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KMID : 0370019970110000057
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Chung-Ang Journal of Pharmacal Sciences
1997 Volume.11 No. 0 p.57 ~ p.66
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The Production and Isolation of Mouse Immunoglobulin against RNase A and B
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Abstract
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Ribonuclease A, B (RNase A, B) are the enzymes catalyse he hydrolysis of 3´,5´-phosphodiester linkages of ribonucleic acids. RNase B(M.W. 15.5KDa) differs from RNase A(M.W. 13.6KDa) in that RNase B has five glycoforms consisting of Man_9GlcNAc_2, to Man_9GlcNAc_2 at the single glycosylation site (Asn-34). It has not been fully studied that the difference of glycosylation between RNase A and RNase B would make difference in production of polyclonal antibody when it was used as antigen. In the present study, male Balb/c mice aged 6-7weeks were used, and three mice per group were immunized intraperitoneally with 0.2ml of emulsion mixture containing 100ug RNase A or B in 0.01ml of PBS and 0.18ml of Freund´s adjuvant, and immunized with PBS as a control. To identify the production of polyclonal antibody, total protein amount was detected by use of UV- spectrophotometer and tested 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In order to know the affinity of polyclonal antibody to RNase A, B and to measure the concentration of polyclonal antibody. ELISA method had been applied using the mouse monoclonal antibody 6 isotypes (IgG1, IgG2a, IgG2b, IgG3, IgA, IgM) and monoclonal anti-goat/sheep IgG peroxidase. Protein A column was prepared to test the possibility of separation of the polyclonal antibody to each immunoglobulin class. As a result, the glycosylation between RNase A and B did not have an effect on production pattern of polyclonal antibody from ascites of mice immunized with RNase A or B. Second, in the present study, the polyclonal antibody made from mice immunized with RNase A or RNase B should be isolated to immunoglobulin class or subclass by use of various binding or elution buffer.
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