ASCA IgG ELISA Assay Kit

$340.00

The Eagle Biosciences ASCA IgG ELISA Assay Kit is used for the qualitative and semi-quantitative determination of IgG antibodies to Saccharomyces cerevisiae in human serum. The ASCA IgG ELISA is for research use only and not for use in diagnostic procedures.

SKU: ASG31-K01 Categories: , ,

ASCA IgG ELISA Assay Kit

For Research Use Only

Size: 1×96 wells
Sensitivity: 1 U/ml
Dynamic Range: 1 – 300 U/ml
Incubation Time: 2.5 hours
Sample Type: Serum
Sample Size: 10 µL

Additional Information

Assay Background

Non-specific inflammatory bowel diseases includ-ing Crohn’s disease (Enteritis regionalis) and ul-cerative colitis (UC) are characterized by unknown etiology as well as chronic-remitting inflammatory processes of the intestine. Whereas the inflamma-tion of ulcerative colitis is restricted to the mucosa and submucosa of colon and rectum, Crohn’s dis-ease (CD) shows a wide spread inflammation of the gastro-intestinal tract with granuloma for-mation.  The risk developing one of these diseases is strongly influenced by immunologic, genetic, in-fectious and environmental factors. The differential diagnosis of inflammatory bowel diseases to chronic diarrhea, recurrent abdominal dolor, infectious colitis, anorexia as well as the differentiation of CD to ulcerative colitis is still a high challenge. The determination of IgA and IgG antibodies to Saccharomyces cerevisiae (baker’s yeast) has been described as one important serological marker for the differential diagnosis of Crohn’s disease re-cently. Up to 70 % of patients with CD show anti-body levels to Saccharomyces cerevisiae. Although the cause for their occurrence has been unclear, antibodies to Saccharomyces cerevisiae (ASCA) are strongly associated with inflammatory pro-cesses of the intestine. In combination with the detection of autoanti-bodies to atypical anti-neutrophil cytoplasmic antigens (aANCA) which are mainly found in patients with ulcerative colitis, ASCA are a valid pa-rameter for the differentiation of Crohn’s disease and ulcerative colitis.

Assay Principle

The Eagle Biosciences ASCA IgG ELISA Assay Kit is an enzyme immunoassay for the qualitative and quantitative determination of IgG antibodies to Saccharomyces cerevisiae in human serum.

Autoantibodies of the diluted patient samples, calibrators, and the control react with mannan (cell surface component of baker’s yeast) immobilized on the solid phase of a microtiter plate. ASCA IgG guarantees the specific binding of anti-Saccharomyces cerevisiae IgG antibodies of the specimen under investigation by employing purified mannan of Saccharomyces cerevisiae for coating. Following an incubation period of 60 min at room temperature, unbound sample components are removed by a wash step.

The bound IgG antibodies react specifically with anti-human-IgG conjugated to horseradish perox-idase (HRP). Within the incubation period of 30 min at RT, excessive conjugate is separated from the solid-phase immune complexes by the follow-ing wash step.

HRP converts the colorless substrate solution of 3,3’,5,5’-tetramethyl¬benzidine (TMB) added into a blue product. The enzyme reaction is stopped by dispensing an acidic solution into the wells after 15 min at room temperature turning the solution from blue to yellow.

The optical density (OD) of the solution at 450 nm is directly proportional to the amount of specific antibodies bound. The standard curve is established by plotting the antibody concentrations of the calibrators (x-axis) and their corresponding OD values (y-axis) measured. The concentration of antibodies of the specimen is directly read off the standard curve. Evaluating the test by a semi-quantitative method is also possible.

Assay Procedure

  1. Bring all reagents to room temperature (18…25°C) before use. Mix gently without causing foam.
  2. Dispense 100 µl calibrators 1 – 4 (CAL 0 optionally, quantitative) or 100 µl calibrator 1 (semi-quantitative) 100 µl control P (N optional) 100 µl diluted patient samples into the respective wells.
  3. Cover plate, incubate 60 min at room temperature (18…25°C).
  4. Decant, then wash each well three times using 300 µl wash solution (made of B).
  5. Add 100 µl of conjugate (D) solution to each well.
  6. Cover plate, incubate 30 min at room temperature (18…25°C).
  7. Decant, then wash each well three times using 300 µl wash solution (made of B).
  8. Add 100 µl of substrate (E) to each well.
  9. Cover plate, incubate 15 min protected from light at room temperature (18…25°C).
  10. Add 100 µl of stop solution (F) to each well and mix gently.
  11. Read the OD at 450 nm versus 620 or 690 nm within 30 min after adding the stop solution.

Typical Standard Curve

Manual

Product Manual


Publications

Citations

  • Conrad K, Schmechta H, Klafki A, Lobeck G, Uhlig HH, Gerdi S, Henker J: Serological differentiation of inflammatory bowel diseases. Eur J Gastrol & Hepatol. 2002 14:129-135
  • Vermeire S: Serological Diagnosis in IBD. IBDM 2002 3:82-89