Development Of An Enzyme-linked Immunosorbent Assay For The Determination Of Indomethacin In Different Environmental Water Samples

A highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ELISA) is successfully developed for the analysis of indomethacin in different environmental water samples(tap water, drinking water, surface water and wastewater).Indomethacin is a commonly used nonsteroidal anti-inflammatory drug (NSAID). The immunogen and coating antigen were prepared by covalently linking indomethacin to bovine serum albumin(BSA) and ovalbumin (OVA).Indomethacin antiserum was obtained by immunization of two adult New Zealand rabbits for 5 times and the superior antibody was characterized in terms of sensitivity, specificity, precision, accuracy and stability. Under optimal experimental conditions, the standard curve was constructed in the concentration range of 0.01 - 10 ng/mL(0.01,0.03,0.1,0.3,1.0,3.0,10ng/mL). For ten consecutive standard curves run in two weeks, IC50 values (the concentration of analyte producing 50% of inhibition) were found within 0.10 - 0.25 ng/mL, and the detection limit (DL) at a signal-to-noise ratio of 3 (S/N=3) was about 0.01 ng/mL. The specificity of the ELISA method is presented by cross-reactivity. Among all the tested compounds, as a pro-drug of indomethacin, acemetacin is structurally similar to indomethacin and therefore its cross-reactivity ratio with indomethacin antibody is 92.3%. However, the cross-reactivity values of the antibody with other tested NSAIDs were very low. Precision, accuracy and stability were confirmed by the spiking experiments, the recoveries were found within 98%-123%.In our assay, HPLC method was also applied in the determination of indomethacin in different water samples and the testing results were compared with ELISA method. The correlation coefficient of 0.988 was obtained, demonstrating a good correlation of ELISA with HPLC.