Base specificity and binding of anti-ss/dsDNA monoclonal antibody. Application to the determination of ssDNA.
An anti-DNA murine hybridoma was generated and selected in a fusion of Sp2/0 myeloma cells with splenocytes derived from BALB/c mice immunized with DNA/protein complex. The monoclonal antibody designated as 4C7 was assessed by competitive immunoassay for binding to denatured, structurally single-stranded (ss)DNA, double-stranded (ds)DNA and oligodeoxynucleotides. The monoclonal antibody exhibited a marked preference for the ss conformation. Competitive inhibition assay performed with nucleosides and homopolynucleotides indicated that the deoxyadenosine residue was essential for antibody recognition and binding. The monoclonal antibody was purified by poly(A)-Sepharose chromatography and biotinylated. The ssDNA detection limit in the enzyme-linked immunosorbent assay with the biotinylated antibody ranged from 1 to 16 ng/ml. A dot immunobinding test was developed which was sensitive for the quantitation of picogram amounts of single-stranded DNA deposited on nitrocellulose filters and applied to the immunoenzymatic detection in human sera of target DNA.
Batova
I N
IN
Department of Immunobiology, Bulgarian Academy of Sciences, Sofia.
Kyurkchiev
S D
SD
Kehayov
I R
IR
D000225:Adenine / Q000378:metabolism; D000818:Animals; D000974:Antibodies, Antinuclear / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000918:Antibody Specificity; D001483:Base Sequence; D001666:Binding Sites, Antibody; D002417:Cattle; D004247:DNA / Q000097:blood; D004277:DNA, Single-Stranded / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007114:Immunization; D007700:Kinetics; D008180:Lupus Erythematosus, Systemic / Q000097:blood; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009711:Nucleotides / Q000378:metabolism / Q000494:pharmacology; D012474:Salmon; D012680:Sensitivity and Specificity
D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009711:Nucleotides; D004247:DNA; D000225:Adenine