CeliCheck (Tissue Transglutaminase IgA/IgG) ELISA

Transcription

1 CeliCheck (Tissue Transglutaminase IgA/IgG) ELISA For the quantitative determination of IgA and IgG antibodies against neoepitopes of tissue transglutaminase in human serum. For Research Use Only. Not For Use In Diagnostic Procedures. Catalog Number: 35-CELHU-E01 Size: 96 wells Version: 005: ALPCO January 16, 2018 Page 1 of 10

2 1. Intended Use The CeliCheck (Tissue Transglutaminase IgA/IgG) ELISA is a solid phase enzyme immunoassay for the combined quantitative and qualitative detection of IgA and IgG antibodies against neoepitopes of tissue transglutaminase (ttg) in human serum. The assay employing human recombinant ttg crosslinked with gliadin-specific peptides displays neoepitopes of ttg which ensures a significantly increased analytical sensitivity and specificity of the assay. 2. Research Application Gluten-sensitive enteropathy or celiac disease is characterized by atrophy of the small intestinal villi leading to a so-called flat mucosa. It is caused by a pathological intolerance to gliadin, the alcohol-soluble fraction of gluten in wheat, rye and barley. Research shows that as celiac disease is caused by the uptake of gluten, consequently a gluten-free diet cures the disease completely and thus must be maintained over a life-time. Renewed consumption of gliadin leads to a return of the symptoms. Studies indicate that the disease is HLA-associated (>95% of subjects have DQ2 enrefd by DQA1*0501 and DQB1*0201) and manifests at any age with a peak onset in early childhood, even in neonates. Research demonstrates that antibodies against gliadin and anti-endomysium antibodies (EMA) are of major significance in celiac disease. They are detected so far by indirect immunofluorescence, which is restricted to subclass IgA only. However, the identification of tissue transglutaminase (ttg) as a major target antigen of EMA provided the opportunity of a more easy and reliable way to research celiac disease. Tissue transglutaminase is an enzyme that upon wounding is released from cells where it is thought to aid in tissue repair. Research shows that anti-ttg antibodies show higher analytical sensitivity and specificity than anti-gliadin antibodies. Furthermore, evidence suggests that anti-ttg antibodies may correlate with the activity of celiac disease and thus are especially useful for researching diet. The crosslink of ttg with gliadin-specific peptides results in neoepitopes of ttg. As these neoepitopes are structurally closer to the physiological antigens, the new generation ttg and CeliCheck assay shows a markedly increased analytical sensitivity and specificity. These epitopes show no crossreactivities with gliadin. 3. Principle of the Assay Serum samples diluted 1:101 are incubated in the microplates coated with the specific antigen. Antibody s in the sample bind to the antigen. The unbound fraction is washed off in the following step. Afterwards anti-human immunoglobulins conjugated to horseradish peroxidase are incubated and react with the antigen-antibody complex of the samples in the microplates. Unbound conjugate is washed off in the following step. Addition of TMB-substrate generates an enzymatic colorimetric (blue) reaction, which is stopped by diluted acid (color changes to yellow). The intensity of color formation from the chromogen is a function of the amount of conjugate bound to the antigen-antibody complex and this is proportional to the initial concentration of the respective antibodies in the sample. Page 2 of 10

3 4. Kit Contents To Be Reconstituted Item Quantity Cap Color Solution Color Sample Buffer (5x) 1 x 20 ml White Yellow Wash Buffer (50x) 1 x 20 ml White Green Ready to Use Item Quantity Cap Color Solution Color Negative Control 1 x 1.5 ml Green Colorless Positive Control 1 x 1.5 ml Red Yellow Cut-off Calibrator 1 x 1.5 ml Blue Yellow Calibrators 6 x1.15 ml White Yellow* Conjugate, IgA/G 1 x 15 ml White Red TMB Substrate 1 x 15 ml Black Colorless Description/Contents 5x concentrated Tris, sodium chloride (NaCl), bovine serum albumin (BSA), sodium azide < 0.1% (preservative) 50x concentrated Tris, NaCl, Tween 20, sodium azide < 0.1% (preservative) Description/Contents Stop Solution 1 x 15 ml White Colorless 1M Hydrochloric Acid Microtiter Plate 12 x 8 well strips N/A N/A *Color increasing with concentration Control material (diluted), bovine serum albumin (BSA), sodium azide < 0.1% (preservative) Control material (diluted), bovine serum albumin (BSA), sodium azide < 0.1% (preservative) Control material (diluted), bovine serum albumin (BSA), sodium azide < 0.1% (preservative) Concentration of each calibrator: 0, 3, 10, 30, 100, 300 U/mL; Calibrator material (diluted), bovine serum albumin (BSA), sodium azide < 0.1% (preservative) Containing: Immunoglobulins conjugated to horseradish peroxidase, bovine serum albumin (BSA) Stabilized tetramethylbenzidine and hydrogen peroxide (TMB/H2O2) With breakaway microwells. Refer to paragraph 1 for coating details Materials Required, but Not Provided Microtiter plate reader with 450 nm reading filter and recommended 620 nm reference filter ( nm). Glassware (cylinder mL), test tubes for dilutions. Vortex mixer, precision pipettes (10, 100, 200, 500, 1000 µl) or adjustable multipipette ( mL). Microplate washing device (300 µl repeating or multichannel pipette or automated system), adsorbent paper. The assay is designed to be used with purified water according to the definition of the United States Pharmacopeia (USP 26 - NF 21) and the European Pharmacopeia (Eur.Ph. 4th ed.). 5. Storage and Shelf-Life Store all reagents and the microplate at 2-8 C/35-46 F, in their original containers. Once prepared, reconstituted solutions are stable for at least 1 month at 2-8 C/35-46 F. Reagents and Page 3 of 10

