PRODUCTION AND CHARACTERIZATION OF ANTI-DNA-RNA MONOCLONAL-ANTIBODIESAND THEIR APPLICATION IN LISTERIA DETECTION

Murine monoclonal antibodies (MAbs) specific for DNA-RNA hybrids were successfully produced with two different heteropolymers as antigens, c DNA-mRNA and phiX174 DNA-RNA heteroduplexes. The former was simpler to prepare. Both had shown similar immunogenicities. Two different immun oglobulin M MAbs were isolated. The 20D3 MAb, generated with the phiX1 74 DNA.RNA hybrid, showed association constants of 1.05 x 10(12), 2.12 x 10(10), and 1.68 x 10(7) for the antigens phiX174 DNA-RNA, cDNA-mRN A, and poly(rA)-poly(dT), respectively. The 6B5 MAb, obtained with the cDNA-mRNA hybrid, showed association constants of 1.59 x 10(5), 5 X 1 0(12); and 7.1 x 10(8) for the above-described antigens, respectively. With the 20D3 MAb, an immunoassay was developed for the detection of Listeria DNA-RNA hybrids. In brief, a biotinylated rRNA gene probe spe cific for the genus Listeria was hybridized witb rRNA in the solution phase. The hybrids thus formed were then captured in microtiter plate wells precoated with the purified 20D3 MAb, and the probe-target hybri ds were detected with a streptavidin-alkaline phosphatase conjugate. T his assay was shown to be specific for the genus Listeria and highly s ensitive, allowing the detection of as little as 2.5 pg of target rRNA .