Abstract

Polyclonal antibodies are mixtures of monoclonal antibodies that were produced against different epitops. The goal of this project is to know the production, purification and horseradish peroxidase (HRP) conjugation of polyclonal antibodies against hamster immunoglobulins in rabbits. 300 µg/300 µl of ten hamster immunoglobulins was mixed with the same volume (300 µl) of adjuvant and injected into three 6-month-old white New Zealand rabbits. Anti hamster rich rabbits serums were isolated from whole blood and precipitated with ammonium sulfate in the final concentration of 50%. The precipitate was dialysed against phosphate buffered saline (PBS) (pH: 7.4) and applied to ion exchange chromatography (IEC) on diethylaminoethyl (DEAE)-sepharose 6B with tris-phosphate (pH: 8.1), and tris-phosphate contain 50 mM NaCl buffer. The purity of produced antibody was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reduced condition. Then purified immunoglobulin G (IgG) was conjugated with HRP. For exact measurement of conjugated IgG titer and evaluating of cross reaction, enzyme linked immunosorbent assay (ELISA) test was designed. Since IEC is a more simple and inexpensive method for the purification of IgG, we obtained a protein with approximate purity of 95%. Produced IgG showed high titer and high specificity in the designed ELISA. Purified antibody and its conjugation with HRP are used in research and diagnosis of hamster disease.

Key words: Production, purification, hamster immunoglobulins.

Abbreviation

Abbreviations: PAbs, Polyclonal antibodies; ELISA, enzyme linked immonosorbent assay; HRP, horseradish peroxidase; IgG, immunoglobulin G;PBS, phosphate buffered saline; IM, intra muscularly; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CFA, complete Freund’s adjuvant.