Large scale generation and characterization of anti-human IgA monoclonal antibody in ascitic fluid of BALB/c mice (original) (raw)
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Journal of Immunological Methods, 1996
5 1 monoclonal antibodies (McAb) with putative specificity for human IgA, the IgA subclasses, Am allotypes or secretory component (SC) were evaluated for immunoreactivity and specificity by nine laboratories employing immunodiffusion, agglutination, immunohistological assays, immunoblotting and direct binding and competitive inhibition enzyme immunoas-Abbreviations: Am allotype, genetically determined antigenic differences in IgA proteins; AP, alkaline phosphatase; BSA, bovine serum albumin: C u, heavy chain constant region domains (e.g.. C, 1, hinge, C n 2. C u 3. C n 41; Fc, immunoglobulin fragment crystallizable; HRP, horseradish peroxidase: IgA PAN. an antibody that binds to al/ allotypic forms and subclasses of IgA: Ig. immunoglobulin;
Sequences encoding the 27K and 25K nefgene products (Nef 27 and Nef 25) were amplified by PCR from a human immunodeficiency virus type 1 infectious clone and subcloned directly into Escherichia coli, yeast and baculovirus expression vectors. The yeast-and baculovirus-derived Nef had native N termini but the expression levels were low. The expression levels of the E. col#derived glutathione S-transferase-Nef fusion proteins were very high and a major portion was soluble. Large-scale production orE. coli-derived Nef27 and Nef 25 was carried out by growing recombinant cells in a fermenter under fed-batch conditions followed by affinity purification on glutathione-Sepharose before and after thrombin cleavage. Large quantities of highly purified recombinant Nef proteins have been produced for functional and structural studies. Under nonreducing conditions both Nef 27 and Nef 25 existed as a mixture of monomers, dimers and small amounts of higher oligomers, but when reduced were monomeric. The highly purified Nef proteins had no G protein activities, however Nef27 was biologically active. When electroporated into uninfected CD4 + T lymphocytes both E. coli-derived Nef 27 and yeast-derived myristylated Nef 27 down-regulated the surface expression of CD4, demonstrating that this method can be used to assess the biological activity of purified recombinant Nef.
Background: The ability of polyclonal antibodies to react with many epitopes of an antigen makes them valuable reagents in research and diagnosis. The aim of this study was purification of mouse IgG2a and production of polyclonal antibody against purified mouse IgG2a subclass. Materials and Methods: Mouse IgG2a was purified by ProA affinity. Verification method of the purified antibody was SDS-PAGE and ELISA by a mouse isotyping Kit. Rabbit was immunized with purified IgG2a. The production of antibody in rabbit was investigated by direct ELISA method. Rabbit serum was collected and precipitated at the final concentration of 50% ammonium sulfate. Polyclonal antibody was purified by ion-exchange chromatography and labeled with HRP. The titre and cross reactivity of product was detected by direct ELISA method. Results: The results of SDS-PAGE in reduced and non-reduced conditions showed bands with 50-KDa, 25-30 KDa MW and a distinct band with 150 KDa MW. Isotype determination showed the presence of mouse IgG2a in related fraction. The titer of Anti-mouse polyclonal antibody was 200000. The optimum titer of prepared HRP conjugated IgG was 4000. Conjugated rabbit IgG has more cross reactivity with mouse IgG2b. Conclusion: Taking together, affinity chromatography and ion-exchange chromatography are appropriate techniques for purification of mouse IgG subclasses and rabbit IgG, respectively.
An ELISA has been set up for quantifying mouse monoclonal antibodies in culture supernatant. The assay includes rabbit anti-mouse IgG antibodies chromatographycally purified. This preparation was used as coating and as conjugated antibodies in the ELISA. The assay can detect IgGl with sensitivity of 0.2 ng/mL, IgG2a (0.85 ng/mL), IgG2b (0.13 ng/mL), and IgG3 (3.19 ng/mL) in culture supernatants. The effective working range was from subnanogram per mL quantities to 30 ng/mL by using a computer statistical program. Variation coefficient of ELISA was below 7%. Correlation estimates with a similar ELISA using commercial reagents were performed for each mouse antibody subclass. The assay was able to detect the four mouse mon-oclonal antibody subclasses in pure human serum as compared with the same ELISA using commercial an-tibodies. A 24-h pharmacokinetic profile of 1 patient treated with an IgG2a monoclonal antibody is presented.