4 the microplate shall be used within the expiry date indicated on each component, only. Avoid intense exposure of TMB solution to light. Store microplates in designated foil, including the desiccant, and seal tightly. 6. Precautions of Use Health hazard data This product is for Research Use Only. Not For Use In Diagnostic Procedures. Thus, only staff trained and specially advised in methods may run the assay. Although this product is not considered particularly toxic or dangerous in conditions of normal use, refer to the following for maximum safety. Recommendations and precautions This kit contains potentially hazardous components. Though kit reagents are not classified as being irritant to the eyes and skin, it is recommended to avoid contact with eyes and skin and wear disposable gloves. WARNING! Calibrators, Controls and Buffers contain sodium azide (NaN 3) as a preservative. NaN 3 may be toxic if ingested or adsorbed by skin or eyes. NaN 3 may react with lead and copper plumbing build-up. Please refer to decontamination procedures as outlined by CDC or other local/national guidelines. Do not smoke, eat or drink when manipulating the kit. Do not pipette by mouth. All biological source material used for some reagents of this kit (e.g., controls, standards) has been tested by approved methods and found negative for HbsAg, Hepatitis C and HIV 1. However, no test can guarantee the absence of viral agents in such material completely. Thus, handle kit controls, standards and samples as if capable of transmitting infectious diseases and according to national requirements. The kit contains material of animal origin as stated in the Materials Table, handle according to national requirements. 7. General Directions for Use In case that the product information, including the labeling, is defective or incorrect please contact ALPCO. Do not mix or substitute Controls, Calibrators, Conjugates or microplates from different lot numbers. This may lead to variations in the results. Allow all components to reach room temperature (20-32 C/ F) before use, mix well and follow the recommended incubation scheme for an optimum performance of the assay. Incubation: It is recommended run the assay at 30 C/86 F for automated systems. Never expose components to higher temperature than 37 C/ 98.6 F. Always pipette substrate solution with brand new tips only. Protect this reagent from light. Never pipette conjugate with tips used with other reagents. Page 4 of 10

5 8. Sample Collection, Handling, and Storage Preferentially use freshly collected serum samples. Blood withdrawal must follow national requirements. Do not use icteric, lipemic, hemolyzed or bacterially contaminated samples. Sera with particles should be cleared by low speed centrifugation (<1000 xg). Blood samples should be collected in clean, dry and empty tubes. After separation, the serum samples should be used during the first 8 hours, respectively stored tightly closed at 2-8 C/35-46 F up to 48 hours, or frozen at -20 C/-4 F for longer periods. 9. Assay Procedure 9.1. Preparation prior to starting Preparation of Reagents: Dilute concentrated reagents: Dilute the concentrated sample buffer 1:5 with distilled water (e.g. 20 ml plus 80 ml). Dilute the concentrated wash buffer 1:50 with distilled water (e.g. 20 ml plus 980 ml). To avoid mistakes it is suggested to mark the cap of the different calibrators. Preparation of Samples: Dilute serum samples 1:101 with sample buffer (1x) e.g µl sample buffer (1x) + 10 µl serum Mix well! Washing: Prepare 20 ml of diluted wash buffer (1x) per 8 wells or 200 ml for 96 wells, e.g. 4 ml concentrate plus 196 ml distilled water. Automated washing: Consider excess volumes required for setting up the instrument and dead volume of robotic pipette. Manual washing: Discard liquid from wells by inverting the plate. Knock the microwell frame with wells downside vigorously on clean adsorbent paper. Pipette 300 µl of diluted wash buffer into each well, wait for 20 seconds. Repeat the whole procedure twice again. Microplates: Calculate the number of wells required for the test. Remove unused wells from the frame, replace and store in the provided plastic bag, together with desiccant, seal tightly (2-8 C/35-46 F). Page 5 of 10

