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

• Published in: Applied and Environmental Microbiology Vol. 59; no. 8; pp. 2698 - 2705
• Main Authors: FLISS, I, SAINT LAURENT, M, EMOND, E, LEMIEUX, R, SIMARD, R. E, ETTRIKI, A, PANDIAN, S
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.08.1993
• Subjects: acide nucleique; acidos nucleicos; adn; alimentos; Animals; Antibodies, Monoclonal - biosynthesis; Antibody Affinity; anticorps monoclonal; anticuerpos monoclonales; Antigens, Bacterial; arn; arn mensajero; arn messager; arn ribosomal; arn ribosomial; Bacteria; Bacteriological methods and techniques used in bacteriology; Bacteriology; biodegradacion; biodegradation; Biological and medical sciences; Biology; biotin; biotina; biotine; cell counting; contaminacion; contamination; conteo de celulas; Deoxyribonucleic acid; DNA; DNA Probes; DNA, Bacterial - genetics; DNA, Bacterial - immunology; elisa; Enzyme-Linked Immunosorbent Assay - methods; Evaluation Studies as Topic; Female; foods; Fundamental and applied biological sciences. Psychology; gene; genes; Listeria; Listeria - genetics; Listeria - immunology; Listeria - isolation & purification; listeria monocytogenes; messenger rna; Mice; Mice, Inbred BALB C; Microbiology; monoclonal antibodies; Nucleic Acid Heteroduplexes - isolation & purification; Nucleic Acid Hybridization; nucleic acids; numeration cellulaire; Polymers; produit alimentaire; Ribonucleic acid; ribosomal rna; ribosomas; ribosome; ribosomes; RNA; RNA, Bacterial - genetics; RNA, Bacterial - immunology; RNA, Messenger - genetics; RNA, Messenger - immunology; Species Specificity; test elisa; Immunization; Immunoglobulins; Rodentia; DNA RNA hybrid; Monoclonal antibody; Immunological method; Antigen; Vertebrata; Mammalia; Immunogenicity; Listeria; Mouse; Bacteria; Detection; ELISA assay
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.59.8.2698-2705.1993

Murine monoclonal antibodies (MAbs) specific for DNA-RNA hybrids 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.