| Vitamin deficiency is an important nutritional problem,especially in developing countries.On the other hand,excessive intake of vitamins affects the internal environment of the body and even leads to poisoning.Therefore,vitamin analysis is essential for disease diagnosis,nutritional evaluation,pharmaceutical and food production,especially functional foods,health foods and infant foods.Immunoassay has been paid more and more attention due to its unique sensitivity and selectivity,excellent timeliness and portability,and the possibility of on-site detection.In this study,vitamin B1（VB1）,vitamin B3（VB3）,vitamin B5（VB5）,vitamin B6（VB6）namely pyridoxine（PN）,pyridoxamine（PM）,pyridoxal（PL）,vitamin B9（VB9）,vitamin B12(VB12),vitamin A（VA）,vitamin E（VE）,Vitamin K1（VK1）,Vitamin K3（VK3）was selected as the research objects.The structural modification of hapten was used as the entry point to prepare specific monoclonal antibodies and corresponding immunological analysis methods were developed.According to the basic principles of hapten design and the chemical structures of the target analyte,the haptens of vitamins were designed and synthesized.To obtain artificial antigens,the haptens were coupled to the carrier protein using the carbodiimide method,glutaraldehyde method or active ester method.And the hapten-protein conjugates were characterized by ultraviolet-visible spectrophotometry and polyacrylamide gel electrophoresis.The vitamin monoclonal antibodies were prepared by the classical hybridoma technology.Ten kinds of monoclonal antibodies were prepared in this study,namely:Anti-VB1 m Ab 1F5,Anti-VB3 m Ab 2D9,Anti-VB5 m Ab 2D5,Anti-VB6 m Ab 2F9 and 1E3,Anti-VB9 m Ab 2E7,Anti-VB12 m Ab 2E7,Anti-VA m Ab 3D4,Anti-VE m Ab 5C2,Anti-VK1 m Ab 1B5 and Anti-VK3 m Ab 3C8.The subtypes of the prepared m Abs were Ig G2b,Ig G3,Ig G2b,Ig G2a,Ig G3,Ig G2a,Ig G2a,Ig G3,Ig G2a,Ig G2b and Ig G2b,respectively.The affinity constant of the prepared m Abs,Ka values,were 1.35×1010,1.41×109,6.05×109,1.40×109,6.77×109,3.37×109 and 8.33×109,2.33×109,3.06×108,5.83×109,1.14×1010L/mol,respectively.Based on the ten prepared anti-vitamin m Abs,indirect competitive enzyme linked immunosorbent assays（ic-ELISAs）for the detection of vitamins in food were established.We optimized the concentration of antigen and m Abs,and p H,ionic strength and organic solvent content of working solution were optimized.Under optimal conditions,the IC50 of the ic-ELISA for VB1,VB3,VB5,PN,PM,PL,VB9,VB12,VA,VE,VK1,VK3 detection was20.71,603.41,201.31,106.6,250.57,400.11,0.119,0.416,493.68,1,002.36,1,005.77 and0.50 ng/m L,respectively.And the LOD of the ic-ELISA for VB1,VB3,VB5,PN,PM,PL,VB9,VB12,VA,VE,VK1,VK3 detection was 4.51、65.71、32.22、13.11、29.70、39.46、0.019、0.064、69.77、99.92、110.20 and 0.11 ng/m L,respectively.The developed ic-ELISA has good specificity and no cross-reactivity with other vitamins.The average recovery in energy drinks spiked with VB1,VB3,VB5,VB6,VB9 and VB12 was 89.35%～116.0%,and the coefficient of variation（CV）was 1.53%～9.43%.The average recovery in milk powder spiked with VB1,VB3,VB5,VB6,VB9 and VB12 was 91.00%～120.0%,the CV was 1.99%～8.30%.The average recovery in milk powder spiked with VA,VE,VK1 and VK3 was 86.62%～111.1%,the CV was 2.08%～6.78%.The average recovery in multivitamins spiked with VB1,VB3,VB5,VB6VB9 and VB12 was 80.70%～120.0%,and the CV was 3.14%～8.90%.The average recovery in multivitamins spiked with VA,VE,VK1 and VK3 was 90.00%～110.1%,and the CV was3.05%～6.60%.Based on the monoclonal antibodies against water-soluble vitamins,a colloidal gold immunochromatographic assay（GICA）was constructed for the rapid detection of six water-soluble vitamins in food.Quantitative analysis showed that the calculated limit of detection（c LOD）of VB1,VB3,VB5,PN,PM,PL,VB9,and VB12 detection were 5.33,794.1,39.22,14.10,55.58,56.25,0.165,and 0.215 ng/m L,respectively.Confirmed by positive samples,the cut-off value of VB1,VB3,VB5,VB6,VB9 detection in energy drink samples was250,20,000,2,500,2,400,and 5 ng/m L,respectively.The cut-off value of VB1,VB3,VB5,VB9,VB12detection in milk powder samples was 500,20,000,5,000,5 and 10μg/kg.The cut-off value of VB1,VB3,VB5,VB6,VB9,and VB12 in multivitamin samples was 250,20,000,2,500,2,500,5 and 10 ng/m L respectively.The established GICA could be used for the rapid detection of six water-soluble vitamins in foods.The obtained monoclonal antibodies against fat-soluble vitamins were further used to construct a GICA detection method for the rapid detection of four fat-soluble vitamins in food.The visual limit of detection（v LOD）of the test strips of VA,VE,VK1,VK3 detection in milk powder was 5.0 mg/kg,10.0 mg/kg,1.0 mg/kg and 10μg/kg,respectively.The v LOD of test strips of VA,VE,VK1,VK3 detection in the multivitamin tablets was 2.5μg/m L,10.0μg/m L,1.0μg/m L and 10 ng/m L,respectively,and the cut-off values were 25μg/m L,50μg/m L,20μg/m L and 200 ng/m L,respectively.The depicted GICA have been successfully used for the semi-quantitative detection of VA,VE,VK1,and VK3 in milk and multivitamin tablets samples.These results show that the employment of ic-ELISA for the determination of vitamins in food samples exhibited remarkable ability in terms of sensitivity and selectivity,which conforms its great potential in real sample analysis.In addition,colloidal gold immunochromatographic test strips could be applied in rapid,high-throughput and on-site detection of vitamins in food samples,providing a convenient detection tool for vitamin analysis. |
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