ELISA Kit for IgM Antibody to Hepatitis A Virus is an in vitro enzyme immunoassay for the detection of HAV-IgM in human serum or plasma.
Principle:This kit uses capture ELISA method to detect anti-HAV IgM. The purified rabbit anti human IgM monoclonal antibody (Anti-mu-chain) is coated on the solid phase of multi-wells. Serum sample, Horseradish peroxidase labeled HAVAg are added to coated wells. After incubation, if HAV-IgM is present in the sample, a complex of Anti-μ-chain-HAV-IgM-HAVAg labeled with HRP will form. Wash wells to remove other unbounded serum components, incubate with substrate (TMB) to form a colored product, and measure the absorbance at 450nm to indicate the presence or absence of HAV-IgM in the sample. The test is special, sensitive, reproducible and easy to operate.
Storage and Stability:Store all components at 4°C. Stable for 6 months For maximum recovery of product, centrifuge the original vial prior to removing the cap.
Sample Collection And Preservation:Blood serum samples are routinely prepared form vein. Blood plasma sample are routinely prepared with routine amount of anticoagulant such as heparin or sodium citrate. Sample can be stored at 4°C if tested within five days. Sample can be stored at -20°C at least for 3 months. Avoid hemolysis and repetitive freeze and thaw of samples. Samples with cloud or precipitation should be centrifugated or filtered before test. Prevent serum from bacteria contamination during collection and storage.
Test Procedure:1. Bring ELISA Kit for IgM Antibody to Hepatitis A Virus (all reagents), and samples to room temperature before use (approximately 30 minutes)
2. Dilute concentrated Wash Buffer 1:20 with ddH2O 3. For each test, set one blank, two positive and three negative controls. Add 50ul positive and negative control serum into positive and negative control wells respectively without sample diluent. 4. Add 50ul test serum into test wells. 5. Cover wells with seal paper, incubate for 30 minutes at 37°C. 6. Discard the liquid in all wells and fill the wells with wash solution (300ul per well). Lay aside for 15 seconds, discard the liquid in all wells and fill the wells with wash solution. Repeat 5 times and dry wells after last wash. 7. Add 100ul Enzyme Conjugate in each well except the blank well. 8. Cover wells with seal paper, incubate for 30 minutes at 37°C. 9. Wash 5 times the same as step 6. 10. Add 50ul substrate A and B respectively to each well, mix gently, protect from light and lay aside for 15 minutes at 37°C
11. Add one drop of stop solution (50ul) into each well to stop the reaction, including blank well. 12. Measure the absorbance at 450nm against the blank, or measure the absorbance at 450nm/630-690nm.
Interpretation of Results:Colorimetric Method Cut Off Value Calculation:COV = 0.07 + the average OD of negative controls Positive OD450 of sample ≥ COV Negative OD450 of sample < COV Note: If the absorbance of negative controls is below 0.05, calculate it as 0.05. If the absorbance of negative controls is above 0.05, calculate it as its original value.
Precautions:1. The samples should be fresh, avoid hemolysis, bacteria growing, and repetitive freeze and thaw cycles. 2. Do not interchange reagents between kit lots. The seal paper can’t be used repeatedly. 3. Mix reagents well before use. If crystal forms in certain reagents, such as the wash buffer, it can be used without problems after warming up and mixing well. 4. Follow instruction exactly during assay, especially in temperature and time for reactions. All pipetting devices should be used with care and calibrated regularly following the manufacturer’s instructions. 5. Unused coated wells should be returned to the sealed pouch, and stored at 4°C. 6. To prevent cross-contamination, wear gloves and working suits throughout the procedure, and execute the disinfection and isolation regulations strictly. Dispose of all samples and materials used to perform the test. The 5g/L liquid sodium hypochlorite solution or 121°C high pressure steam may be used to disinfect samples and materials before disposal (positive control serum in the kit has been inactivated).
