Enhanced ultrasensitive detection of structurally diverse antigens using a single immuno-PCR assay protocol.

Our studies of DNA damage and repair in autoimmune disease, lymphomagenesis, and carcinogenesis, require identification of an immunoassay approach that is capable of ultrasensitive detection in a routine human tissue biopsy of several physicochemically diverse antigens, some of which will be present at very low level. Immuno-polymerase chain reaction (immuno-PCR) is a recently described method for ultrasensitive antigen detection that combines the amplification power of PCR with a method similar to a standard antibody capture, enzyme-linked immunosorbent assay (ELISA). As a test of the universality 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 forms of the following physicochemically diverse antigens: oligomeric pyruvate dehydrogenase complex (PDC; Mr 8.5 x 10(6)), the promutagenic DNA base adduct O(6)-methylguanosine (Mr 298) and its monomeric repair enzyme, O(6)-methylguanine-DNA methyltransferase (MGMT; Mr 22,000), and a peptide from the N-terminus of MGMT (Mr 2310). We found that all antigens could be ultrasensitively assayed using the single immuno-PCR protocol. Assay limits observed using antigen-specific (primary) antibodies at 1 microg/ml, were in the approximate range of 10(2)-10(9) molecules, with O(6)-methylguanosine being detected most sensitively. Sensitivity of the antigen assay appeared to positively correlate with primary antibody titres determined by ELISA. Furthermore, we observed a substantial increase in detection sensitivity for all antigens by the use of primary antibodies at the higher level of 10 microg/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 assay sensitivity of up to four orders of magnitude greater than those previously reported through the use of this assay to measure other antigens. This represents up to a nine order of magnitude increase in immunoassay sensitivity compared to ELISA. Our findings provide compelling evidence that immuno-PCR is indeed a universal ultrasensitive antigen detection method. Using the indicated assay enhancements. immuno-PCR performed as detailed here can offer greatly increased sensitivity for antigen measurement compared to other methods. Thus, our findings suggest that parallel quantitation of several different antigens in very small samples of human tissue will be readily attainable using immuno-PCR.

Suicidal dephosphorylation of thiamine pyrophosphate coupled with pyruvate dehydrogenase complex.

Earlier it was noted that purified pyruvate dehydrogenase complex (PDC) produced by "Sigma" usually contains almost saturating amounts of thiamine pyrophosphate (ThPP). In this communication we present the observation that the endogenous ThPP coupled to PDC is dephosphorylated while staying at -10 degrees C, because in the enzyme preparation thiamine monophosphate and un-phosphorylated thiamine appear (HPLC determination). Under the same conditions exogenous ThPP is not dephosphorylated despite contact with the PDC preparation. This may suggest that interactions of some active groups of the enzyme with molecules of endogenous ThPP leads to break-up of the phosphoesters bonds, and destruction of the coenzyme. Decrease of PDC activity during storage is not in proportion with the degree of ThPP dephosphorylation. However the observed instability of PDC activity may be a consequence of the spontaneous process of its coenzyme autodestruction.