Molecular Nutrition

Preparation, Purification and Quantitative Identification of Bovine Lactoferrin Polyclonal Antibody

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  • State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received date: 2017-06-12

  Online published: 2018-01-04

Abstract

This study aimed at preparing polyclonal antibody against lactoferrin in milk with high purity and specificity to provide experimental materials for the identification and quantification of lactoferrin synthesized in milk and mammary gland. Four healthy New Zealand white rabbits were immunized with bovine lactoferrin and challenged again after 4 weeks. Thereafter, the rabbits were immunized once every 2 weeks until antibody titer was desirable. Then the blood was collected from the heart and the serum was obtained. The antibody was purified by the saturated ammonium sulfate method and protein A resin. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blot were used to identify the purity and specificity of the purified antibody. Antibody titer was determined and the curve of antibody inhibition was obtained. Finally, we used the obtained antibody to quantitatively determine lactoferrin in commercial liquid milk, milk powder and the homogenate of mammary gland tissue of dairy cows. The results showed that the obtained rabbit anti-bovine lactoferrin polyclonal antibody had high purity and specificity, the antibody concentration was 11.02 mg/mL, and the titer was 1:128 000; lactoferrin concentration in mammary gland tissue of dairy cows was 16.13 μg/g, that in commercial liquid milk was close to 0, and that in milk powder was 5.28 μg/g. In conclusion, lactoferrin polyclonal antibody with high purity and specificity was prepared by saturated ammonium sulfate method and protein A resin in the present study, which can be used to identify lactoferrin in products like milk.

Cite this article

WANG Jian, HAO Liyuan, WEI Jingya, SUN Peng . Preparation, Purification and Quantitative Identification of Bovine Lactoferrin Polyclonal Antibody[J]. Chinese Journal of Animal Nutrition, 2018 , 30(1) : 100 -106 . DOI: 10.3969/j.issn.1006-267x.2018.01.014

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