Preparation Of Monoclonal Antibody And Establishment Of Immunoassay For Analysis Of Vitamin B₁₂

B₁₂ vitamins,or cobalamins,are organic complexes containing central cobalt ions in a corrin ring.As an essential nutrient for human health,B₁₂ vitamins play an important role in the synthesis of DNA and development of the myelin sheath,especially in the blood delivery system and nervous system.So it’s necessary to develop an sophisticated,rapid and simple way for analysis of vB₁₂,which is important for protecting consumers’interests.In the current work,vB₁₂ was coupled to keyhole limpet hemocyanin（KLH）and Bovine Serum Albumin（BSA）by a CDI method to produce artificial antigens.The ratio of their conjugations were confirmed by UV spectrophotometer,which were6:1 and 4:1,respectively.Balb/c female mice（6 weeks old）were immunized with KLH-vB₁₂ to produce antibodies specific to vB_12.The monoclonal antibody named 4A1 was identified as IgG 2a subtype,κlight chain,then it was inject to abdominal cavity of mice to churn out lots of antibodies.The antibodies were purified using protein A and the concentration were 4.97 mg/mL.With the aim to develop a highly sensitive ELISA,several factors such as pH value,ionic strength,surfactant,and organic solvent concentration that could affect the sensitivity of the assay were estimated.Under the optimum conditions,Cross reactivity was found to be less than 0.01%with other compounds of vitamin B,except for three analogs of cobalamins which showed a cross reactivity from 0.27%to 2.31%.The limit of detection was 0.2 ng/m L with a linear working range of 2–100 ng/mL（R²=0.993）.The spiked samples were detected with recovery ranging from 86.02–110.54%,and the coefficients of variation were from 2.62%to 13.7%.Chemiluminescenceenzyme-linkedimmunosorbentassay（CL-ELISA）provided an more sensitive way for analysis of vB₁₂ in energy drinks,tablets and infant formula.The limit of detection（LOD）was lower than 0.2 ng/mL with the linear working range of 0.5–20 ng/mL（R²=0.991）.Recoveries from tablets,drinksand milk were 91.5–102.9%,81.6–92.2%and 78.7–84.5%,respectively.After HPLC validation,the method proposed had been proved to be a trustworthy tool to quantify vB₁₂ in daily samples.Besides,acridinium ester was coupled with anti-vB₁₂ antibody to obtained more simple way for the determination of vB₁₂.For this CLIA analysis,the linear working range was found to be 2.5–25 ng/m L（R²=0.989）.The LOD of this method was 1 ng/mL with IC₅₀ of 10.2 ng/mL.We also explore a new assay for analysis vB₁₂by bispecific antibody,and the the limit of detection（LOD）was found to be 0.1ng/mL with the linear working range of 1-25 ng/mL,IC₅₀ of 5.21 ng/m L.