Monoclonal anti-idiotype antibodies recognizing the variable region of a high-affinity antibody against 11-deoxycortisol. Production, characterization and application to a sensitive noncompetitive immunoassay.
Anti-idiotype antibodies recognizing the variable regions of a particular anti-hapten antibody are valuable tools, which can be used in sensitive hapten immunoassays based on a noncompetitive format. Here, we describe the production and characterization of monoclonal anti-idiotype antibodies against idiotopes on the variable regions of an antibody showing high affinity and specificity to 11-deoxycortisol (11-DC). 11-DC is the biosynthetic precursor of cortisol and a diagnostic index for the assessment of pituitary-adrenal function. BALB/c or A/J mice were repeatedly immunized with the anti-11-DC antibody conjugated with keyhole limpet hemocyanin and their spleen cells were then fused with P3/NS1/1-Ag4-1 myeloma cells. Seven kinds of anti-idiotype antibodies were generated, one of which was a beta-type antibody recognizing the paratope and others which were alpha-type antibodies recognizing the framework region. A noncompetitive ELISA based on idiotype-anti-idiotype reactions was established using one of these alpha-type antibodies in combination with the beta-type antibody and with the anti-11-DC antibody. This noncompetitive assay system provided improved sensitivity (detection limit: 1.0 pg=2.9 fmol), which is approximately 10 times higher than the corresponding competitive enzyme immunoassay, and offered a practical specificity for clinical use. Appropriate serum 11-DC levels were obtained for normal subjects [0.16+/-0.09 (S.D.) microg/l (n=6), ranging from 0.086 to 0.316 microg/l] using the present assay system.
Kobayashi
Norihiro
N
Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Aoba-ku, 980-8578, Sendai, Japan.
Shibusawa
Kazuki
K
Kubota
Kazuyuki
K
Hasegawa
Noriko
N
Sun
Pi
P
Niwa
Toshifumi
T
Goto
Junichi
J
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000888:Antibodies, Anti-Idiotypic / Q000096:biosynthesis / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity; D003350:Cortodoxone / Q000097:blood / Q000737:chemistry / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006241:Haptens / Q000276:immunology; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D012680:Sensitivity and Specificity
D000906:Antibodies; D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D006241:Haptens; D007135:Immunoglobulin Variable Region; D003350:Cortodoxone