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KMID : 0525619970020030093
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1997 Volume.2 No. 3 p.93 ~ p.104
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Development of ELISA System for the Detection of Chitoolingosaccharides in Food
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김순영/Kim SY
손동화/이계호/Shon DH/Lee KH
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Abstract
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To determinate COS contents in Doenjang, immunoassay system was developed. Chitooligosaccharides mixture(COSM) for ELISA was produced by the treatment of chitosanase I from Aspergillus fumigatus KH-94. COSM was conjugated to BSA using EDPC(1-ethyl-3,3`-dimethylamino-propyl-carbodiimide). By immunization of the COSM-BSA conjugate into rabbits anti-COS antibodies were produced and enzyme-linked immunosorbent assay(ELISA) was established. By the use of COSM-horse radish peroxidase(HRP) conjugate and specific antibodies purified through protein A column, competitive direct ELISA(cdELISA) was developed and the detection limit was 0.1 yg/ml. In the cdELISA IC50S of glucosamine, chitobiose, chitotriose, chitotetraose, chitopentaose, and chitohexaose and N-acetylchitobiose, N-acetylchitotetraose, and N-acetylchitohexaose were 2000, 10,2.4, 1.8,0.75, and 0.9yM, and 70, 109, and 412yM, respectively. However, the antibodies almost didn`t cross-reacted with N-acetylchitotriose and N-acetylchitopentaose. In addition, there were no cross-reactivities ofthe antibody toward raffinose, cellobiose, maltotriose, and isomaltooligosaccharides. For the assay of COS in Doenjang cdELISA was unapplicable without sample clean-up and concentration of COS in Doenjang, immunoaffinity columns were prepared by packing of Sepharose beads attached with anti-COS antibodies. When COS in Doenjang extract was eluted tough immunoaffinity column and then quantified by cdELISA, average recoveries for Doenjang spiked with COSM at levels of 2, 6, 20, and 60yg/g were 115, 91.7, 91, and 73.3%, respectively with a mean of 92.8% and a coefficient of variation was 16.0%. By the tandem immunoaffinity chromatography-cdELISA, COS was detected in home made Doenjang at average of 14.0yg/g(n=25; positive=24;range=0~51.2yg/g) and in commercial Doenjang at average of 4.lyg/g(n=14; positive=13; range=0~ 18.4yg/g).
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