title: Development and validation of sensitive sandwich ELISAs for two investigational nonapeptide metastin receptor agonists, TAK-448 and TAK-683.
abstract: TAK-448 and TAK-683, investigational agents with potential utility in the treatment of prostate cancer, are potent low molecular weight metastin receptor agonists consisting of nine amino acids. Monoclonal antibodies (mAbs) against these agents were developed to facilitate their evaluation in preclinical studies. Six mAbs were obtained from four immunogens. Three mAbs recognized the C-terminal of TAK-683 and TAK-448, two recognized the N-terminal of TAK-683, and one recognized the N-terminal of TAK-448. Using various combinations of these six mAbs, sandwich ELISAs for TAK-448 and TAK-683 were developed. These assays were highly sensitive, specific, and accurate. The detection limit for TAK-448 and TAK-683 was 3 and 5 pg/mL, respectively, and there was no interference from rat plasma, rat metastin, or analogs of TAK-448/TAK-683. Recovery achieved ≤±10% with intra-/inter-day assay precision coefficient of variation <10%. The assay demonstrated high stability and sample pre-treatment was not required. Each assay detected the dose-dependent concentration of TAK-448 and TAK-683 in blood 24h after a single intravenous administration of 0.1 and 1mg/kg doses. In conclusion, sensitive sandwich ELISAs were developed to detect the small peptides TAK-448 and TAK-683. The novel assays reliably quantified these nonapeptides in rat plasma, and thus will be useful for preclinical studies of these agents. This methodology may be applicable to the development of similar assays for other short peptides.
authors: Advanced Science Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Co., Ltd., 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan. nobuyo.yoshida@takeda.com
authors: Nobuyo
authors: N
authors: Yoshida
authors: Naoki
authors: N
authors: Nishizawa
authors: Hisanori
authors: H
authors: Matsui
authors: Yuu
authors: Y
authors: Moriya
authors: Chieko
authors: C
authors: Kitada
authors: Taiji
authors: T
authors: Asami
authors: Hirokazu
authors: H
authors: Matsumoto
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000970:Antineoplastic Agents / Q000008:administration & dosage* / Q000097:blood* / Q000276:immunology; D002138:Calibration; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D005260:Female; D007275:Injections, Intravenous; D059648:Kisspeptins / Q000008:administration & dosage* / Q000097:blood* / Q000276:immunology; D057230:Limit of Detection; D016014:Linear Models; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D043562:Receptors, G-Protein-Coupled / Q000819:agonists*; D000074502:Receptors, Kisspeptin-1; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; C118688:KISS1R protein, human; D059648:Kisspeptins; D043562:Receptors, G-Protein-Coupled; D000074502:Receptors, Kisspeptin-1; C578072:TAK-683; C577479:metastin (46-54), acetyl-tyrosyl(46)-hydroxypropyl(47)-threonyl(49)-azaglycyl(51)-methylarginyl(53)-tryptophyl(54)-