Production and characterization of anti-DNA-RNA monoclonal antibodies and their application in Listeria detection.

摘要

Murine monoclonal antibodies (MAbs) specific for DNA-RNA hybrids were successfully produced with two different heteropolymers as antigens, cDNA-mRNA and phi X174 DNA-RNA heteroduplexes. The former was simpler to prepare. Both had shown similar immunogenicities. Two different immunoglobulin M MAbs were isolated. The 20D3 MAb, generated with the phi X174 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 phi X174 DNA-RNA, cDNA-mRNA, 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 10(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 specific for the genus Listeria was hybridized with 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 hybrids were detected with a streptavidin-alkaline phosphatase conjugate. This assay was shown to be specific for the genus Listeria and highly sensitive, allowing the detection of as little as 2.5 pg of target rRNA.