The measurement of staphylococcal protein A by ELISA in immunoglobulin preparations (original) (raw)
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Biochemical and Biophysical Research Communications, 2003
The production of recombinant hepatitis B virus surface antigen (rHBsAg) purified by immunoaffinity chromatography with monoclonal antibodies is used to obtain a vaccine against this virus. Monoclonal antibodies to rHBsAg from mouse ascites have been purified by Staphylococcal Protein A (SpA)—prior coupling to Sepharose CL-4B (Amersham-Bioscences, Uppsala, Sweden). A high sensitivity immunoassay has been developed for the quantification of part-per-million of SpA contaminants likely to co-purify with monoclonal antibodies obtained by Protein A affinity chromatography, in the presence of immunoglobulins. Specific sheep polyclonal Abs against SpA (SpAc1) were used as plate coating and the SpA detection was possible thanks to the conjugates of sheep Ab fragments F(ab)2 (fSpAc1) and horseradish peroxidase (fSpAc1-peroxidase), reducing the possible unspecific interaction between SpA and Fc fragments. The immunoassay was shown to be specific for SpA contaminants. The quantification limit of the assay was 0.39 ng/ml spreading to the measurement of contamination levels less than 2 ppm of SpA in final preparations of monoclonal antibodies used for the immunopurification of pharmaceutical products, which is quite low for this application.
Journal of Immunological Methods, 1992
Immunoglobulin G (IgG) of Indian soft-furred rat, Millardia meltada, was purified by an immunoaffinity chromatography and antibodies against it was raised in rabbit. Using this rabbit anti-M. meltada IgG antibody, sensitivity of enzyme-linked immunosorbent assay (ELISA) to measure parasite-specific antibodies in the sera of M. meltada was markedly enhanced than the previous method using rabbit anti-mouse IgG and rabbit anti-rat IgG antibodies, which could cross-react to M. meltada IgG. Since M. meltada could effectively produce circulating antibodies against two intestinal helminths, Strongyloides venezuelensis and Nippostrongylus brasiliensis, the high susceptibility of this animal to an array of parasites seems to be not due to general immunological deficiency.-KEY WORDS: ELISA, IgG, Millardia meltada.
Staphylococcal protein A binding to the Fab fragments of mouse monoclonal antibodies
The Journal of Immunology
Two mouse IgG1 monoclonal antibodies specific for the Lewis(a) human blood group antigen were purified on protein A-Sepharose by using buffers of decreasing pH for elution. Unlike other IgG1 antibodies that eluted at pH 7.0 to 6.0, these antibodies could only be eluted at pH 4.0 to 3.0. The Fab and F(ab')2 fragments of these antibodies also eluted at pH 4.0 to 3.0, although the Fc fragment of one eluted at pH 6.0. This interaction of protein A with Fab was not due to anti-protein A antibody activity, because the presence of Lewis(a) trisaccharide did not prevent the binding of Fab to protein A-Sepharose and because Fab that had bound to solid phase hapten could still be recognized by protein A. Thus, certain mouse IgG1 antibodies possess determinants in their Fab portion recognized by protein A, allowing for the purification of such Fab fragments on protein A-Sepharose.
European Journal of Nuclear Medicine, 1993
The present study was untertaken to compare the technetium-99m labelled non-specific polyclonal human immunoglobulin (Ig) with 99mTc-labelled monomeric human immunoglobulin (m-Ig), 99mTc-labelled, protein A-purified, human immunoglobulin (A-Ig) and 99mTc-labelled monomeric, protein A-purified, human immunoglobulin (mA-Ig) as tracer agents for the detection of a thigh infection with Staphylococcus aureus. In vitro the binding of the various tracer agents to bacteria at various intervals was determined. For the in vivo evaluation, mice were infected and received one of the various labelled proteins. Scintigrams were made 0.25, 1, 4 and 24 h later. All 99mTc-labelled Igs bound to bacteria in vitro: the percentages of binding for the m-Ig (from 1 h onwards) and A-Ig and mA-Ig (from 3 h onwards) were significantly higher than that for Ig. The in vivo target-tonon-target (T/NT) ratios were significantly higher from 4 h onwards for all purified Igs than for Ig. Protein A-purified Igs yielded higher T/NT ratios than m-Ig. Furthermore, the amount of activity in the liver was significantly lower 24 h after administration of m-Ig, A-Ig and mA-Ig than after administration of Ig. It is concluded that in this experimental infection 99mTc-labelled monomeric Ig localizes a staphylococcal thigh infection better and faster than 99mTc-labelled unpurified Ig. However, the accumulation obtained with protein A-purified Ig or protein Apurified monomeric Ig was the highest of all tracer agents tested.
Journal of Chromatography & Separation Techniques, 2012
Immunoglobulin Y (IgY) is the major protein present in the avian egg yolk. This antibody fulfils important functions in the protection of Ostrich birds against infections. The aim of this study was to demonstrate the binding capacity of Staphylococcal proteins A (SpA) to Ostrich IgY and assess purification of the IgY by SpA affinity chromatography. Chloroform polyethylene glycol (Polson), affinity chromatography, Enzyme-Linked Immunosorbent Assay (ELISA) and Western blotting methods were used in the process. Results obtained revealed that Ostrich IgY has heavy chain of 70 kDa and light chain of 30 kDa confirming results by Western blot. In addition livetins (egg yolk proteins) were shown in the protein electrophoresis that preceded the Western blot. The binding capacity between SpA and Ostrich IgY is important because SpA can be used as a reagent in immunoassays for antibody detection against microbial agents that usually infect livestocks. This is the first time the use of SpA for purification of Ostrich IgY is being reported in literature.
Journal of Immunological Methods, 1989
Studies were performed to detect and isolate trace contaminants of staphylococcal enterotoxin B (SEB) in various protein A preparations isolated by affinity chromatography employing human IgG covalently bound to Sepharose 4B. Utilizing an ELISA technique, trace amounts (0.018-0.138%) of SEB could be detected in protein A preparations after separation of the SEB employing a molecular sizing column in a high pressure liquid chromatography (HPLC) system. Trace contamination by SEB could be removed from protein A preparations by an additional DEAE ion exchange chromatography step employing a low ionic strength buffer system (0.005 M NaC1 in 0.01 phosphate buffer, pH 7.50). The resulting protein A preparations possessed a purity higher than that observed prior to the final purification step. Polyacrylamide gel electrophoresis (PAGE) analyses of the trace contamination removed from protein A preparations by ion exchange chromatography revealed, in addition to SEB, several additional contaminating polypeptides of an unknown nature. These studies indicate that protein A preparations of high purity can be prepared by employing DEAE ion exchange chromatography in addition to affinity chromatography utilizing immobilzed human IgG.