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Prospect 難辨梭菌毒素A/B微孔板檢測(cè)試劑盒

Prospect 難辨梭菌毒素A/B微孔板檢測(cè)試劑盒

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直接定性酶免檢測(cè)新鮮的、凍存的或Cary-Blair培養(yǎng)基保存的糞便樣本中的難辨梭菌毒素A/B

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ProSpecT C. DIFFICILE TOXIN A/B MICROPLATE ASSAY

Code: R244596

Use
ProSpect™ C. difficile Toxin A/B Microplate Assay is a qualitative enzyme immunoassay (EIA) for the detection of C. difficile Toxin A and B in human faecal specimens from patients suspected of havingClostridium difficile disease. The test is intended for use as an aid in diagnosis of Clostridium difficile-associated disease (CDAD).

Background
Clostridium difficile, a Gram-positive, anaerobic, spore-forming bacillus, is the most common identifiable cause of antibiotic-associated diarrhoeal disease1,2. The disease occurs when treatment with broad-spectrum antibiotics suppresses bacteria in the normal intestinal flora, allowing opportunistic growth of toxigenic strains of C. difficile. The toxins produced by C. difficile, designated Toxin A and Toxin B, have potent enterotoxic and cytotoxic effects, respectively. The severity of the disease may range from uncomplicated diarrhoea to conditions  known as pseudomembranous colitis (PMC), characterised by nausea, abdominal pain, watery diarrhoea, dehydration, low grade fever and the appearance of raised yellow plaques over the colorectal mucosa. Fulminating colitis may be fatal if untreated. Nosocomial outbreaks of C. difficile  gastrointestinal illness and relapses may occur11. Antibiotic treatment directed towards C. difficile can help resolve the disease.

Diagnosis is usually performed through the detection of one or both C. difficile toxins3-12. A cell culture based cytotoxin assay is considered the reference method, but is relatively time-consuming to perform. Immunoassays detecting either Toxin A, alone, or Toxin A and B, together, have become established tools in the diagnosis of C. difficile disease4,5. The existence of non-toxigenic variants of C. difficilesupports the use of toxin based assays for definitive diagnosis. Nucleic acid probe based assays have been used experimentally, but may be complicated by the fact the organism may present asymptomatically in about 50% of infants, 20-30% of hospitalised patients and in 2-3% of healthy adults6,7. The clinical significance of detecting both Toxin A and B is not fully understood. While most disease-causing strains produce both toxins, which may act synergistically, documented cases of disease caused by Toxin B only strains of C. difficile suggest that it is clinically important to assay for both Toxin A and B8,9,10.

Description
The ProSpecT C. difficile Toxin A/B test detects the presence of Toxin A and B in clinical stool specimens through the use of specific antibodies. Microwell strips are coated with mouse monoclonal anti-Toxin A and rabbit anti Toxin B antibodies. A stool specimen  can  be diluted in Sample Diluent or used directly if pre-diluted in modified Cary-Blair medium. The sample is added to a microwell, allowing the toxins, if present, to bind to the immobilised antibodies. After washing to remove unbound components, a conjugate reagent containing goat anti-Toxin A-HRP and rabbit anti-Toxin B-HRP is added to each well. Unbound conjugate is removed by washing and a chromogenic substrate solution is added to detect the presence of bound toxin. A stop reagent is added and the test results are read visually or spectrophotometrically. The presence of a yellow colour indicates the presence of toxin.

For further information about the test and its use, including limitations, please see the IFU.

Material Supplied

Microplate (12 strips of 8 wells)Coated with mouse anti-Toxin A and Rabbit anti-Toxin B
Enzyme conjugate (25ml)Peroxide labelled goat anti-Toxin A and rabbit anti-Toxin B
Positive control (4ml)C. difficile Toxin A and Toxin B supernatant with 0.01% thimerosal
Negative control (4ml)Buffered solution with 0.1% sodium azide
Sample diluent (110ml)Buffered solution with 0.1% sodium azide
Wash buffer (110ml)10X concentrate buffered solution with 0.1% thimerosal
Colour substrate (25ml)TMB in buffer
Stop Solution (6ml)1.0N sulphuric acid (corrosive)
Disposable plastic transfer pipettes (100) 
Procedure card (1) 
Instructions for use (1) 
Plate cover (1) 

 

Materials Required but Not Supplied
(1) Stool specimen sample containers (2) Disposable test tubes (3) Timer (measuring minutes) (4) Wash bottle or dispenser for Wash Buffer (5) Distilled or deionised water.

Optional material not provided
(1) Microplate reader (spectrophotometer) capable of reading bichromatically 450/620-650nm (2) Cotton or rayon tipped applicator sticks (3)Micropipette to deliver 200μl volumes (5) Vortex mixer with plate adapter or shaker (6) Modified Cary-Blair transport medium.

Ref

  1. Bartlett, J.G., 2002 N. Engl. J. Med. 346(5):334-339.
  2. Kelly, C.P. and LaMont, L.T., 1988. Ann. Rev. Med. 49:375-390.
  3. Wilkins, T. and Lyerly, D.M., 2003. J. Clin. Microbiol. 41:531-534.
  4. O’Connor, D., Haynes, P., Cormican, M., Collins, E., Corbette-Feeney, G. and Cassidy, M., 2001. J. Clin. Microbiol. 39:2846-2849.
  5. Turgeon, D.K., Novicki, T.J., Quick, J., Carlson, L., Miller, P., Ulness, B., Cent, A., Ashley, R., Larson, A., Coyle, M., Limaye, A.P., Cookson, B.T. and Fritsche, T.R., 2003. J. Clin. Microbiol. 41:667-670.
  6. Gumerlock, P.H., Tang, Y.J., Weiss, J.B. and Silva Jr, J., 2003. J. Clin. Microbiol. 31:507-511.
  7. Belanger, S.D., Boissinot, M., Clairoux, N., Picard, F.J. and Bergeron, M.G., 2003. J. Clin. Microbiol. 41:730-734.
  8. Limaye, A.P., Turgeon, D.K., Cookson, B.T. and Fritsche, T.R., 2000. J. Clin. Microbiol. 38:1696-1697.
  9. Alfa, M.J, Kabani, A., Lyerly, D., Moncrief, S., Neville, L.M. Al-Barrak, A., Harding, G.K.H., Dyck, B. Olekson, K. and Embil, J.M., 2000. J. Clin. Microbiol. 38:2706-2714.
  10. Barbut, F., Lalande, V., Burghoffer, B., Thien, H.V., Grimprel, E. and Petit, J., 2002. J. Clin. Microbiol. 40:2079-2083.
  11. Lyerly, D.M., Neville, L.M., Evans, D.T., Fill, J., Allen, S., Greene, R., Sautter, R., Hnatuck, P., Torpey, D.J. and Schwalbe, R., 1998. J. Clin. Microbiol. 36:184-190.
  12. Nicholson, G. and Jones, M., 1999. Br. J. Biomed. Sci. 56:204-208.
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