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 in producing recombinant proteins and this technology is increasingly being used to produce antigens for use in immunoassays to measure antibodies. To circumvent the requirement to purify such antigens before use, we developed a method for coupling glutathione to microtitre plates so that GST-containing recombinant proteins could be purified and immobilised in one step in a suitable state for immunoassays. This procedure involves covalent linkage (using the heterobifunctional cross-linker sulphosuccinimidyl 4-(p-maleimidophenyl)butyrate) of reduced glutathione through its sulphydryl group to lysine residues of haemoglobin previously immobilised on microtitre plates. Haemoglobin was superior over other proteins tested in giving the lowest non-specific binding; in this regard it was also important to limit the amount of cross-linker 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 antigen 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 capture recombinant proteins containing functional GST--a significant point for some recombinant expression systems in which a large proportion of the protein product is insoluble because of incorrect folding.
Murray
A M
AM
Department of Cellular and Molecular Sciences, St George's Hospital Medical School, London, UK.
Kelly
C D
CD
Nussey
S S
SS
Johnstone
A P
AP
D001323:Autoantibodies / Q000032:analysis*; D003432:Cross-Linking Reagents; D048909:Diabetes Complications; D003920:Diabetes Mellitus / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004800:Enzymes, Immobilized / Q000276:immunology; D005968:Glutamate Decarboxylase / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry*; D005982:Glutathione Transferase / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry; D006801:Humans; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D016750:Stiff-Person Syndrome / Q000150:complications / Q000276:immunology
D001323:Autoantibodies; D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D006454:Hemoglobins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D005968:Glutamate Decarboxylase; D005978:Glutathione