Beijing Kinghawk CO.,LTD

ELISA Kit for Hepatitis C Virus antibody
(Indirect immunoassay)
INTENDED USE
The Kinghawk anti-HCV ELISA Kit is an indirect ELISA designed to detect antibodies to HCV in human serum or plasma. It is used for blood screening and aided diagnosis for Hepatitis C virus infection.
PRINCIPLE
The Kinghawk anti-HCV ELISA Kit is an indirect ELISA designed to detect antibodies to HCV in human serum or plasma. The test procedure involves three incubation steps:
1.Test serum or sample (properly diluted) is incubated in multi-wells coated with the genetic engineering antigens (HCV core, NS3, NS4 and NS5). Any HCV specific antibodies in the sample will bind to the immobilized antigen. The plate is washed to remove unbound antibodies and other serum components.
2.Anti-human-IgG -HRP conjugated antibody is added to the wells and the plate is incubated. The Conjugate will react with HCV antibodies immobilized on the solid phase in Step1. The wells are washed to remove unreacted Conjugate.
3.The multi-wells containing immobilized peroxidase conjugate are incubated with peroxidase substrate solution. Hydrolysis of the substrate by peroxidase produces a color change. After a period of time, the reaction is stopped and the resultant photons are counted.
REAGENTS

NOTE: Kit also contains:
Two Plate Sealers/ One self-seal plastic bag /One User Guide
SPECIMEN REQUEST
1. Fresh Serum or plasma sample is suitable for this kit (anticoagulant like Sodium Citrate,heparin,etc.)
2. Not test severe hemolytic sample or the sample containing ***
3. Before testing, the sample should be balanced to room temperature (15-30°C), frozen sample need to thaw and adequately mix.
4. If the sample will not be tested immediately after collection, should be stored at 2-8°C for 3 days. Long-term storage should be placed at -20°C or lower temperature.
5. No repeated freezing and thawing sample as well as heating sample be used
PROCEDURE
1.The kit should be brought to room temperature (18-25ºC)before starting the assay. Dilute the 50ml of Wash Buffer (20×) with 950ml of distilled or deionized water.
2.Label one reagent blank well, two negative control wells and two positive control wells. Add 100ul of Sample Diluent to reagent blank well and sample wells. Add 100ul of Control to control wells. Add 10ul of Sample to sample wells. Put the plate on the shaker for 5-10 seconds. The unused plate strips will be stored at 2-8ºC in the self-seal plastic bag.
3.Cover the plate with a Plate Sealer and incubate at 37ºC for 60 minutes. Wash the plate 5 times with the Wash Buffer, each time stewing for 15~20 seconds and blotting dry.
4.Add 100ul of Conjugate to each well except the reagent blank well. Cover the plate with a Plate Sealer and incubate at 37ºC for 30 minutes. Wash the plate 5 times with the Wash Buffer, blot dry.
5.Add 50ul of Preoxidase Substrate Buffer and 50ul of TMB Substrate to each well, and shake the plate. Incubate at 37ºC for 30 minutes. Add 50ul of stop solution to each well, carefully shake the plate and wipe the plate bottom.
6.Blank the EIA Multi-well reader at 450nm with the blank well and read the absorbance (A) of each well under determination wavelength 450nm within 30min. Or read the absorbance (A value) of each well under the reference wavelength 630nm and determination wavelength 450nm within 30min.
INTERPRETATION OF RESULTS
1.Cutoff A value=0.13×P+ N (P = Mean A value of Positive Control. P must be >0.50. When P is >2.00, it is considered as 2.00. N = Mean A value of Negative Control. N must be <0.10. When N is <0.02, it is considered as 0.02)
2.A value of negative must be < 0.10. A value of positive must be >0.50. Otherwise, the assay will be considered false and should be redone.
3.Test Specimen A value < Cutoff A value is considered negative results. Test Specimen A value ≥ Cutoff A value is considered positive results.
4.Preservatives like sodium azide and severe hemolytic samples (containing hemoglobin,bilirubin,etc) will disturb the test. Recommend to use fresh Serum or plasma
5.If appearing positive result, resample and redo. If positive again, the sample be sent to HIV determination laboratory.
PERFORMANCE
1.Precision: CV(%) ≤15% (N=10)
2.Sensitivity and Specificity have to accord with National Reference
Rules of national reference:
Positive Reference(≥29/30).Negative Reference(≥29/30).Sensitivity Reference(≥2/4), L4 is Negativity.Coefficient of Variation:CV≤15% (n=10).
PRECAUTIONS
1.Wear disposable gloves when handling samples and reagents.
2.All samples should be treated as if they contain infectious components.
3.Do not use beyond the expiration date and do not mix reagents from different lots.
4.Check the pipettes and other equipment for accuracy and correct operation
REFERENCE
1.China pharmacopoeia,3rd version  2010
2.In Vitro Diagnostic Kit Specification Guide,SFDA(2007-04-28)
PACKAGE:  96T, 48T
STORAGE:  The kit must be stored at 2-8ºC. Do not freeze Conjugate
SHELF LIFE: 12months
BEIJING KINGHAWK PHARMACEUTICAL CO., LTD