Development and validation of a two-site immunoradiometric assay for human atrial natriuretic factor in unextracted plasma.
This two-site immunoradiometric assay (IRMA) for human atrial natriuretic factor (ANF)99-126 in plasma utilizes a mouse monoclonal antibody raised against rat (r) ANF103-125 with specificity directed towards the ring structure of ANF, and a rabbit antiserum to human (h) ANF99-126. The monoclonal antibody is radioiodinated, and the IgG fraction of the antiserum is coated onto wells of a microtiter plate. Plasma or standard hANF99-126 (150 microL) is incubated with the radioligand in coated wells for 24 h. The detection limit is 0.9 pg per well, corresponding to 2 pmol/L, with a working range (CV less than 10%) from 4.5 to 540 pmol/L. Intra- and interassay precision are 7% and 9%, respectively, and the assay is unaffected by plasma matrix. In humans, the IRMA is specific for hANF99-126, the major circulating form of ANF, and does not cross-react with metabolites having deletions at the carboxy terminus. Plasma IRMA values in normal seated subjects were 6.6 +/- 1.5 (SEM) pmol/L and results correlated with those of an extraction RIA (r = 0.81, P less than 0.001).
Lewis
H M
HM
Department of Pharmacology, University of Birmingham, U.K.
Ratcliffe
W A
WA
Stott
R A
RA
Wilkins
M R
MR
Baylis
P H
PH
D000911:Antibodies, Monoclonal; D009320:Atrial Natriuretic Factor / Q000097:blood*; D006152:Cyclic GMP / Q000652:urine; D006801:Humans; D007158:Immunologic Techniques; D011863:Radioimmunoassay; D011874:Radiometry
D000911:Antibodies, Monoclonal; C472955:NPPA protein, human; D009320:Atrial Natriuretic Factor; D006152:Cyclic GMP