Purification and analysis of goat lactoferrin concentration and the study of its correlation with the methylene blue reduction test

• Main Author: 陳文志
• Other Authors: 毛嘉洪
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/89952796134907158173

碩士 === 國立中興大學 === 生命科學院碩士在職專班 === 91 === ABSTRACT Lactoferrin (LF) is a multi-functional glycoprotein in non-specific immune defense mechanism. The secretions in exocrine of human and animals have a lot of LF, which especially exist in the milk for protecting breast and supporting partially acquired immunity in newborn animals. Therefore, to analyze the content of LF concentration in unprocessed milk may be help to monitor the health condition of animals mammary gland and the quality of milk in early lactation stage. The purpose of these studies was to purify LF from caprine colostrum and to construct the competitive enzyme-linked immunosorbent assay (ELISA) of caprine LF for examine the change of LF concentration in unprocessed milk, as well as to discusse the correlation of LF and quality of caprine milk. In these studies, firstly, we use heparin affinity column to purify caprine LF of Alpine goat colostrum. The LF was used to be the LF-biotin conjugate and to be the standard for ELISA. The agar-plate precipitation reaction involved the reaction of caprine LF with rabbit anti-cattle LF antibody, which have apparent precipitin line between them. Sequentially, the molecular weight of both bovine and caprine LF was identified as 80 kDa by SDS-PAGE and Western blotting, and the caprine LF contained specific affinity reaction with rabbit anti-cattle LF antibody. The result showed they have highly homology in bovine and caprine LF, therefore we could use rabbit anti-cattle LF antibody as coating antibody for develop ELISA of caprine LF. Secondly, we prepared LF-biotin conjugate to decide optimal concentration of antibody and LF-biotin conjugate were 1:30,000 and 1:25,000, respectively by lattice competitive method and make the standard curve from ELISA of LF and LF-biotin conjugate. The result shown the range of detection sensitivity of LF between 3 ng/ml and 10 mg/ml using ELISA. Finally, we applied the ELISA to examine the change of LF concentration of unprocessed caperine milk in bucket per month, which had been the priced by methylene blue reduction test (MBRT) from milk processing factory from September, 2002 to February, 2003. The results showed that when milk sample of MBRT ≦ 6 hours, the average of LF concentration were apparently higher than milk sample of MBRT ＞ 6 ~ 8 hours ( p ＜ 0.01) in 172 high quality unprocessed caprine milk (MBRT ≧ 5.5 ~ 8 hours). The preliminary data showed the negative correlation between LF concentration and MBRT time in high quality unprocessed caprine milk. Comprehensive above mentioned showed these studies constructed a purification method and ELISA for caprine LF. The ELISA could not only be used to examine LF content in caprine milk but also be used to judge high and low quality of unprocessed caprine milk.