• 대한미생물학회지
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• 제21권1호
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• pp.151-161
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• 1986

In order to develop sensitive and sepcific assay methods for E. coli heat labile enterotoxin(LT) hybridoma cell lines secreting LT specific monoclonal antibody were obtained. LT was purified from cell lysate of E. coli O15H11. The steps included disruption of bacteria by French pressure, DEAE Sephacel ion exchange chromatography, Sephadex G200 gel filtration, and second DEAE Sephacel ion exchange chromatography, successively. Spleen cells from Balb/c mice immunized with the purified LT and $HGPRT^{(-)}$ plasmacytomas, $P3{\times}63Ag8.V653$ were mixed and fused by 50% (w/v) PEG. Hybrid cells were grown in 308 wells out of 360 wells, and 13 wells out of them secreted antibodies reacting to LT. Among these hybridoma cell 1G8-1D1 cell line was selected since it had produced high-titered monoclonal antibody continuously. By using culture supernatant and ascites from 1G8-1D1 cells the monoclonal antibody was characterized, and an assay system for detecting enterotoxigenic E. coli was established by double sandwich enzyme-linked immunosorbent assay (ELISA). The following results were obtained. 1. Antibody titers of culture supernatant and ascites from 1G8-1D1 hybridoma cells were 512, and 102, 400, respectively by GM1-ELISA and its immunoglobulin class was IgM. 2. The maximum absorption ratio of 1G8-1D1 cell culture supernatant to LT was 90% at $300\;{\mu}g/ml$ of LT concentration. LT concentration shown at 50% absorption ratio was $103.45{\mu}g$ and the absorption ratio was decreased with tile reduction of LT concentration. This result suggests that monoclonal antibody from 1G8-1D1 hybridoma cell bound with LT specifically. 3. The reactivities of 1G8-1D1 cell culture supernatant to LT and V. cholerae enterotoxin(CT) were 0.886 and 0.142(O.D. at 492nm) measured by the GM1-ELISA, indicating 1G8-1D1 monoclonal antibody reacted specifically with LT but not with CT. 4. The addition of 0.1ml of ascites to 0.6mg and 0.12mg of LT decreased the vascular permeability factor to 41% and 44% respectively, but it did not completely neutralize LT. 5. By double sandwich ELISA using monoclonal antibody, as little as 75ng of the purified LT per ml could be detected. 6. The results by assay of detecting LT in culture supernatants of 14 wild strains E. coli isolated from diarrhea patients by the double sandwich ELISA were almost the same level as those by reverse passive latex agglutination.

• Journal of Animal Science and Technology
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• 제47권4호
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• pp.573-582
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• 2005

본 시험은 미역분말의 첨가가 홀스타인 젖소의 유생산과 내분비계에 미치는 영향을 규명하기 위하여 실시하였다. 기초사료에 미역분말을 1일 두당 800g 수준으로 급여하여 대조구와 비교하였다. 사료건물섭취량은 처리에 의한 영향을 받지 않았고 비유말기 산유량은 대조구에 비해 일일 6.25kg 이나 많게 유의하게 증가하였다(p<0.05). 하지만 유성분은 처리구간에 유의한 차이를 나타내지 않았다. 대조구의 체세포수는 유동적이었지만 처리구의 체세포수는 시험 종료 시 $10^5$개/ml로 시험개시에 비해 무려 53.77%의 감소효과를 보여 탁월한 체세포 감소효과를 나타내었다. 미역분말의 첨가가 혈중 면역글로부린의 함량에는 영향을 주지 않은 것으로 나타났다. 비유에 직접적인 영향을 미치는 IGF-1의 혈장농도는 미역처리구에서 유의성 있게 증가하였으며 비유와 직접적인 관련이 있는 또 다른 호르몬인 $T_3$ 와 $T_4$의 농도도 유의적으로 증가하는 양상을 나타내었다. 지질대사산물인 TC, HDL-C와 LDL-C의 농도는 모두 처리구에서 증가하는 양상을 나타냈다. 이와 같이 미역부산물의 사료 중 첨가는 유생산량의 증가를 보였는데 이는 혈중 비유관련 호르몬의 분비에 영향을 준 결과로 확인된 바 젖소사료자원으로서의 응용가치가 클 것으로 사료된다.