Two new murine monoclonal antibodies rised against human IgG
Journal of the Serbian Chemical Society
Many pathological conditions are accompanied with changes in the concentration of the total IgG or some of its fraction. For this reason there is great interest in the production of reagents specific for IgG. In this paper, the binding characteristics of two new murine monoclonal antibodies (MoAb), assigned MoAb 15 and MoAb 22, are reported. These MoAbs were produced by hybridoma technology. By performing ELISAs and Western blots analyzes, it was demonstrated that both MoAbs interact specifically with human IgG. Cross reactivity with other sera proteins was not observed. In order to precisely localize the epitopes recognized by MoAb 15 and MoAb 22, the Western blots interactions of these MoAbs with electrophoreticaly separated IgG-fragments, obtained by the action of proteolytic enzymes (papain, pepsin, trypsin), were analyzed. According to the results of these experiments, both MoAbs interacted with epitopes in the Cg3 domain. The affinity constants, calculated from Scatchard plots...
Journal of Immunological Methods, 1985
A single-step method for purification of mouse monoclonal antibodies directly from ascitic fluids using hydroxylapatite column chromatography is described. The procedure yields highly purified IgG or lgM antibodies. The purified immunoglobulin is essentially free of contaminating mouse albumin, transferrin, and other ascites proteins, as determined by SDS-polyacrylamide gel electrophoresis. Hydroxylapatite chromatography can also separate monoclonal IgG antibodies from contaminating IgG antibodies found in ascites fluid of animals that have been immunosuppressed prior to ascites induction. Furthermore, the evidence presented here suggests that some hybridomas of SP2/0 origin synthesize an extraneous light chain resulting in the secretion of hybrid antibody molecules.
Biochemical and Biophysical Research Communications, 2004
In this paper we describe an immunoenzymatic assay based on a rat monoclonal antibody (Ram j) developed to determine mouse IgG concentration, which is widely used for samples obtained on purification processes, like supernatant waste and the content of IgG in the vaccine (rHBsAg). This assay involves the use of a rat antibody-horseradish peroxidase-conjugated for the revealing of the antigen-antibody reaction. The rat antibody was produced in cell culture using a dialysis tube (DT). The immunoassay was standardized following several concepts, such as specificity, precision, and linearity. The result obtained permitted us to replace the use of polyclonal antibodies to determine the j light chain mouse antibodies by a rat monoclonal antibody of high sensibility and reproducibility. The assay permitted a reliable measurement of murine j Ig up to 0.68 ng/ml and was capable of quantifying 6.25 ng/ml. Due to the high frequency of the j light chain in mouse antibodies this system acquires a great application.
Evaluation of protocols for purification of mouse monoclonal antibodies
Journal of Immunological Methods, 1986
Protocols for purification of mouse monoclonal antibodies (MAbs) from nude mice ascites were investigated in order to assess the yield and to compare the purified products in two-dimensional gel electrophoresis (2DGE). Three MAbs (one IgG2 and two IgG1), selected for their differing behaviours towards protein A, were purified by ammonium sulphate precipitation and/or gel filtration, anion exchange (DEAE), hydroxylapatite and affinity (protein A) chromatography, or by a combination of these methods. Protein A constantly provided the highest purity whatever the IgG subclass. The best results in terms of yields and purity were a function of the optimization of the protein A protocol. In our study, they were obtained in a 3 h protocol (IgG2), a 16 h protocol with discontinuous pH gradient method (IgG1 with sufficiently high affinity for protein A) or a multi-step protocol involving DEAE and protein A (IgG1 with low affinity for protein A). DEAE chromatography alone provided a slightly better yield, but only moderate purity. Hydroxylapatite chromatography appeared to be less potent in terms of yield, purity and day-today reproducibility. Salt precipitation and gel filtration enabled only relative enrichment of the MAb solution. Some degradation products of both heavy and light chains clearly appeared in the 2DGE patterns of antibodies purified by different protocols, and seem to be partly related to the elution pH and to the duration of the purification procedure. Finally, this work highlights considerable heterogeneity not only between two different MAbs of the IgG1 subclass but also within a monoclonal population of immunoglobulins.
Polyclonal Antibody Production against Mouse Purified IgG2a towards Use in Basic Research
Research in Molecular Medicine, 2016
1 Immunology Research Center, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. 2 Tabriz Autonomous University of Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran. 3 Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. 4 Immunology Laboratory, Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz-Iran. 5 Pharmacology Department, Faculty of pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
In this study, the effects of different sample preparation techniques on the separation of monoclonal antibody IgG1 were investigated experimentally. Monoclonal IgG1 was obtained from hybridoma cell line TB/C3 transfected with bcl-2 carrier plasmid, which was grown in serum-free medium. Three different pre-treatment techniques prior to Protein G affinity chromatography have been used in order to concentrate and partial purify the monoclonal antibody. The pre-treatments researched in this paper are precipitation of the antibody by ammonium sulfate, dilution of the antibody in the binding buffer of affinity chromatography and ultrafiltration through an Amicon Ultra-15 filter with molecular weight cut-off at 100 kDa. Purification through direct application of the antibody onto the Protein G affinity column without pre-treatments was used as a control method. The results indicate that the ultrafiltration through an Amicon filter was an effective method for both concentration and partial...