Intended use

The Wieslab^? ASCA test kit is an enzyme-linked immunosorbent assay (ELISA) for detection and semi-quantitation of IgG antibodies to mannan of Saccharomyces cerevisiae in human sera. The assay is used to detect antibodies in a single serum specimen. The results of the assay are to be used as an aid to the diagnosis of Crohn’s disease.

FOR IN VITRO DIAGNOSTIC USE.

Background

Antibodies to the mannan structure of Saccharomyces cerevisiae has been known for several years and their association to Crohns disease discussed. Their diagnostic use have, however, not been appreciated until the ANCA test was established and it was shown that ANCA occurred in ulcerative colitis. Thus, it became possible to use these two tests in combination to differentiate Crohns disease from UC. The clinical presentation of these patients are often similar and then diagnosis depend on clinical, radiographic, endoscopic and laboratory data and especially in pediatric patients a serological test can be of use to avoid biopsy. The exact sensitivity and specificity is not yet settled. Studies indicate that 40-80% of UC patients have ANCA and 0-20% of Crohn′s patients have ANCA, while 0-10% of UC patients have ASCA and 50-70% of Crohn′s patients have ASCA. In one study the combination of positive ANCA with negative ASCA for diagnosis of UC had a sensitivity of 57% and specificity of 97%, while the combination of negative ANCA with positive ASCA for the diagnosis of Crohn′s had a sensitivity of 49% and a specificity of 97%.

Technical information

The wells of the microtitre strips are coated with mannan structure of Saccharomyces cerevisiae. During the first incubation, specific antibodies in diluted serum, will bind to the antigen coating.

The wells are then washed to remove unbound antibodies and other components.

A conjugate of alkaline phosphatase-labelled antibodies to human IgG binds to the antibodies in the wells in this second incubation.

After a further washing step, detection of specific antibodies is obtained by incubation with substrate solution. The amount of bound antibodies correlates to the colour intensity and is measured in terms of absorbance (optical density (OD)). The absorbance is then calculated against a calibrator curve and the results are given in arbitrary U/mL.

Kit components and storage of reagents

- One frame with grey coloured break apart wells (12x8) coated with mannan structure of Saccharomyces cerevisiae and one lid sealed in a foil pack with a dry pack.

- 1.5 mL negative control (NC) containing human serum in diluent.

- 1.5 mL positive control (PC) containing human serum in diluent.

- 13 mL conjugate containing alkaline phosphatase-labelled antibodies to human IgG (colour blue).

- 2 x 32 mL Diluent (Dil) containing PBS (red colour).

- 13 mL Substrate pNPP-solution.

- 30 mL wash solution 30x concentrated.

- Five calibrators containing human serum in diluent. 1.5 mL Cal 1 = 160 U/mL, 1.5 mL Cal 2 = 80 U/mL, 1.5 mL Cal 3 = 40 U/mL, 1.5 mL Cal 4 = 20 U/mL, 1.5 mL Cal 5 = 10 U/mL.

All reagents in the kit, except the wash solution, are ready for use and should be stored at 2-8° C.

Remove only the number of strips needed for testing, resealing the aluminium package carefully.