6 9.2. Pipetting Scheme It is suggested to use the following pipetting scheme for the calibrator, controls, and samples: For Quantitative Interpretation For Qualitative Interpretation A Cal A Cal E S1 A NC S2 B Cal A Cal F S1 B NC S2 C Cal B Cal F S2 C CC S3 D Cal B Cal F S2 D CC S3 E Cal C PC S3 E PC F Cal C PC S3 F PC G Cal D NC G S1 H Cal D NC H S Assay Steps Step Number Description of Step 1 Ensure preparations from section 7.1 have been carried out prior to pipetting 2 Use the following steps in accordance with required quantitative/qualitative results: Controls and Samples Pipette into the designated wells as described in section 9.2 above, 100 µl of either: a. For Quantitative Assay: Calibrators (A to F) b. For Qualitative Assay: Cut-off Calibrator (CC) 3 and 100 µl of each of the following: -Negative Control (NC) and Positive Control (PC) -Diluted serum samples (S1, S2...) Incubate for 30 minutes at C/ F 4 Wash 3 times with 300 µl prepared Wash Buffer (diluted 1:50) 5 Conjugate Pipette 100 µl of conjugate into each well 6 Page 6 of 10

7 Incubate for 30 minutes at C/ F 7 Wash 3 times with 300 µl prepared Wash Buffer (diluted 1:50) 8 Substrate Pipette 100 µl TMB Substrate into each well 9 Incubate for 30 minutes at C/ F, protected from intense light 10 Stop Solution Pipette 100 µl stop solution into each well, using the same order as pipetting the substrate 11 Incubate for 5 minutes minimum Agitate plate carefully for 5 seconds Read absorbance at 450 nm (recommended 450/620 nm) within 30 minutes 14 Page 7 of 10

8 10. Calculation of Results Quantitative and Qualitative Interpretation For quantitative interpretation establish the standard curve by plotting the optical density (OD) of each calibrator (y-axis) with respect to the corresponding concentration values in U/mL (x-axis). For best results, it is recommended to use log/lin coordinates and 4-Parameter Fit. From the OD of each sample, read the corresponding antibody concentrations expressed in U/mL. Example of a standard curve It is recommended to pipette calibrators in duplicate for each run. Calibrators IgA/G OD 450/620 nm CV % (Variation) 0 U/mL U/mL U/mL U/mL U/mL U/mL Samples above the highest calibrator range should be reported as >Max. They should be diluted as appropriate and re-assayed. Samples below calibrator range should be reported as <Min. For lot specific data, see enclosed quality control leaflet. Laboratories may perform in-house Quality Control by using their own controls and/or internal pooled sera, as foreseen by national regulations. In case that the values of the controls do not meet the criteria, the assay is invalid and has to be repeated. The following technical issues should be verified: expiration dates of reagents, storage conditions, pipettes, devices, photometer, incubation conditions, and washing methods. For qualitative interpretation, read the optical density of the cut-off calibrator and the samples. Compare sample ODs with the OD of the cut-off calibrator. For qualitative interpretation, it is recommended to consider sera within a range of 20% around the cut-off value as equivocal. 11. Technical Data Sample material: Sample volume: Total incubation time: Calibration range: Analytical sensitivity: Storage: Number of wells: serum 10 µl of sample diluted 1:101 with 1x sample buffer 90 minutes at C/ F U/mL 1.0 U/mL at 2-8 C/35-46 F use original vials only 96 wells Page 8 of 10

9 12. Performance Data Analytical sensitivity Testing sample buffer 30 times on CeliCheck (Tissue Transglutaminase IgA/IgG) ELISA gave an analytical sensitivity of 1.0 U/mL. Cross-Reactivity The microplates are coated with recombinant human tissue transglutaminase and gliadin-specific peptides. No cross-reactivities to other autoantigens have been found, in particular the gliadinspecific peptides do not cross-react with gliadin. Linearity Chosen sera have been tested with this kit and found to dilute linearly. However, due to the heterogeneous nature of human autoantibodies there might be samples that do not follow this rule. Sample No. Dilution Factor Measured Concentration (U/mL) Expected Concentration (U/mL) Recovery (%) 1 1 / / / / / / / / Precision To determine the precision of the assay, the variability (intra- and inter-assay) was assessed by examining its reproducibility on three serum samples selected to represent a range over the standard curve. Sample No. Intra-Assay Mean (U/mL) CV (%) Sample No. Inter-Assay Mean (U/mL) CV (%) Calibration Due the lack of international reference calibration this assay is calibrated in arbitrary units (U/mL). Page 9 of 10

10 13. References 1. Dietrich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO, Schuppan D (1997). Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med 1997; 3: Dietrich W, Laag E, Schöpper H, Volta U, Ferguson A, Gillett H, Riecken EO, Schuppan D (1998). Autoantibodies to tissue transglutaminase as predictors of celiac disease. Gastroenterology 1998; 115: Mäki M, Collin P (1997). Coeliac disease. Lancet : Shan L, Molberg O, Parrot I, Hausch F, Filiz F, Gray GM, Sollid LM, Khosla C (2002). Structural basis for gluten intolerance in Celiac Sprue. Science 297: Page 10 of 10