Heterogeneity of antibodies to metallothionein isomers and development of a simple enzyme-linked immunosorbent assay.
A competitive enzyme-linked immunosorbent assay (ELISA) for the measurement of metallothionein (MT) in tissues and body fluids has been developed. The ELISA employs the IgG fraction of a rabbit antiserum to rat liver Cd-MT-2 polymer, a biotinylated secondary antibody, and peroxidase conjugated avidin. With a 1:4000 dilution of the immunoglobulins, typical standard curves (logit-log regression) provide a linear range of 0.1-100 ng for MT-2 and 10-1000 ng for MT-1. Fifty percent inhibition is accomplished with 15 ng and 250 ng for MT-2 and MT-1, respectively. Rat liver MT-1 and MT-2 containing different metals (Ag, Cu, and Zn) inhibited the antibodies as effectively as CdMT. However, the antibodies exhibited greater affinity for both Apo-MT isoforms. Previously reported discrepancies between results obtained by metal binding assays (e.g., Ag-hem binding) and radioimmunoassay for MT levels in tissues have been largely resolved. By addition of 1% Tween 20 to samples, the ELISA routinely estimated the total MT in samples of rat, mouse, and human liver and kidney at 88% of the value obtained by the silver-hem binding assay. Specific antibodies to MT-2 were purified from our antiserum by affinity purification using CH-Sepharose 4B coupled with rat liver MT-1. Estimation of MT in samples using purified MT-2 antibodies provided slightly lower values (72%) for MT in tissues as compared to the Ag-hem method. The predominant form of MT in tissues of control animals was found to be MT-2.(ABSTRACT TRUNCATED AT 250 WORDS)
Chan
H M
HM
Department of Pathology, University of Western Ontario, London, Canada.
Pringle
G A
GA
Cherian
M G
MG
D000818:Animals; D000906:Antibodies; D003600:Cytosol / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007668:Kidney / Q000737:chemistry; D008099:Liver / Q000737:chemistry; D008297:Male; D008668:Metallothionein / Q000032:analysis* / Q000276:immunology; D051379:Mice; D008809:Mice, Inbred C3H; D008817:Mice, Mutant Strains; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity
D000906:Antibodies; D008668:Metallothionein