A generic capture ELISA for recombinant proteins fused to glutathione S-transferase: validation for HPV serology.
An enzyme-linked immunosorbent assay (ELISA) system has been developed that uses glutathione crosslinked to casein as capture protein to bind recombinant protein antigens fused to N-terminal glutathione S-transferase (GST). The method allows simple and efficient immobilization and one-step purification of overexpressed recombinant antigens from crude lysates on ELISA plates coated with glutathione casein. Several antigens can be tested in parallel under the same conditions without the need to biochemically purify or renature the proteins. An additional undecapeptide epitope fused to the C-terminus of each antigen permits the detection and quantification of any full-length protein antigen bound to the ELISA plate with one single monoclonal antibody. The ELISA system was applied with four antigens to detect antibodies against E6 and E7 proteins of human papillomavirus types 16 and 18. Antibody reactivities of 164 sera from patients with cervical carcinoma and healthy individuals were in good agreement with those determined using a previously established capture ELISA with biochemically purified and renatured proteins as antigens although the GST capture ELISA was more sensitive with no loss of specificity. The GST capture ELISA could be adapted to provide standardized antibody assays for many protein antigens.
Sehr
P
P
Applied Tumor Virology, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, D-69120, Heidelberg, Germany.
Zumbach
K
K
Pawlita
M
M
D000914:Antibodies, Viral / Q000276:immunology; D000957:Antigens, Viral, Tumor / Q000235:genetics / Q000276:immunology; D002277:Carcinoma / Q000276:immunology* / Q000821:virology; D002364:Caseins / Q000737:chemistry; D004268:DNA-Binding Proteins; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D005978:Glutathione / Q000737:chemistry; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000276:immunology; D027383:Papillomaviridae / Q000276:immunology*; D050725:Papillomavirus E7 Proteins; D030361:Papillomavirus Infections / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012097:Repressor Proteins; D012680:Sensitivity and Specificity; D014412:Tumor Virus Infections / Q000276:immunology*; D002583:Uterine Cervical Neoplasms / Q000276:immunology* / Q000821:virology
D000914:Antibodies, Viral; D000957:Antigens, Viral, Tumor; D002364:Caseins; D004268:DNA-Binding Proteins; C068651:E6 protein, Human papillomavirus type 16; C052603:E6 protein, Human papillomavirus type 18; C052633:E7 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16; D005982:Glutathione Transferase; D005978:Glutathione