Enhanced ultrasensitive detection of structurally diverse antigens using asingle immuno-PCR assay protocol

Our studies of DNA damage and repair in autoimmune disease, lymphomagenesis , and carcinogenesis, require identification of an immunoassay approach tha t is capable of ultrasensitive detection in a routine human tissue biopsy o f several physicochemically diverse antigens, some of which will be present at very low level. Immuno-polymerase chain reaction (immuno-PCR) is a rece ntly described method for ultrasensitive antigen detection that combines th e amplification power of PCR with a method similar to a standard antibody c apture, enzyme-linked immunosorbent assay (ELISA). As a test of the univers ality of immuno-PCR, and as an assessment of the suitability of this method for our studies, we used a single immuno-PCR protocol to assay purified fo rms of the following physicochemically diverse antigens: oligomeric pyruvat e dehydrogenase complex (PDC; M-r 8.5 X 10(6)), the promutagenic DNA base a dduct O-6-methylguanosine (M-r 298) and its monomeric repair enzyme, O-6-me thylguanine-DNA methyltransferase (MGMT; M-r 22,000), and a peptide from th e N-terminus of MGMT (M-r 2310). We found that all antigens could be ultras ensitively assayed using the single immuno-PCR protocol. Assay limits obser ved using antigen-specific (primary) antibodies at 1 mu g/ml, were in the a pproximate range of 10(2)-10(9) molecules, with O-6-methylguanosine being d etected most sensitively. Sensitivity of the antigen assay appeared to posi tively correlate with primary antibody titres determined by ELISA. Furtherm ore, we observed a substantial increase in detection sensitivity for all an tigens by the use of primary antibodies at the higher level of 10 mu g/ml. The latter approach permitted antigen assay within the approximate range of 10(0)-10(7) molecules. The combination of higher titre primary antibodies and their use at higher input level, produced an increase of immuno-PCR ass ay sensitivity of up to four orders of magnitude greater than those previou sly reported through the use of this assay to measure other antigens. This represents up to a nine order of magnitude increase in immunoassay sensitiv ity compared to ELISA. Our findings provide compelling evidence that immuno -PCR is indeed a universal ultrasensitive antigen detection method. Using t he indicated assay enhancements, immuno-PCR performed as detailed here can offer greatly increased sensitivity for antigen measurement compared to oth er methods. Thus, our findings suggest that parallel quantitation of severa l different antigens in very small samples of human tissue will be readily attainable using immuno-PCR. (C) 1999 Elsevier Science B.V. All rights rese rved.