellular matrix protein were tested; their molecular masses were 19 and 32 kDa. Analysis of the N-terminal amino acid sequence of the purified 32-kDa mycelial protein showed substantial homology with P. brasiliensis, Histoplasma capsulatum, and Neurospora crassa hypothetical proteins. Additionally, a monoclonal antibody (MAb) produced against this protein recognized the 32-kDa protein in the SDS extracts of both fungal forms for immunoblot. Immunofluorescence analysis revealed that this MAb reacted not only with mycelia and yeast cells, but also with conidia, indicating that this protein was shared by the three fungal propagules. By immunoelectron microscopy, this protein was detected in the cell walls and in the cytoplasm. Both the 32-kDa purified protein and MAb inhibited the adherence of conidia to the three extracellular matrix proteins in a dose-dependent manner. These findings demonstrate the presence of two polypeptides capable of interacting with extracellular matrix proteins on the surface of P. brasiliensis propagules, indicating that there may be common receptors for laminin, fibronectin, and fibrinogen. These proteins would be crucial for initial conidial adherence and perhaps also in dissemination of paracoccidioidomycosis.

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Journal articles on the topic 'MAB PURIFICATION'

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