Monoclonal anti-nucleoside antibodies. Characterization and application in an enzyme immunoassay of single-stranded DNA.
Two mouse monoclonal antibodies were raised against adenosine and guanosine coupled to bovine serum albumin (BSA) by periodate oxidation. They were named A-16 and G-K21 respectively and selected for their ability to recognize single-stranded DNA. Their epitope specificities were assessed and their dissociation constants determined by an indirect ELISA method. The KD values for adenosine and guanosine coupled to BSA were 9.9 X 10(-7) M and 1.1 X 10(-10) M for G-K21 respectively, and 2.5 X 10(-8) M and 1.0 X 10(-6) M for A-16. These monoclonal anti-nucleoside antibodies were used to develop a sandwich enzyme immunoassay for single-stranded DNA. The purified IgG antibodies were coupled to beta-galactosidase and alkaline phosphatase by the one-step glutaraldehyde method and used in a test optimized for pH, saturating proteins, coating antibody, nature of the conjugate and protein concentrations. Less than 100 pg/well of single-stranded DNA could be detected, and the detection was linear over a DNA concentration ranging from 0.34 to 34 ng/ml. The assay could quantitate single-stranded DNAs of differing origin, but not RNAs. The test was compared to another titration method, and used to calibrate target DNA amounts in non-radioactive hybridization experiments.
Traincard
F
F
Laboratoire des Sondes Froides, Institut Pasteur, Paris, France.
Chevrier
D
D
Mazie
J C
JC
Guesdon
J L
JL
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D004247:DNA / Q000032:analysis; D004277:DNA, Single-Stranded / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D005453:Fluorescence; D051379:Mice; D008807:Mice, Inbred BALB C; D009691:Nucleic Acid Denaturation; D009705:Nucleosides / Q000276:immunology*; D012313:RNA / Q000032:analysis
D000911:Antibodies, Monoclonal; D004277:DNA, Single-Stranded; D009705:Nucleosides; D012313:RNA; D004247:DNA