Eliciting DNA Photoproduct-Specific Antibodies with a Dinucleotide Photoproduct Antigen

DNA photoproduct-specific antibodies have proven to be very useful for the quantification of DNA photodamage induction and repair in cells by radio immunoassays (RIAs) and enzyme-linked immunoassays (ELISAs). RIAs and ELISAs for quantifying cis-syn, (6-4), and Dewar photoproducts of dipyrimidine sites have been reported that utilize both polyclonal and monoclonal antibodies, though nothing has yet been reported for quantifying trans-syn dimers. Photoproduct-specific monoclonal antibodies are attractive analytical agents because they have the potential advantage of being able to differentiate between photoproducts of the four possible dipyrimidine sites and their degradation products. With few exceptions, heterogeneous mixtures of photoproduct-containing DNA obtained by irradiation of DNA have been traditionally used as the antigens, and as a consequence the polyclonal and monoclonal antibodies elicited cannot discriminate well between photoproducts of different dipyrimidine sequences. A possible solution to this problem is to raise monoclonal antibodies against individual photoproducts of dinucleotide sites. To investigate this idea, a set of dinucleotide photoproduct antigens were prepared by covalently linking the four major photoproducts of thymidylyl-(3'→5')-thymidine, the cis-syn, trans-syn-I, (6-4), and Dewar products, to bovine serum albumin through a flexible aliphatic chain. Mice were immunized against the (6-4) dinucleotide antigen and the serum assayed by ELISA against photoproducts of both dinucleotides and oligonucleotides. Photoproduct-containing DNA for the assays was prepared by ligating photoproduct-containing octamers to biotinylated oligonucleotides to form 49-mers that could be bound to microtiter plates via avidin. The serum exhibited very high titers (≈100 000) for the (6-4) product of both the dinucleotide and the oligonucleotide and was highly selective for the (6-4) product, demonstrating that the (6-4) photoproduct of the dinucleotide is an effective antigen for eliciting antibodies that can recognize the (6-4) product in DNA.