Objective To establish the ELISA kit of monoclonal antibodies to Clostridium difficile toxin A.Methods A sandwich ELISA was used.Flat-bottomed 96 well polystyrene microtitre plates were coated with 100 ?l of purified rabbit monospecific antitoxin(8 ?g/ml, capturing antibody) in carbonate buffer(pH9.6) and incubated overnight at 4℃, the plates were washed once in PBS containing 0.05% Tween-20, pH7.4 (PBST). After 200 ?l of 10% BSA in PBS-T was added to the wells and incubated at 37℃ for 2h, washed 5 times in PBS-T with 3 min incubation at room temperature between each wash, 100 ?l of C. difficile toxin A or test samples in PBS-T were added to each well and incubated for 1h at 37℃, washed 5 times. Then 100 ?l of 1:1000 diluted monoclonal antibodies IgG-Horseradish peroxidase conjugate(detecting antibody) was added for 1h at 37℃, wells were washed five times with PBS-T, and 0.1ml of tetramethylbenzidines substrate was added to each well. After 15 min at 37℃ in dark, the reactions were stopped by the addition of 1 drop of 2M sulfuric acid and the A450 was measured.Results The tested specimens included culture filtrates of 2 strains of toxigenic C. difficile, 2 non-toxigenic strains of C. difficile, 26 strains of E. coli, 2 strains of S. dysenteriae, 1 strains of Bifidobacterium, 5 strains of V. cholera, 2 strain of S. typhi, 7 strains of C. botulinum, 1 strain of toxigenic C. sordllii, and 1 strain of C. butyricum. The ELISA demonstrated high specificity and good sensitivity, it detected amounts of toxin A as low as 0.1ng/ml.Conclusion An ELISA kits with high specificity and good sensitivity for the rapid detection of C. difficile toxin A was presented. It will be a beneficial tool to clinical detection of Clostridium difficile toxin A.

Aim: To study the effects of Clostridium difficile toxin A on the cytotoxity and apoptosis of human hep-atoma cell line SMMC-7721. Methods: Clone inhibiting experiment and MTT calorimetric assay were used to assay SMMC-7721 proliferation. Morphological changes relevant to the apoptosis and the DNA damage were analyzed by transmission electron microscope and single cell gel assay. The number of apoptosis cells and the expression of Bcl-2 and p53 protein were detected by the flow cytometry. Results: 0. 018- 4. 690 mg/L toxin A significantly decreased the colony information of SMMC-7721 cells, and greatly inhibited SMMC-7721 proliferation in a time- and concentration-dependent manner. Morphological changes related to apoptosis were evident under transmission electron microscope and DNA damage was detected by single cell gel assay when SMMC-7721 cultured with 4. 690 mg/L toxin A for 48 h. Toxin A 0. 0734. 690 mg/L induced apoptosis in SMMC-7721 cells from 6. 8% to 41. 8%. In addition, Toxin A 0. 293-4. 690 mg/L significant decreased Bcl-2 protein expression and increased p53 protein expression in SMMC-7721. Conclusion: These results showed that clostridium difficile toxin A had a significantly cytotoxicity in human SMMC-7721 which was attributed to toxin A induced apoptosis.

Objective To establish the quality standard of Compound Kangganling Granules. Methods TLC method was used for qualitative identification of Lonicerae Japonicae Flos and Forsythiae Fructus, and HPLC method was used for determination of chlorogenic acid in the preparation. The HPLC separation was performed on Diamonsil C18 column. The mobile phase was consisted of acetonitrile-0.4%phosphoric acid (20∶80, V/V), and the flow rate was 1.0 mL/min. Results TLC identified chlorogenic acid in Lonicerae Japonicae Flos and phillyrin in Forsythiae Fructus. The linear relationship of chlorogenic acid in the preparation measured by HPLC was A=30461C-5938.8, r=0.9998, RSD=1.01%, showing that the linear range of chlorogenic acid was 10.2-102.0μg/mL. Conclusion The method is accurate and rapid, with good stability, reliability and reproducibility, and can be used for the quality control and evaluation of the preparation.

