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Purification and Characterization of Rabies Anti-Idiotype Antibodies Isolated from Chicken Egg Yolks
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| Author:
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SAYU YUNI, KHOMAINI HASAN, EKA NONENG NAWANGSIH, IIS INAYATI RAKHMAT
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| Abstract:
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Rabies is a public health problem in the world, including Indonesia. There are 34 provinces in Indonesia, only 10 provinces are free from rabies [1]. Chicken immunoglobulin can be used as a surrogate antigen for rabies virus for the preparation of anti-idiotype (Ab2) vaccines, even though the antibody titer induced is lower when compared to a viral vaccine [2]. As a substitute antigen candidate, the anti-idiotype antibody immunoglobulin Y (IgY) must be separated from the other components, so it needs to be purified. This study aims to obtained pure IgY. Purification of IgY derived from freeze dried egg yolks was carried out using Precipitaion Solution A and Solution B kits, followed by dialysis using a dialysis membrane against 25 mM phosphate buffer solution, pH 7.4 (Buffer A) for 16 hours. Anion exchange chromatography is carried out at a flow rate of 0.5mL/minute. To release the Ab2 component bound to the matrix, the column was eluted linearly with a 25 mM phosphate buffer solution, pH 7.4 containing 500 mM NaCl (Buffer B). Purified anti-idiotype Ab2 antibodies are then analyzed to obtain amino acid sequences. There are two protein peaks that appear, before the process of elution and after elution 5-10% with buffer B. Protein peaks from the results of the anion exchange chromatography purification were then examined by SDS PAGE Technique, result Ab2 rabies (~ 180 kDa) has been successfully purified with high purity. The heavy chain (~ 60 kDa) and the light chain (~ 30 kDa) from Ab2 have also been successfully verified with SDS PAGE in the reduction state. Determination of Ab2 activity with AGPT technique has shown positive results, and the amino acid sequence can be determined.
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| Keyword:
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Rabies, Antibodies, Egg yolks
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| EOI:
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-
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| DOI:
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https://doi.org/10.31838/ijpr/2020.12.03.223
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| Download:
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