Synthesis Of Choramphenicol Immunogen And Purification Of Immunoglobulin Yolk Against Choramphenicol

Chloramphenicol is an antibiotic with a broad spectrum. It is danger to human health due to its toxicity, so the residual chloramphenicol must be stirictly controlled and detected in food and animal production. Enzyme-linked immunosorbent assay (ELISA) methods offer the possibility of highly sensitive, relatively rapid and cost-effective measurements. ELISAs are extensively studied and applied in pesticide residue and toxin monitoring, which is an area with enormous potential for growth.In order to establish an enzyme-linked immunosorbent assay (ELISA) for rapidly examing chloramphenicol (CAP), the antibody with high titer and specificity must be firstly prepared. So it is necessary for antibody production to synthesize an immunogen by conjugating the hapten (CAP) with a carrier protein. CAP was coupled with bovine serum albumin (BSA) to prepare a complete antigen CAP-BSA by glutaraldehyde (GA) and N-N'-carbonyldiimidazole (CDI) in this paper. After dialysis or ultrafiltration, the products were purified with Superdex 75. The target product was obtained by collecting the right first fraction in the wash curve from the gel filtration. The successful linkage of the CAP-BSA was identified by the ultraviolet spectrum (UV) and the infrared spectrometry (IRS). The residual amount of amino groups was determined by the TNBS spectrometry, and the amount of CAP linked to BSA was achieved. A large amount of CAP was coupled in the complete antigen by glutaraldehyde method , while an appropriate amountby N-N'-carbonyldiimidazole (CDI) method, mostly between ten and twenty. SDS-polyacrylamide gel electrophoresis was used to analyse the purified products. There existed only one zone in SDS-PAGE and the products can be considered as an immunogen. The content of CAP in the CAP-BSA had been determined by an ELISA kit purchased from market.All the above results indicated that the immunogen CAP-BSA was successfully synthesized, which was going to be injected in the hens to obtain immunoglobulin of yolk (IgY) against CAP. A constant specific activity of IgY was found to be remaining for ten months after the intensify immunization schedule. A preliminary purification was conducted according to several kinds of methods, e.g. PEG and ammonium sulphate precipitations. A relatively high purity and specific activity of IgY were obtained by an optimizing method of capralic acid-ammonium sulphate precipitation, and a plenty ofphospholipid was got rid of well, which led to benefit to store for a long time.Using bis (3-aminopropyl) amine as a spacer between the gel and the CAP ligand, a separation material was synthesized for immunoaffinify chromatography to purify the specific IgY against CAP. The specific IgY was about 3.5% in the total IgY. The antibody activity was increased by ten times after affinity separation.