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Published on: July 11, 2015
Analysis of colostral proteins in calf serum by enzyme-linked immunosorbent assay
H Kiriyama1, E Harada, B Syuto
1Department of Physiology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo.
Abstract:
Enzyme-linked immunosorbent assay method was improved to monitor the concentrations of colostral proteins in the range of 10 to 10(3) ng/ml in calf serum. Colostral proteins were purified from fat-free colostrum, and antibodies against them were prepared from the rabbit anti-colostrum protein sera. Concentration of each protein was determined by enzyme-linked immunosorbent assay without interference by calf serum proteins in a mixture of colostrum and precolostral calf serum. Changes in the colostral protein concentrations in the sera of five postcolostral calves were monitored by enzyme-linked immunosorbent assay. After feeding colostrum to the neonatal calf, serum IgG concentration increased rapidly within 16 h to 8.1 to 36.8 mg/ml and gradually declined until 3 d to the steady levels, 4.7 to 23.6 mg/ml. The concentrations of casein and P2 (colostral small proteins, which were eluted at the second peak in Sephadex G-100 gel filtration) also increased more rapidly within 16 h to 9.6 to 264.0 micrograms/ml and 31.5 to 1600 micrograms/ml, respectively, and steeply decreased to near the detection limit on 3 d after feeding. These results indicate that enzyme-linked immunosorbent assay is useful to measure and monitor the absorbed colostral proteins and also to survey calves receiving and not receiving colostrum.
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Western Blotting
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.
Enzyme-Linked Immunosorbent Assay
There are many different types of ELISAs, but they all involve an antibody molecule whose constant region binds an enzyme, leaving the variable region free to bind its specific antigen. Enzyme-substrate reaction allows the antigen to be visualized or quantified.