Development Of A Competitive Inhibition ELISA For Detection Of Folic Acid

Folic acid,a member of the B vitamins,is an important component of cell division and maintenance of cellular activity,which has the function of auxiliary transport of one carbon unit.Folic acid deficiency is an important risk factor of induced megaloblastic anemia,neural tube defects,congenital heart defects,and some cardiovascular diseases.Therefore,developing a rapid,convenient and accurate method for detecting the level of folic acid,means a lot to human health and product regulation.In this study,the folate immunogen(FA-BSA,FA-BSA-ACA)and the coating antigen(FA-OVA)were synthesized by using the carbodiimide method.The protein concentration were 12.59 mg/mL,10.8 mg/mL and 9.71mg/mL.The UV spectra of the conjugates showed thecharacteristic absorption of FA.The conjugation ratio,measured by OPA,were 13.16:1,8.90:1 and 4.22:1 respectively.Mice were immunized with a mixture of FA-BSA and adjuvant by subcutaneous injection.After the fourth immunization,antisera titre from mice immunized with FA-BSA reached 1:60000,IC50 was 0.695μg/mL,which is much better than the FA-BSA-ACA immunized mice.Took splenocytes from FA-BSA immunized mice and SP2/0 cell to fused into hybridomas.7 days after fusion,hybridomas were found and the supernatant was tested by indirect ELISA at 14th day.The results showed that there were 60 positive holes.Limiting dilution was used to cloning positive cell,but there were no differences between supernatant and complete medium.Hybridomas may lost the function of secreting antibodies.Rabbits were immunized by FA-BSA in order to eliciting anti-FA polyclonal antibody.Antisera titre reached 1:128000 and IC50 was 59.91μg/mL.Affinity chromatography was used to purify the antibody,The protein concentration was 0.63mg/mL,measured by BCA method.The optimal anti-antigen and antibody working condition was confirmed by the checkerboard method.The coating concentration was 2μg/mL,antibody concentration was 1.27μg/mL,best secondary antibody dilution was 1:2000.On the basis of optimization,the indirect ELISA method and standard curve was established.The equation was Y =-0.4924X + 0.3819,the R2 was 0.993 and the IC50 was 5.965μg/mL.The recoveries ranged from 83.333%to 98.627%,and the intra-assay coefficients of variation were between 5.677%and 8.553%,and the inter-assay coefficient of variation was between 5.217%and9.392%.Compared with the antisera detection,these results showed that the method of indirect competition ELISA for FA detection was improved by antibody purification.