Journal of the Korean Society of Food Science and Nutrition (한국식품영양과학회지)

The Korean Society of Food Science and Nutrition (한국식품영양과학회)
권호 표지

Development of Enzyme-Linked Immunosorbent Assay for Rapid and Sensitive Analysis of Biotin

Biotin의 분석을 위한 효소면역측정법(ELISA)의 개발

  • Published : 1998.12.01
Download PDF
⟨ Previous Next ⟩

Abstract

In order to develop more rapid and reproducible analysis of biotin known as vitamin H, attempts were made to establish the condition for enzyme linked immunosorbent assay(ELISA) compared with traditional microbiological assay(MBA). Antibiotin and antiserum were obtained from the immunized rabbits injected with emulsion of biotin KLH conjugate and Freund's adjuvant. The antiserum showed cross reactivity on biocytin, a derivative of biotin, which is converted to biotin in intestine, at the rate of 177%(median inhibitory concentration(IC50)=12.58ppb), but not on other derivatives such as desthiobiotin, diaminobiotin and 2 imino biotin. Specific antibody for biotin was purified from the antiserum through protein A column and desalting column. The conditions of competitive direct ELISA (cdELISA) were established. Detection range of biotin concentration by cdELISA was 0.01∼300ng/ ml(ppb). In the spike test with milk, fruit flake and pine carrot juice, the correlation coefficient between two methods of MBA and ELISA was reliably consistent at the value of r=0.992. But detection of biotin by microbiological assay(MBA) was rather restricted in range and nonspecific. Detection range of biotin by MBA was 0.1∼0.5ng/ml(ppb). It showed cross reactivities on biocytin and desthiobiotin at the rate of 80.1% and 66.7%, respectively. In conclusion, ELISA revealed a significant improvement compared with MBA for the biotin detection in terms of sensitivity, detection range and cross reactivity. In addition, a variety of samples could be analyzed rapidly and conveniently at one time by using ELISA. These results strongly suggest that the ELISA is very promising for the practical application to detect biotin contents in a wide range of food stuffs.

Keywords

References

  1. Recommended Dietary Allowances(10th ed.) The National Research Council
  2. Handbook of vitamins(2nd ed.) Machlin, L. J.
  3. Vitamins(2nd ed.) Friedrich, W.
  4. Recommended Dietary Allowances for Koreans Korea Institute for Population & Health
  5. Food Composition Table(4th ed.) Rural Nutrition Institute
  6. Methods in molecular biology v.10 Avidin-biotin technology Bayer, E. A.;Wilchek, M.
  7. Anal. Biochem. v.163 The use of biotinylated monoclonal antibodies and streptavidin affinity chromatography to isolate herpesvirus hydrophobic proteins or glycoproteins Gretch, D. R.;Suter, M.;Stinski, M. F.
  8. Microchem. J. v.41 Some observation on a new filter pad technique for the estimation of avidin and biotin Cox, J. C.;Longoria, C. C.
  9. Nature v.227 Cleavage of structural protein during assembly of the head of bacteriophage T₄ LaemmLi, U. K.
  10. Official methods of analysis(15th ed.) AOAC
  11. Int. J. Vit. Nutr. Res. v.59 Biotin assessment in foods and body fluids by a protein-binding assay (PBA) Bitsch, R.;Salz, I.;Hotzel, D.
  12. Can. Inst. Food Sci. Technol. J. v.23 Total folate, pantothenic acid and biotin content of yoghurt products Hoppner, K.;Lampi, B.
  13. Kor. J. Appl. Microbiol. v.24 Development of an enzyme-linked immunosorbent assay for the detection of fumonisin Shon, D. H.;Han, S. M.;Lee, Y. W.;Cho, S. H.;Kang, S. Y.;Lee, K. A.
  14. Anal. Biochem. v.79 The use of linear regression analysis in the isotope dilution assay of biotin Hood, R. L.
  15. J. Micronutr. Anal. v.7 Rapid biospecific methods of vitamin analysis Lee, H. A.;Mills, E. N. C.;Finglas, P. M.;Morgan, M. R. A.