Immunoassay for the detection of E. coli proteins in recombinant DNA derived human growth hormone.
An enzyme-linked immunosorbent assay (ELISA) has been developed for the quantitation of part-per-million levels of the most probable E. coli polypeptide (ECP) contaminants of E. coli produced biosynthetic human growth hormone (hGH). The antibody preparation, used for both coat and conjugate in this ELISA, was demonstrated to be reactive with the reference ECPs (a collection of the most probable protein contaminants) by both affinity chromatography and immunoblot analysis. Affinity purification of this antibody preparation using immobilized reference ECPs resulted in an assay with a higher signal-to-noise ratio and also 'normalized' the antibody population to approach stoichiometric equivalence with the immobilized ECPs. Reference ECPs, size fractionated by gel filtration, were quantitated in agreement with their absorbance at 280 nm. The assay was demonstrated to be specific for ECPs obtained from the hGH purification process. Since the purification of each recombinant DNA derived protein from E. coli requires its own unique process, this means that no generic ECP assay can be developed. It is felt that the criteria established for this assay provide a comprehensive approach to the development of quantitative multiple antigen immunoassays.
D000918:Antibody Specificity; D001426:Bacterial Proteins / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D004926:Escherichia coli / Q000032:analysis; D013006:Growth Hormone / Q000032:analysis*; D006801:Humans; D007158:Immunologic Techniques; D007163:Immunosorbent Techniques; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000032:analysis*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013006:Growth Hormone