| Methotrexate(MTX)is a folic acid analogue that inhibits dihydrofolate reductase and thymidylate synthetase.This inhibition prevents the reduction of dihydrofolic acid to tetrahydrofolic acid and disrupts the synthesis of purine bases(adenine and guanine)and a pyrimidine base,thymidine,which are constituents of DNA and cellular RNA.It is widely used in the treatment of lymphocytic leukemia,rheumatoid arthritis and psoriasis.When administered with a high dose of MTX,patients can cause oral mucosal inflammation,liver function and renal dysfunction,leukopenia,trigger miscarriage and other side effects.Calcium folate is known to rescue the side effect caused by high doses of MTX,but it is necessary to monitor the content of MTX at all times.Therefore,it is particularly important to establish a rapid,simple and accurate method to monitor the concentration of MTX in the serum.In this study,MTX immunogens(MTX-BSA and MTX-ACA-BSA)and coated antigens(MTX-OVA)were prepared by carbodiimide method.The coupling results were analysed by the UV scan pattern.The protein concentrations of MTX-BSA,MTX-ACA-BSA and MTX-OVA were determined to be 9.85,8.22 and 8.75 mg/mL separately,using the BCA protein assay.The coupling ratios were determined 36.34:1,21.59:1 and 10.18:1 separately by OPA method.Mice was immunized with MTX-BSA and MTX-ACA-BSA to obtain anti-MTX mice antibodies.The titers were 1:40000 and 1:80000 separately.Rabbits were immunized with MTX-BSA to obtain anti-MTX rabbit serum with a titer of 1:256,000.Anti-MTX serum was purified by affinity purification,and the protein concentration of purified antibody was 0.3 mg/mL using the BCA protein assay.The best dilution of MTX-OVA and purified antibody were 0.125 μg/mL and 2 μg/mL determined by checkerboard method,The dilution of 1:3000 was the best dilution of the enzyme labeled antibody with the inhibition rate.Based on this,the MTX standard solution was diluted with human serum of 1:10.Then we established a standard curve of IcELISA for MTX.Based on the optimal conditions,the MTX kit was developed with a detection range of 0.04μM to 2.00 μM.The MTX kit had an analytical sensitivity of 0.0188 μM and a functional sensitivity of 0.05 μM,a good analytical intra-assay precision(less than 10%)and inter-assay precision(less than 14%),and the recovery rate between 91%～105%.The accelerated test of the kit showed that the kit could still be used for one year at 4℃. |
