Antibody-based approaches to coumarin analysis.
7-Hydroxycoumarin (7-HC) was chemically conjugated by diazo coupling to carrier proteins such as bovine serum albumin (BSA), thyroglobulin and ovalbumin. These conjugates were characterised by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC). Rabbits were immunised using the 7-HC-BSA conjugate. The highest antibody titre achieved was 1:10,000, as determined by competitive enzyme-linked immunosorbent assay (ELISA). The resulting antibodies were purified by ammonium sulphate precipitation, followed by protein A affinity chromatography. Their purity was assessed by SDS-PAGE and HPLC. These antibodies have been used in the development of a competitive ELISA, an amperometric biosensor and an electrochemical immunoassay. Both the ELISA and amperometric biosensor have been successfully applied to the analysis of 7-HC and its glucuronide conjugate in human urine samples. Each of these antibody-based methods provides a novel approach to the analysis of the main metabolites of coumarin.
Egan
D A
DA
School of Biological Sciences, Dublin City University, Ireland.
Deasy
B
B
Dempsey
E
E
Smyth
M R
MR
O'Kennedy
R
R
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D003374:Coumarins / Q000276:immunology / Q000378:metabolism / Q000652:urine*; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008297:Male; D010047:Ovalbumin / Q000378:metabolism; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013954:Thyroglobulin / Q000378:metabolism; D014468:Umbelliferones / Q000276:immunology / Q000652:urine
D000906:Antibodies; D003374:Coumarins; D014468:Umbelliferones; D012710:Serum Albumin, Bovine; C031477:7-hydroxycoumarin; D010047:Ovalbumin; D013954:Thyroglobulin; C030123:coumarin