Human Anti-Hepatitis C Virus Antibody, Anti-HCV ELISA Kit INQUIRY

Cat.No: EHCV-130
Cat. No. EHCV-130
Size 96T
Intended use This kit is an enzyme-linked immunosorbent assay for qualitative detection of antibodies to hepatitis C virus in human serum or plasma. It is intended for screening blood donors and diagnosing patients related to infection with hepatitis C virus.
Principle Of The Test Anti-HCV employs solid phase, indirect ELISA method for detection of antibodies to HCV in two-step incubation procedure. Polystyrene microwell strips are pre-coated with recombinant, highly immunoreactive antigens corresponding to the core and the non-structural regions of HCV (third generation HCV ELISA). During the first incubation step, anti-HCV specific antibodies, if present, will be bound to the solid phase pre-coated HCV antigens. The wells are washed to remove unbound serum proteins, and rabbit anti-human IgG antibodies (anti-IgG) conjugated to the enzyme horseradish peroxidase (HRP-Conjugate) are added. During the second incubation step, these HRP-conjugated antibodies will be bound to any antigen-antibody(IgG) complexes previously formed and the unbound HRP-conjugate is then removed by washing. Chromogen solutions containing Tetramethylbenzidine (TMB) and urea peroxide are added to the wells and in presence of the antigen-antibody-anti-IgG (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 it is proportional to the amount of antibody captured in the wells, and to the amount of antibody in the sample respectively. Wells containing samples negative for anti-HCV remain colorless.
Reagents And Materials Provided Microplate: 96 well polystyrene microplate (12 strips of 8 wells) coated with recombinant HCV antigens;
Specimen Diluent: 12ml, 1 vial;
Negative Control: 1ml, 1 vial;
Positive Control: 1ml, 1 vial;                                                         
HRP-Conjugate: 7ml, 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:  1 sheet;
  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 the reagents and samples to reach room temperature 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 stock wash Buffer 1 to 20 with distilled or deionized water. Use only clean vessels to dilute the Wash 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 Specimen Diluent into each well except the  blank.  Add 100µl of Positive control, Negative control, and 10 µl Specimen into their respective wells. Cover the plate with the plate cover and incubate for 20 minutes at 37.
4. Washing (1): After the end of the incubation, remove and discard the plate cover. Wash each well 5 times with diluted Wash buffer. Each time, allow the microwells to soak for 30-60 seconds. After the final washing cycle, turn the strips plate onto blotting paper or clean towel, and tap it to remove any remainders.
5. Add 100 µl HRP-Conjugate to each well except the Blank. Cover the plate with the plate cover and incubate for 20 minutes at 37.
6. Washing (2): At the end of the incubation, remove and discard the plate cover. Wash each well 5 times with diluted Wash buffer as in Step 5.
7. Dispense 50 µl of TMB solution A and 50 µl of TMB solution B  into each well including the Blank and mix by tapping the plate gently. Incubate the plate at 37 for 10 minutes avoiding light. The enzymatic reaction between the TMB solution A/B solutions produces blue color in Positive control and anti-HCV 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 Positive control and HCV positive 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.) = *Pc × 0.13+*Nc 
*Nc = the mean absorbance value for three negative controls
*Pc = the mean absorbance value for three positive controls
Important: The OD value of the positive control should be lower than 0.1, If the mean OD value of the negative control is lower than 0.02, take it as 0.02. The OD value of the positive control should be higher than 0.5,  If the mean OD value of the positive control is higher than 2.0, take it as 2.0.
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 Positive control must be equal to or greater than 0.500 at 450/630 nm, or at 450 nm after blanking.
2) The OD value of the Negative control must be less than 0.100 at 450/630 nm or at 450 nm after blanking.
3. Interpretations of the results:
(S = the individual optical density (OD) of each specimen)
Negative Results (S/C.O.<1): samples giving an optical density less than the Cut-off value are negative for this assay, which indicates that no antibodies to hepatitis C virus have been detected with anti-HCV ELISA kit. Therefore, the patient is probably not infected with HCV.
Positive Results (S/C.O.1): samples giving an optical density greater than, or equal to the Cut-off value are considered initially reactive, which indicates that antibodies to hepatitis C virus have probably been detected using anti-HCV ELISA kit. Retesting in duplicates of any initially reactive sample is recommended. Repeatedly reactive samples could be considered positive for antibodies to HCV and therefore the patient is probably infected with hepatitis C virus. Blood unit positive for HCV antibodies should be immediately discarded.
Borderline: Samples with optical density O.D.Cut-off ×2 are considered borderline and retesting of those samples in duplicates is recommended. Repeatedly positive samples could be considered positive for hepatitis C virus infection.
Follow-up and supplementary testing of any anti-HCV positive samples with other analytical system (e.g. RIBA, WB) is required to confirm the diagnosis.
Limitations 1. Non-repeatable positive result may occur due to the general biological characteristics of ELISA assays. The assay is design to achieve very high performance characteristics of sensitivity and specificity and the “indirect model” minimizes the unspecific reactions, which can occur due to interference between unknown meters in sample and the rabbit anti-human IgG used as a conjugate. Antibodies may be undetectable during the early stages of the disease and in some immunosuppresed individuals.
2. Positive results must be confirmed with another available method. Any positive result must be interpreted together with the patient clinical information and other laboratory results.
3. Common sources for mistakes: kits beyond the expiry date, bad washing procedures, contaminated reagents, incorrect assay procedure steps, insufficient aspiration during washing, failure to add samples or reagents, equipment, timing, volumes, sample nature and quality.
4. The prevalence of the marker will affect the assay’s predictive values.
5. This kit is intended ONLY for testing of individual serum or plasma samples. Do not use it for testing of cadaver samples, saliva, urine or other body fluids, or pooled (mixed) blood.
6. This is a qualitative assay and the results cannot be use to measure antigens 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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