Quantitation of monoclonal antibodies by ELISA. The use of purified mouse IgG and mouse IgM monoclonal antibodies as standards in a quantitative ELISA measuring monoclonal antibodies produced by cell culture.
Murine monoclonal antibodies (MAbs) of IgG1, IgG2a and IgG2b subclasses and IgM class in hybridoma culture supernatants were quantified using a sensitive, reliable, optimized indirect double antibody sandwich ELISA. In the ELISA, the MAb in the culture supernatants was sandwiched between affinity isolated heavy chain specific polyclonal antibodies used for capture and detection. Quantitation was achieved by comparison with a standard curve produced by a purified MAb of the same class, subclass or ideally the same clone as the MAb to be quantified. These quantitative results were compared with those obtained using purified IgG and IgM polyclonal serum samples as standards and those obtained by total protein estimation using measurement at OD280nm. The IgG subclass MAbs used as standards were purified using protein G and the IgM class MAb was purified by ion exchange followed by gel filtration chromatography. Bovine IgG contamination of the MAb supernatants and the purified MAbs was also measured by a double antibody sandwich ELISA.
International Blood Group Reference Laboratory (IBGRL), Bristol, UK.
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Mushens
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Guest
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Scott
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D007074:Immunoglobulin G / Q000032:analysis*; D007143:Immunoglobulin Heavy Chains / Q000032:analysis; D007075:Immunoglobulin M / Q000032:analysis* / Q000302:isolation & purification; D051379:Mice; D011506:Proteins / Q000032:analysis; D012015:Reference Standards
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007075:Immunoglobulin M; D011506:Proteins