A novel competitive ELISA for both free and protein-bound nitrotyrosine.
3-Nitro-L-tyrosine (nitrotyrosine) has recently been considered to be useful as a biomarker of endogenous production of several reactive nitrogen species including peroxynitrite. In the present study, nitrotyrosine was coupled to human serum albumin (HSA) using a two-step glutaraldehyde method and immunized mouse with multifocal intradermal injections. Using a conventional immunization protocol, 12 stable monoclonal antibodies (MAbs) producing cell lines recognizing nitrotyrosine were obtained. Six MAbs were selected for further characterization. A study of cross-reactions with nitrotyrosine-like compounds showed that the antibodies had a high specificity for nitrotyrosine, but no detectable reactivity with L-tyrosine, p-nitro-L-phenylalanine, o-phospho-L-tyrosine or 3-amino-L-tyrosine. Using these high titer and affinity antibodies, a competitive inhibition ELISA was developed with a lower detection limit of approximately 20 nmol/L to detect both free and protein-bound nitrotyrosine in biological systems.
Qu
Li-Na
LN
13th Department, Institute of Space Medico-Engineering, 1 West Yuanmingyuan Road, Beijing, China.
Yang
Tang-Bin
TB
Yuan
Yan-Hong
YH
Zhong
Ping
P
Yang
Bin
B
Zhao
Hai
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005976:Glutaral / Q000737:chemistry; D006801:Humans; D051379:Mice; D030421:Peroxynitrous Acid / Q000737:chemistry; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry; D019000:Phosphotyrosine / Q000737:chemistry; D011485:Protein Binding; D026361:Reactive Nitrogen Species; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D026361:Reactive Nitrogen Species; D030421:Peroxynitrous Acid; C040239:4-nitrophenylalanine; D019000:Phosphotyrosine; C019253:3-aminotyrosine; C002744:3-nitrotyrosine; D014443:Tyrosine; D010649:Phenylalanine; D005976:Glutaral