Polymerase chain reaction products containing 5-methyldeoxycytidine: a microplate immunoquantitation method.

Numerous non-isotopic techniques have been developed in the last decade to detect and quantitate hybridization products (1). Since the polymerase chain reaction (PCR) was introduced this technique has become increasingly important for the diagnosis of bacterial and viral infections. The need for simple methods allowing quantitative evaluation of PCR products has resulted in the development of alternatives to gel electrophoresis and blotting. The most sensitive techniques rely on the incorporation of biotinylated dUTP or of a digoxigenin-dUTP complex. The products containing the modified nucleotide are recognized by an avidin-enzyme conjugate or an anti-digoxygenin antibody. The use of 5-methylcytidine (5-MeCyd) offers an alternative to the labeling of nucleic acid probes with biotin-dUTP. It is possible to raise antibodies against this hapten. We obtained such antibodies, either polyclonal or monocional, and used them to detect and quantitate the presence of 5-MeCyd in urine samples from cancer patients (2), in metaphase chromosomes (3) as well as in the nuclei of cells grown in vitro (unpublished results). Following on from this we tested the possibility of using these antibodies to detect and quantitate products generated through PCR made in the presence of 5-MedCTP. The reaction was carried out in the presence of biotinylated primers, specific for the Epstein-Barr virus (EBV), which allowed the PCR products to be retained in a polystyrene microtitration plate to which avidin had been covalently linked.