Selection and optimisation of monoclonal antibodies for a two-site immunoradiometric assay for ACTH.
Four monoclonal antibodies with predominant specificities towards different sequences within the ACTH molecule were investigated in a 2-site immunoradiometric assay (IRMA) for human ACTH. Antibody 3H9 recognises the extreme N-terminal sequence, antibodies 1A12 and 1D1 are specific for the mid N-terminal sequence but differ in that the former cross-reacts with alpha MSH whereas the latter does not, and antibody 2A3 recognises the C-terminal sequence. Combinations of iodinated antibodies with antibodies covalently linked to Sephacryl S300 were tested for their compatibility and potential for a sensitive assay. Two antibody combinations (1D1 plus 3H9 or 1A12) gave no dose-response curve indicating severe steric inhibition, whereas other combinations yielded assays with widely different detection limits (2-2400 ng ACTH/l). The combination of labelled 1D1 and solid-phase 2A3 gave the most sensitive assay and when optimised for antibody concentrations and incubation times the working range was 10-5 X 10(4) ng/l (CV less than 20%). The optimised sequential 2-step IRMA involves incubation of standard or test sample with labelled 1D1 for 18 h at 4 degrees C followed by incubation with solid-phase 2A3 for 2 h at room temperature, after which the labelled complex is separated by the sucrose layering technique. The detection limit of this IRMA was several 100-fold lower than by RIA using the same antibodies. The IRMA detected large molecular weight precursors containing the full ACTH sequence (22 000, 31 000 and 34 000) but not ACTH fragments (1-18, 1-24, 18-39). It is concluded that selected monoclonal antibodies provide a sensitive and rapid 2-site IRMA for intact ACTH and its precursors.
S H
SH
Dobson
C
C
Gray
H
H
Smith
T
T
Baker
J G
JG
Ratcliffe
A
A
White
D000324:Adrenocorticotropic Hormone / Q000276:immunology*; D000911:Antibodies, Monoclonal; D000915:Antibody Affinity; D000918:Antibody Specificity; D004306:Dose-Response Relationship, Immunologic; D000939:Epitopes; D006801:Humans; D011863:Radioimmunoassay / Q000379:methods*; D012995:Solubility
D000911:Antibodies, Monoclonal; D000939:Epitopes; D000324:Adrenocorticotropic Hormone