PRODUCTION OF GLUTATHIONE-COATED MICROTITRE PLATES FOR CAPTURING RECOMBINANT GLUTATHIONE-S-TRANSFERASE FUSION PROTEINS AS ANTIGENS IN IMMUNOASSAYS

Glutathione S-transferase (GST) is commonly used as a fusion partner i n producing recombinant proteins and this technology is increasingly b eing used to produce antigens for use in immunoassays to measure antib odies. To circumvent the requirement to purify such antigens before us e, we developed a method for coupling glutathione to microtitre plates so that GST-containing recombinant proteins could be purified and imm obilised in one step in a suitable state for immunoassays. This proced ure involves covalent linkage (using the heterobifunctional cross-link er sulphosuccinimidyl 4-(p-maleimidophenyl)butyrate) of reduced glutat hione through its sulphydryl group to lysine residues of haemoglobin p reviously immobilised on microtitre plates. Haemoglobin was superior o ver other proteins tested in giving the lowest non-specific binding; i n this regard it was also important to Limit the amount of cross-linke r used to 0.1 mM. Using glutamic acid decarboxylase as a model antigen , the new affinity capture assay was at least as good as the two-step procedure involving direct adsorption to plates of previously purified antigen; it may have the additional advantage of preserving the antig en in a more native conformation than direct adsorption. The new assay also performed as well as an assay using anti-GST antibodies adsorbed onto plates; glutathione plates, unlike anti-GST plates, will only ca pture recombinant proteins containing functional GST-a significant poi nt for some recombinant expression systems in which a large proportion of the protein product is insoluble because of incorrect folding. (C) 1998 Elsevier Science B.V. All rights reserved.