Production and purification of polyclonal antibody against F(ab')۲ fragment of human immunoglobulin G

Antibodies are essential tools of biomedical and biochemical researches. Polyclonal antibodies are produced against different epitopes of antigens. Purified F(ab')۲ can be used for animal’s immunization to produce polyclonal antibodies. Human immunoglobulin G (IgG) was purified by ion exchange chromatography method. In all stages verification method of the purified antibodies was sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified IgG was digested by pepsin enzyme and F(ab')۲ fragment was purified by gel filtration separation method. For production of polyclonal antibody, rabbit was immunized by purified F(ab')۲ and antibody production was investigated by enzyme-linked immunosorbent assay. Purified anti-IgG F(ab')۲ was conjugated with fluorescein isothiocyanate. Ion exchange chromatography purification yielded ۳۸ mg of human IgG antibody. The results of SDS-PAGE in reduced and non-reduced conditions showed bands with ۲۵-۳۰ kDa molecular weight (MW) and ۵۰-kDa respectively and a distinct band with ۱۵۰ kDa MW. The results of non-reduced SDS-PAGE for determining the purity of F(ab')۲ fragment showed one band in ۹۰ kDa and a band in ۱۵۰ kDa MW position. Purification by Ion exchange chromatography method resulted about ۱۲ mg rabbit polyclonal antibody. Flow cytometry showed generated polyclonal antibody had an acceptable activity compared to commercial antibody. Taking together, purified IgG F(ab')۲ and polyclonal anti-IgG F(ab')۲ are useful tools in biomedical and biochemical researches and diagnostic kits.

کلیدواژه ها:

F(ab&#۰۳۹ ، )۲ fragment ، Immunoglobulin G ، Pepsin digestion ، Polyclonal antibody ، Purification