DiaMetra Anti Tissue Transglutaminase IgG Elisa Kit

PRINCIPLE
Anti Tissue Transglutaminase IgG test is based on thebinding of serum or plasma antibodies on the humanrecombinant tissue transglutaminase coated into themicroplates. The antibodies in calibrators, controls orprediluted patient samples bind into the inner surface of thewells. After a 30 minutes incubation the microplate iswashed with wash buffer for removing non-reactive serumcomponents. An anti-human-IgG horseradish peroxidase conjugatesolution recognize IgG class antibodies bound to theimmobilized antigens. After a 30 minutes incubation anyexcess enzyme conjugate, which is not specifically boundis washed away with wash buffer. A chromogenic substrate solution containing TMB isdispensed into the wells. After 15 minutes of incubation thecolor development is stopped by adding the stop solution.The solutions color change into yellow. The amount ofcolor is directly proportional to the concentration of IgGantibodies present in the original sample.

CLINICAL SIGNIFICANCE
Celiac disease is characterized by chronic inflammation of the intestinal mucosa and flattening of the epithelium (positive villous atrophy). The origin of the celiac disease is the intolerance to gluten, the protein of wheat, rye and barley. The main symptoms are diarrhea, gastrointestinal problems, anemia, fatigue, psychiatric problems and other diverse side effects. In some cases patients may be asymptomatic. Clinical and mucosal recovery after institution of a gluten free diet is objective evidence that the enteropathy is gluten induced. Diagnosis of celiac disease is confirmed by abnormal findings on the small bowel biopsy and later verified by the clinical response to a gluten-free diet, i.e. the avoidance of wheat, barley, rye, oats and triticale. Left untreated patients suffering from celiac disease have an increased risk of lymphoma or gastrointestinal neoplasm. Furthermore, even if clinically silent, longstanding untreated celiac disease predisposes for other autoimmune diseases, like Diabetes mellitus, rheumatoid diseases, autoimmune hepatitis or thyroiditis. The European Society for Pediatric Gastroenterology and Nutrition (ESPGAN) draw the guidelines for the celiac disease diagnosis, including: a) an initial positive gut biopsy, b) 6 months on a gluten-free diet, c) a negative second gut biopsy, d) a gluten challenge for 6 months e) a positive third gut biopsy.

QUALITY CONTROL
The Positive and Negative Control should be run withevery batch of samples to ensure that all reagents andprocedures perform properly.
Because Positive and Negative Control are prediluted,they do not control for procedural methods associatedwith dilution of specimens.
Additional suitable control sera may be prepared byaliquoting pooled human serum specimens and storing at< -20°C.
In order for the test results to be considered valid, all ofthe criteria listed below must be met. If any of these arenot met, the test should be considered invalid and theassay repeated: - The absorbance of the prediluted h-tTG IgG Positivemust be greater than the absorbance of the predilutedNegative Control.

LIMITATIONS OF PROCEDURE
The presence of immune complexes or other immunoglobulin aggregates in the patient sample may cause an increased level of non-specific binding and produce false positives in this assay.
A negative h-tTG IgG result in an untreated patient does not rule out gluten-sensitive enteropathy. The patient may be IgA positive or have no antibody to tTG.

RESULTS
For the test Anti Tissue Transglutaminase IgG a 4- Parameter-Fit with lin-log coordinates for optical density and concentration is the data reduction method of choice. Smoothed-Spline Approximation and log-log coordinates are also suitable. However we recommend using a Lin-Log curve. First calculate the averaged optical densities for each calibrator well. Use lin-log graph paper and plot the averaged optical density of each calibrator versus the concentration. Draw the best fitting curve approximating the path of all calibrator points. The calibrator points may also be connected with straight line segments. The concentration of unknowns may then be estimated from the calibration curve by interpolation.

Preparation of the Sample
Either human serum or plasma samples can be used for the test. All serum and plasma samples have to be diluted 1:100 with sample diluent; for example 10 μL of sample may be diluted to 1,000 μL with sample diluent. Test samples should be clear. Contamination by lipemia is best avoided, but does not interfere with this assay. Specimens may be refrigerated at 2-8°C for up to five days or stored at -20°C up to six months. Avoid repetitive freezing and thawing of samples. This may result in variable loss of autoantibody activity Testing of heat-inactivated sample is not recommended.

RESULTS
For the test Anti Tissue Transglutaminase IgG a 4- Parameter-Fit with lin-log coordinates for optical density and concentration is the data reduction method of choice. Smoothed-Spline Approximation and log-log coordinates are also suitable. However we recommend using a Lin-Log curve.
First calculate the averaged optical densities for each calibrator well. Use lin-log graph paper and plot the averaged optical density of each calibrator versus the concentration. Draw the best fitting curve approximating the path of all calibrator points. The calibrator points may also be connected with straight line segments. The concentration of unknowns may then be estimated from the calibration curve by interpolation.