AIM: To determine whether the eukaryotic initiation factor-4E (eIF-4E) inhibition facilitates the degradation of heparanase mRNA and alters heparanase protein expression in human colon adenocarcinoma cell line, LS-174T. METHODS: A 20-mer antisense s-oligodeoxynucleotide (asODN) targeted against the translation start site of eIF-4E mRNA were introduced into LS-174T cells by lipid-mediated DNA-transfection. eIF-4E protein and mRNA levels were detected by Western blot and RT-PCR, respectively. The mRNA levels of heparanase were determined by Northern blot. The alterations of heparanase expression were confirmed by Western blot analysis. RESULTS: The 20-mer asODN against eIF-4E specifically and significantly inhibited eIF-4E protein expression. Following eIF-4E inhibition, a significant reduction of heparanase mRNA was observed on Northern blot, and at the same time, heparanase protein expression significantly decreased as well. CONCLUSIONS: The results indicate that the inhibition of eIF-4E strongly reduces the stability of heparanase mRNA in colon adenocarcinoma cell line, LS-174T and resultes in an apparent reduction in the expression of heparanase protein. [

Objective: To analyze the effects of Clostridium difficile toxin B (TcdB) on the proliferation and apoptosis of colon cancer cell line SW480 and the possible mechanisms related to cell apoptosis. Methods: SW480 cells were treated with different concentrations of TcdB. Cell proliferation was detected by MTT assay. Cell apoptosis and mitochondrial membrane potential were measured with flow cytometry. Results: TcdB significantly inhibited the proliferation of SW480 cells in a time-concentration dependent manner and the inhibition rate reached 46.36% at 48 h. Flow cytometry results showed that TcdB could induce the apoptosis of SW480 cells in a time-concentration dependent manner and a 20.83% apoptosis rate was induced by 800 ng/ml of TcdB at 48 h. Conclusions TcdB could inhibit the proliferation and induce the apoptosis of colon cancer SW480 cells, and the possible mechanisms might be relate to the initiation of mitochondrial apoptosis pathway.

Objective:To investigate the immune effects of Clostridium difficile toxoid B (CdtB) vaccine formulated with different mucosal adjuvants through microneedle immunization, and to provide ideas for the prevention and treatment of Clostridium difficile infection. Methods:CdtB vaccine was prepared with purified Clostridium difficile toxin B(TcdB) after formaldehyde detoxification. Female BALB/c mice were immunized with different doses of vaccine alone or in combination with mucosal adjuvants. The titers of specific serum IgG and fecal IgA were detected at 0 d, 7 d, 14 d, 28 d and 42 d after immunization. The protective effects of CdtB vaccine were evaluated by cell neutralization assay and Clostridium difficile challenge infection. Results:(1) With the increase of immune dose, the mice immunized with CdtB vaccine alone by microneedle not only produced better serum specific IgG, but also had higher level of IgA in feces. (2) When the mice were immunized with CdtB vaccine containing LT or CTB adjuvant by microneedle, the trend of serum specific IgG titer in each group increased with the increase of immune dose, especially in the group containing LT adjuvant. There were significant differences in the trend of specific IgA titer in feces between the adjuvant groups and the group without adjuvant, but the adjuvant effect was not obvious. (3) No significant difference in serum IgG titer was observed between the mice immunized with 10 μg CdtB by microneedle or intraperitoneal injection, but microneedle immunization significantly increased fecal IgA level. (4) The neutralization titers of specific antibodies in mouse serum after immunization and the test results of challenge protection in mice confirmed that the use of CdtB vaccine had certain protective effects.Conclusions:CdtB vaccine had better immune effects in mice through microneedle immunization, but the adjuvant effects of LT and CTB were not significant.