| The barrier function of intestinal tract always descents at different level after burned, including mechanical barrier, biologic barrier and immunologic barrier. There are some erosion and ulcer on the intestinal mucosa and dysbacteriosis. The level of secretory IgA (sIgA) descents after the immune system function decreases. The endotoxin of the intestine often translocates from the intestine to blood circulation. It is one of the important reasons that systemic infection and multiple organ failure. At present, no special operative drugs antagonizing lipopolysaccharide(LPS) could be applied at clinic. In recent years, it was found that there were abundant IgG sourcing from serum in egg yolk and was named IgY, which could antagonize corresponding antigens perfectly. And the IgY includes F(ab′)2 and Fc,the F(ab′)2 has the complete antibody activity,the Fc can result in allergic reaction . we hope to obtain one kind of drug which can resistant the LPS with few allergic reaction rate. Using the WD antibody purification method, specific Anti-LPS IgY was obtained from the egg yolk of hens immunized with LPS. With the pepsin the Anti-LPS IgY was digestted to the F(ab′)2 and Fc. Then the Fc was cutted to inefficient fragment . After the purification, the Anti-LPS IgY-F(ab′)2 was identificated. The Anti-LPS IgY-F(ab′)2 was found 1.04mg/mL-egg yolk by spectrophotometer . Determined by enzyme labeling staining reaction.(ELISA), the immunochemistry was 1:25600. By SDS-PAGE and Western-blotting, the IgY-F(ab′)2 is 90% purity and its molecular weight was 24KD, it has the same immunity characteristic with the IgG of chicken serum. The IgY-F(ab′)2 against LPS was obtained from the egg yolk of hens immunized with standard LPS. We cultivated four clinical common pathogenic bacteria (P.aeruginosa,E.coli,K.pneumoniae,P.vulgaris)。By phenol-water method,we got four kinds of LPS from the four kinds of bacteria. With lipopolysaccharide quantity detection, we found the The IgY-F(ab′)2 obtained by standard LPS has the effection against the four kinds of LPS. In vitro, the IgY-F(ab′)2 has good dose-effect relation with 0.5mg/mL LPS. By the conjugation of the Lipid A of LPS and effector cell ,the monocyte/macrophage was activated and release Tumor necrosis factor (TNF-α) and Interleukin(IL-6), then results in systemic inflammatory response syndrome and sepsis. TNF-αand IL-6 are major inflammation medium. By the quantity detection of TNF-αand IL-6, the result confirmations the IgY-F(ab′)2 can protect Human periblood monocyte(hPBMC)against lipopolysaccharide. By the fluorescence microscope, we found the IgY-F(ab′)2 can prohibit the conjugate of Fluorescein isothiocyanate-LPS(FITC-LPS)and Human periblood monocyte, (hPBMC). From the experiments, we prepared the IgY-F(ab′)2 against LPS. It had large content,well purity,high valence,strong specificity. In vitro, the IgY-F(ab′)2 has the strong effection against LPS. |
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