Development Of An ELISA Kit For C. Difficile Toxin A And Study On The Preventive And Curative Effects Of Probiotics Against Intestinal Infection Caused By Toxigenic C. Difficile.

Clostridium difficile is one major causative agent of pseudomembranous colitis (PMC) and many cases of antibiotic-associated diarrhea in humans and animals. It has been reported that this organism produces at least two toxins, designated A (enterotoxin) and B (cytotoxin). Toxin A, a tissue-damaging enterotoxin, causes hemorrhagic fluid accumulation in rabbit ileal loops and is cytotoxic for cultured fibroblasts. Toxin B is an extremely potent cytotoxin for many cultured cells. Although considerable advances have been made in our understanding of the pathophysiology of diarrhea and colitis caused by C. difficile infection, and a wide variety of treatments are now available, this tenacious organism continues to infect millions of patients each year and still poses a diagnostic and therapeutic challenge.This paper presents narrower yet significant aspects of an established method for purification of C. difficile toxin, preparation of mAb of C. difficile toxin A, development of an ELISA kit for Clostridium difficile toxin A. We also studied on the preventive and curative effects of probiotics against intestinal infection of mice caused by toxigenic C. difficile.In this report, we first present a modified method for purification of toxin A. Toxingenic C. difficile VPI10463 filtrate was cultured anaerobically by the dialysis bag methods. The toxin A was then purified by precipitation with 50% (NH4)2SO4 and acid precipitation at pH5.5, followed by ion-exchange chromatography on DEAE-Toyopearl 650M. The purified toxin A exhibited only one band on native polyacrylamide gel electrophoresis4(native-PAGE) and Ouchterlony double immunodiffusion, the molecular weight of toxin A was estimated to be 550 kD. The highly purified toxin A was characterized by its biological and immunological properties: protein concentration of 0.881mg/ml, the minimum lethal dose was IxlO6 MLD/ml(i.p.mice), the cytotoxic titer was 107CU/mg, the haemagglutination activity was at a concentration of 1:512, and the ratio of fluid weight (g) accumulated to the length (cm) of the rabbit loop was 2.46. This modified method for purification of toxin A of C. difficile was simple and convenient and offer a successful approach to produceing large volumes of toxin A from C. difficile.Monoclonal antibodies against C. difficile toxin A were then prepared. BALB/c mice were immunized with the purified C. difficile toxin A and the splenocytes from immunized mice were fused with myeloma cells Sp2/0. The hybridoma cells were screened with indirect ELISA and limiting dilution method. Six hybridoma cell lines (2H7, 3E9, 4B5, 5C10, 6G8 and 8A1) secreting monoclonal antibodies against C. difficile toxin A were obtained. The Ig subclasses of mAbs 2H7, 3E9 and 6G8 were IgM, mAbs 4B5 and 8AI were IgGl, and mAb 5C10 was IgG2a. All 6 mAbs had no neutralization activity. Epitope recognized by 5 mAbs(2H7, 4B5, 5C10, 6G8 and 8A1) differed from that by mAb 3E9. Relative affinities of mAbs 8A1 and 4B5 were all above 105, and those of other 4 mAbs were 104. Western blot analysis after no-denatured PAGE showed that all 6 mAbs reacted to C. difficile toxin A with Mr being 550 kD, and under the of denatured condition SDS-PAGE, Western blot analysis showed that all 6 mAbs reacted to subunits of C. difficile toxin A with Mr being 50 kD~240 kD.Thirdly, using the monoclonal antibodies to C. difficile toxin A, an ELISA kit with high specificity and excellent sensitivity for the rapid detection of C. difficile toxin A was established. An indirect sandwich ELISA was described. After the polystyrene microtitre plates with 96 flat-bottomed well were coated with the purified rabbit mono-specific antiserum as capturing antibody, the wells were blocked with 10% BSA in PBS-T. C.?5 ?difficile toxin A or culture filtrates were added to each well and then monoclonal antibodies IgG-Horseradish peroxidase conjugate(HRP-4B5 -mAb) was added as detecting antibody. Tetramethylbenzidines substrate was then added to each well, and the reactions were stopped by the addition of 2 mol/L sulfuric a...