Human Anti-Hepatitis A Virus IgM Antibody / HAV IgM ELISA Kit INQUIRY

Cat.No: EHA-140
Prod. No. EHA-140
Pkg. Size 96T
Product Name Human Anti-Hepatitis A Virus IgM Antibody / HAV IgM ELISA Kit
Intended use The kit is an enzyme linked-immunosorbent assay (ELISA) for qualitative determination of IgM-class antibodies to human hepatitis A virus (HAV-IgM) in serum or plasma. It is intended for use in clinical laboratories for diagnosis and monitoring of patients related to infection with hepatitis A virus.
Principle Of The Test This kit is a solid phase, two-step incubation, antibody capture ELISA assay in which, polystyrene microwell strips are pre-coated with antibodies directed to human immunoglobulin M proteins (anti-μ chain). The patient’s serum/plasma sample is added and during the first incubation, any IgM antibodies will be captured in the wells. After washing out all the other components of the sample and in particular IgG antibodies, the specific HAV IgM captured on the solid phase is detected by the addition of HAV antigens conjugated to horseradish peroxidase (HRP-Conjugate). During the second incubation, the HRP-conjugated antigens will specifically react only with the HAV IgM antibodies and after washing to remove unbound HRP-conjugate, TMB solutions are added to the wells. In presence of the (anti-μ )-(HAV-IgM)-(antigen-HRP) immunocomplex, the colorless TMB are hydrolyzed by the bound HRP conjugate to a blue-colored product. The blue color turns yellow after stopping the reaction with sulfuric acid. The amount of color intensity can be measured and is proportional to the amount of antibody captured in the wells, and to the sample respectively. Wells containing samples negative for HAV-IgM remain colorless.
Reagents And Materials Provided Microplate: 96 well polystyrene microplate (12 strips of 8 wells) coated with anti-IgM antibodies (anti-μ chain);
Negative Control: 0.5ml, 1 vial;
Positive Control: 0.5ml, 1 vial;                                                         
HRP-Conjugate Antibody: 12ml, 1 vial;                                                         
TMB Solution A: 7ml, 1 vial;                                                       
TMB Solution B: 7ml, 1 vial;                                                       
TMB Stop Solution: 7ml, 1 vial;                                                       
Wash Buffer (20×): 50ml, 1 vial;
Microtiter plate sealers:  2 sheets;
 Plastic Sealable Bag: 1 unit.
Materials Required But Not Supplied
1. Validated microplate reader.
2. Eppendorf Tubes for dilution for samples and standards.
3. Deionized or distilled water.
4. Validated adjustable micropipettes, single and multi-channel.
5. Automatic microtiter plate washer or manual vacuum aspiration equipment.
6. 37°C incubator.
Storage Unopened Kit: Store at 2 - 8°C. Do not use past kit expiration date.
Opened/Reconstituted Reagents: TMB Solution A; TMB Solution B; TMB Stop Solution; Wash Buffer; HRP-conjugate antibody
The above mentioned reagents should be stored for up to 1 month at 2 - 8°C.
Microplate Wells: Return unused wells to the foil pouch containing the desiccant pack, reseal along entire edge of zip-seal. May be stored for up to 1 month at 2 - 8°C.
Precautions 1. The kit should be equilibrated to room temperature (20-23) before opening any vials and starting the assay. It is highly recommended that the solutions be used as soon as possible after rehydration.
2. When mixing or reconstituting protein solutions, always avoid foaming.
3. Do not mix or substitute reagents with those from other lots or sources.
4. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
5. Crystals could appear in the 20X wash solution due to high salt concentration in the stock solutions. Crystals are readily dissolved at room temperature or at 37°C before dilution of the buffer solutions.
6. Keep TMB Substrate protected from light.
7. The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.
Specimen Treatment Centrifuge the serum, plasma or cell culture supernatant samples for 10 minutes at 1,000×g. Remove particulates and assay immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles.
ELISA Procedure 1. Allow all the reagents and samples to reach room temperature (18-30) for at least 15-30 minutes. Check the Wash buffer concentrate for the presence of salt crystals. If crystals have formed in the solution, resolubilize by warming at 37 until crystals dissolve. Dilute the Wash Buffer 1 to 20 with distilled or deionized water. Only use clean vessels to dilute the buffer.
 2. Set the strips needed in strip-holder. And number sufficient number of wells including three for the Negative control, two for the Positive control and one Blank (neither samples nor HRP-Conjugate antibody should be added into the Blank well). If the results will be determined by using dual wavelength plate reader, the requirement for use of Blank well could be omitted. Use only number of strips required for the test.
3. Add 100 μl of samples, and 100 μl Positive and Negative controls into their respective wells. Note: Use a separate disposable pipette tip for each specimen, Negative and Positive Control as to avoid cross-contamination
4. Cover the plate with the plate cover and incubate for 20 minutes at 37
5. After the end of the incubation, remove and discard the plate cover. Wash each well 5 times with diluted Washing buffer. Each time allow the microwells to soak for 30-60 seconds. After the final washing cycle, turn down the plate onto blotting paper or clean towel and tap it to remove any remainders.
