Beijing Kinghawk CO.,LTD

ELISA Kit for HIV/1+2 antibody/antigen
(Sandwich immunoassay)
 INTENDED USAGEThis kit is designed to detect antibodies against Human Immunodeficiency Virus (HIV) (subtype M-O of Type I and Type II) and P24 antigens in human serum and plasma. It is applied to test of blood products, to screen donors, and to assist the diagnosis of the infection of HIV.
 PRINCIPLEApplying the sandwich method, ELISA plates are coated with engineered HIV antigens (i.e. gp41 & gp36) and P24 monoclonal antibody which are used to capture the antigen/antibody in serum or plasma. Biotin labeled P24 polyclonal antibody, enzyme labeled engineered antigen and enzyme labeled streptavidin are subsequently added to form the sandwich complex which are developed in TMB substrate system. OD value which is read by spectrophotometer shows whether HIV Type 1+2 Ab/Ag presents in samples.
 REAGENTS

NOTE: Kit also contains:
Two Plate Sealers, One Self-seal Package, desiccant, One User Guide
 INSTRUMENT REQUIREDEIA multi-well reader
 SAMPLE REQUIREDSerum or plasma
 SPECIMEN REQUEST1. 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 sodium azide.
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
The kit should be brought to room temperature (18~25ºC)for 30 minutes before starting the assay. Dilute the 50ml of Wash Buffer (20×) with 950ml of distilled or deionized water, mix well.Label one blank well, two negative control wells, two HIV-1 antibody positive control wells, two HIV-2 antibody positive wells and two p24 antigen positive control wells. Add 75ul of positive and negative controls (mixed well before use) into relative wells. Add 75ul of detect samples into each well except blank well and control wells, mix well. Add 25ul of bio-polyclonal p24 antibody into each well except blank well, mix well. Cover the plate with a Plate Sealer and incubate at 37ºC for 60 minutes.Empty and wash the plate with the Wash Buffer for 5 times, each time let stand 15~20 seconds. Blot dry.Add 100ul of Conjugate into each well except the blank well. Cover the plate with a Plate Sealer and incubate at 37ºC for 30 minutes. Empty and wash the plate with the Wash Buffer for 5 times, each time let stand 15~20 seconds. Blot dry.Add 50ul of Peroxidase Substrate Buffer and 50ul of TMB Substrate to each well, and shake the plate. Incubate at 37ºC for 30 minutes, avoiding light. Add 50ul of stop solution to each well, carefully shake.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 RESULTS1. Cutoff A value=0.10+ N (N = Mean A value of Negative Control.)
2. Mean A value of negative must be ≤ 0.10. Mean A value of positive must be≥1.0. Otherwise, the assay will be considered false and should be redone.
3. Sample A value < Cutoff A value is considered HIV (1+2) antibody and p24 antigen as negative results. Sample A value ≥ Cutoff A value is considered HIV (1+2) antibody and p24 antigen as positive results.
4. If appearing positive result, resample and redo. If positive again, according to National HIV Test Regulation, the sample should be sent to HIV determination laboratory.
5. Preservatives like sodium azide and severe hemolytic samples (containing hemoglobin, bilirubin, etc) will disturb the test. Recommend to use fresh Serum or plasma

PERFORMANCE
1. Precision≤15%
2. Sensitivity and Specificity have to accord with National Reference
Rules of national reference:
Positive  I  Reference (18/18), AP12≥AP11≥2.0
Positive II Reference (2/2)
Positive p24 Reference (10/10)
Negative HIV (1+2) Reference (≥18/20).
  Negative p24 Reference (20/20)
Sensitivity HIV(1+2) Reference (≥3/6), S1 is Negativity
  Sensitivity p24 Reference: ≤10u/ml, L10 is Negativity
Coefficient of Variation:CV≤15%(n=10)
.PRECAUTIONS
1. The kit is for in vitro diagnostic use only.
2. Wear disposable gloves when handling samples and reagents.
3. All samples and liquids should be treated as if they contain infectious components.
4. Do not use beyond the expiration date and do not mix reagents from different lots.
5. The remaining reagents should be store at 2~8ºC. The remaining plates should be store with desiccant in Self-seal Package.
6. 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
STORAGE: The kit must be stored at 2~8ºC
Do not freeze Conjugate
SHELF LIFE: 12months