High-throughput determination of free D-aspartic acid in mammals by enzyme immunoassay using specific monoclonal antibody.
A method for rapid determination of free D-aspartic acid (D-Asp) in mammals has been established using a highly specific mouse monoclonal antibody against D-Asp for the first time. An anti-D-Asp monoclonal antibody was obtained by the immunization of bovine-serum-albumin-conjugated D-Asp to BALB/c mice. The obtained antibody has a high specificity toward D-Asp but shows a slight cross-reactivity to all other D- and L- amino acids including L-Asp. The calibration range of the competitive enzyme linked immunosorbent assay (ELISA) is 0.016-16 micromol/mL D-Asp in rat serum samples. The precisions of this method were evaluated by inter-plate and intraplate assays, and the relative standard deviation values were 4.8% and 4.5%, respectively. The values of D-Asp determined by the present ELISA have a good correlation to those determined by high-performance liquid chromatography with the correlation coefficient of 0.963. Using this ELISA, the time course of D-Asp in the rat serum after intravenous administration was successfully demonstrated. The present method provides a simple and high-throughput determination of D-Asp in mammals, and is a useful tool for clarifying the physiological roles and diagnostic values of this D-amino acid.
Ohgusu
Tomohiro
T
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Fukuoka 812-8582, Japan.
Hamase
Kenji
K
Tanaka
Hiroyuki
H
Shoyama
Yukihiro
Y
Zaitsu
Kiyoshi
K
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001224:Aspartic Acid / Q000097:blood* / Q000737:chemistry; D002138:Calibration; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D007275:Injections, Intravenous; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000737:chemistry; D051381:Rats; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
D000911:Antibodies, Monoclonal; D012710:Serum Albumin, Bovine; D001224:Aspartic Acid; D010047:Ovalbumin