6. Add 100 μl of HRP-Conjugate antibody into each well except for the Blank. Cover the plate with the plate cover and incubate for 40 min. at 37, and Washing as Step 5.
 7. Dispense 50 µl of TMB Solution A and 50 µl TMB Solution B into each well including the Blank, and mix by tapping the plate gently. Incubate the plate at 37 for 15 minutes avoiding light. The enzymatic reaction between the TMB solutions and the HRP-Conjugate antibody produces blue color in Positive control and HAV IgM positive sample wells.
8. Using a multichannel pipette or manually, add 50 µl Stop solution into each well and mix gently. Intensive yellow color develops in Negative control and negative sample wells.
9. Calibrate the plate reader with the Blank well and read the absorbance at 450 nm.
If a dual filter instrument is used, set the reference wavelength at 630 nm. Calculate the Cut-off value and evaluate the results. (Note: read the absorbance within 5 minutes after stopping the reaction.)
Interpretation Of Results And Quality Control
Each microplate should be considered separately when calculating and interpreting results of the assay, regardless of the number of plates concurrently processed. The results are calculated by relating each sample’s optical density (OD) value to the Cut-off value (C.O.) of the plate. If the Cut-off reading is based on single filter plate reader, the results should be calculated by subtracting the Blank well OD value from the print report values of samples and controls. In case the reading is based on dual filter plate reader, do not subtract the Blank well OD from the print report values of samples and controls.
1. Calculation of Cut-off value:
 Cut-off value (C.O.) = *Nc × 2.1
*Nc = the mean absorbance value for three negative controls. Important: If the mean OD value of the negative control is lower than 0.05, take it as 0.05.
If one of the Negative control values does not meet the Quality control range specifications, it should be discarded and the mean value is calculated again using the remaining two values. If more than one negative control OD value does not meet the Quality control range specifications, the test is invalid and must be repeated.
2. Quality control range:
The test results are valid if the Quality Control criteria are verified. It is recommended that each laboratory must establish appropriate quality control system with quality control material similar to or identical with the patient sample being analyzed
1) The OD value of the Blank well, which contains only TMB Solution  and Stop solution, is less than 0.080 at 450 nm.
2) The OD value of the Positive control must be equal to or greater than 0.800 at 450/630 nm, or at 450nm after blanking.
3) The OD value of the Negative control must be less than 0.100 at 450/630 nm or at 450nm after blanking.
3. Interpretations of the results:
(S = the individual optical density (OD) of each specimen)
Negative Results (S/C.O.<1): Samples giving A value less than the Cut-off value are negative for this assay, which indicates that no HAV IgM antibodieshave been detected with WANTAI HAV IgM ELISA kit, therefore there are no serological indications for current infection with HAV.
Positive Results (S/C.O.1): samples giving A value which is equal to, or greater than the Cut-off value are considered initially reactive, which indicates that hepatitis A virus antigens have probably been detected using this 
ELISA kit. Retesting in duplicates of any initially reactive sample is recommended. Repeatedly reactive samples could be considered positive for HAV IgM antibodies and therefore there are serological indications of current or recent infection with hepatitis Avirus.
Borderline:  (A / C.O. = 0.9-1.1): Samples with  A value to Cut-off ratio between 0.9 and 1.1 are considered borderline and retesting of these specimens in duplicates is required to confirm the initial results.
1. The assay is designed to achieve very high performance characteristics of sensitivity and specificity and the “capture model” minimizes the unspecific reactions due to interference with unknown matters in specimen (false positive results).
2. Antibodies may be undetectable during the early stage of the disease. Therefore, negative results obtained with HAV IgM ELISA are only indication that the sample does not contain detectable level of HAV antibodies and any negative result should not be considered as conclusive evidence that the individual is not infected with HAV.
3. The most common sources for mistakes are: kits beyond the expiry date, bad washing procedures, contaminated reagents, incorrect assay procedure steps, insufficient aspiration during washing, failure to add specimens or reagents, improper operation  with the laboratory equipment, timing errors, the use of highly heamolytic specimens or specimens containing fibrin, incompletely clotted serum specimens.
4. The prevalence of the marker will affect the assay’s predictive values.
5. HAV IgM ELISA is a qualitative assay and the results cannot be use to measure antigen concentrations.
Indications Of Instability Or Deterioration Of The Reagents 1. Values of the Positive or Negative controls, which are out of the indicated Quality control range, are indicator of possible deterioration of the reagents and/or operator or equipment errors. In such case, the results should be considered as invalid and the samples must be retested. In case of constant erroneous results classified as due to deterioration or instability of the reagents, immediately substitute the reagents with new ones, or contact our technical support for further assistance.
2. If after mixing of the TMB Solution A and B into the wells, the color of the mixture turns blue within few minutes, do not continue carrying out the testing and replace the reagents with fresh ones.
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