Development of an enzyme-linked immunosorbent assay for metallothionein-I and -II in plasma of humans and experimental animals.
BACKGROUND
An easy and specific enzyme-linked immunoassay (ELISA) for the determination of metallothionein-1 (MT-1) and 2 (MT-2) simultaneously in serum and other biological specimens in humans and experimental animals has not been developed yet.


METHODS
We developed a competitive ELISA, a specific polyclonal antibody against rat MT-2. The epitope mapping of the antibody was conducted using MTs in mouse, rat, rabbit, human and the fragment peptides of human MT-2. MT1/2 and MT-3 knock-out mice and cadmium treated mice were used for the evaluation of the ELISA. Pretreatment method of serum was examined to deplete blocking factors for this assay.


RESULTS
The antibody used for this ELISA had the same cross-reactivity with MT in humans and experimental animals. NH2 terminal peptide of MT with acetylated methionine was proved to be the epitope of this antibody. The reactivity of this ELISA system with liver, kidney and brain in MT1/2 knock-out mice was significantly low, but was normal in MT-3 knock-out mouse. The lowest detection limit of this ELISA was 0.6 ng/ml and the added MT-1 was fully recovered from serum. The mean MT concentration in our preliminary study was 23+/-4.6 ng/ml in human serum. Cadmium treatment to mice induced significantly higher amount of MT in serum, liver, kidney and spleen as reported previously by different established methods.


CONCLUSIONS
The proposed competitive ELISA is an easy and specific method for practical use, determining total MT-1 and -2 simultaneously in serum and other biological specimens of human and experimental animals.
Nakajima
Katsuyuki
K
School of Health Sciences, Faculty of Medicine, Gunma University, Maebashi, Gunma, Japan. nakajimak05@ybb.ne.jp
Kodaira
Tsukasa
T
Kato
Mihoko
M
Nakazato
Kyoumi
K
Tokita
Yoshiharu
Y
Kikuchi
Hiroki
H
Sekine
Hiromi
H
Suzuki
Keiji
K
Nagamine
Takeaki
T
D000328:Adult; D000368:Aged; D000825:Animal Structures / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001769:Blood / Q000187:drug effects; D002104:Cadmium / Q000494:pharmacology; D002138:Calibration; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology; D005260:Female; D006801:Humans; D007564:Japan; D008297:Male; D008668:Metallothionein / Q000032:analysis* / Q000097:blood* / Q000235:genetics / Q000276:immunology; D000090023:Metallothionein 3; D051379:Mice; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000276:immunology; D010446:Peptide Fragments / Q000276:immunology; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000276:immunology; D055815:Young Adult
D000906:Antibodies; D000939:Epitopes; D000090023:Metallothionein 3; C000715430:Mt3 protein, mouse; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D002104:Cadmium; D008668:Metallothionein