Preparation Of Polyclonal Antibody For Minoxidil, Development Of An ELISA Method For The Detection Of Minoxidil

Minoxidil is a vasodilator, and can also stimulate hair growth when locally and chronically used. The FDA approved minoxidil in treatment of male androgen alopecia for external use in 1988. Although minoxidil is only for local external application, it could cause adverse reaction such as mild allergic dermatitis, dizziness, irritating allergies, face and limbs hirsutism, and careless use may lead to eye burns and stimulation. The drug is not allowed to be added in daily shampoo or hair care cosmetics, and "Cosmetics health standard"(2007) prohibits adding minoxidil in cosmetics. However, for profiteering, some manufacturers still add minoxidil in cosmetics violations, which will make hair care cosmetics achieve a certain effect.At present, there is not yet a clear shampoo and hair care cosmetics minoxidil standards on detection methods. Existing literatures determinates the content of minoxidil in lotion and plasma by using UV spectrophotometer and HPLC. Enzyme-linked immunosorbent assy(ELISA) is widely used in the field of drug residues, for advantage of detecting fast, convenience, high sensitivity and specificity, it is developed very rapidly.This study aims at the preparation of the Minoxidil artificial antigen and polyclonal antibody which has good immunogenicity. Because of minoxidil is small molecules, it does not have immunogenicity, The Protein Connection Technology applies to couple Minoxidil and bovine serum albumin(BSA), in order to prepare artificial antigen Minoxidil-BSA. UV spectrum analysis and SDS-PAGE electrophoresis method could be used to carry on the appraisal; The conjugate of minoxidil-BSA was used to immunize BALB/C mice for preparing polyclonal antibody(pAb), and it`s titer and specificity of antiserum could be detected by indirect ELISA and competitive ELISA method. Thereby establishing a complete Minoxidil enzyme-linked immunosorbent assay method.Experimental data showed that, compared with UV scan results and electrophoretic bands, the results of both have undergone significant change between Minoxidil–BSA and BSA, which shows successful synthesis of artificial antigen. The conjugate of minoxidil-BSA was used to immunize BALB/C mice. And the produced antiserum showed high titer of 1:12800 in direct ELISA. The best working concentration determined by phalanx titration method is that, for coating antigen concentration of 1:200,and for antibody dilution of 1:3200. Its ELISA curve equation: y=-0.0823x+0.938(R2=0.9811), Minoxidil mass concentration and the absorbance in 0.001-10μg/ml showed a good linearity.This study has been successfully preparing minoxidil artificial antigen and polyclonal antibodies, And the enzyme-linked immunoassay for minoxidil laid the foundation for colloidal gold detection method of minoxidil residual, and provides a new method of rapid and accurate detection.So far, China has not reported minoxidil residual ELISA detection method. Therefore, this method has a strong social and economic significance.