Determination of metallothionein-3 by a competitive enzyme-linked immunosorbent assay in experimental animals.
An easy and specific enzyme-linked immunoassay (ELISA) for the determination of metallothinein-3 (MT-3) in experimental animals for the research of heavy metal and chemical toxicity has not been reported yet. Therefore, we have developed a competitive ELISA, using a specific monoclonal antibody raised against human recombinant MT-3 (rMT-3). The epitope mapping of the antibody was conducted using mouse, rat, and human MT-3s and peptide fragments of human MT-3. MT-1/2, MT-3 knock-out (KO) mice and human brain and liver were used for the evaluation of the ELISA. A pretreatment method of the tissue homogenates was also examined. The antibody used for the ELISA had the same cross-reactivity with MT-3 in humans and experimental animals. The human MT- 3 NH(2) terminal peptide (Fr. 1-17) was the demonstrated epitope of this antibody. The reactivity of this ELISA in brain homogenate of MT-3 KO mouse was significantly low compared with the wild type and MT-1/2 KO mice. The lowest detection limit of the ELISA was 10 ng/ml and over 80% of the spiked rMT-3 was recovered in the brain homogenate. The assay linearity was intact with a 5-fold dilution in the brain homogenate. The inter- and intra-assay CV was 6.5%, respectively. An effective pretreatment procedure of the tissue homogenate was also established for this MT-3 ELISA. In conclusion, this competitive ELISA is an easy and specific method for measuring the brain MT-3 level in experimental animals.
Frontier Institute Co., Ltd., Hokkaido, Japan.
Hidetoshi
H
Saito
Kyoumi
K
Nakazato
Mihoko
M
Kato
Tsukasa
T
Kodaira
Taito
T
Akutsu
Yoshiharu
Y
Tokita
Keiji
K
Suzuki
Takeaki
T
Nagamine
Katsuyuki
K
Nakajima
D000818:Animals; D001923:Brain Chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007668:Kidney / Q000737:chemistry; D008099:Liver / Q000737:chemistry; D000090023:Metallothionein 3; D051379:Mice; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology*; D010446:Peptide Fragments / Q000276:immunology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology
D000939:Epitopes; D007074:Immunoglobulin G; D000090023:Metallothionein 3; C000715430:Mt3 protein, mouse; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D011994:Recombinant Proteins