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Product Name

Diagnostic Kit for Antibodies to Avian Influenza Virus (ELISA)

Specification

96T×1/kit       

Introduction

Avian influenza is also known as real chicken plague or European chicken plague, is caused by the Avian Influenza Virus( AIV ), An acute, highly fatal infectious disease caused by poultry, characterized by acute sepsis death to asymptomatic toxicity.                                                                 

Principle of Test

The kit is for the qualitative determination of AIV in the sample, adopt AIV antigen to coat microtiter plate, make solid-phase antigen, and then pipette samples to the wells, with anti- goat AIV-ab conjugated Horseradish Peroxidase (HRP). Wash and remove non-combinative antibody and other components. Antibodies specific for the antigen will bind to the pre-coated antigen. After washing completely, add TMB substrate solution and color develops according to the amount of AIV-ab. Reaction is terminated by the addition of a stop solution and the intensity of the color is measured at a wavelength of 450 nm. Compared with the CUTOFF value to judge if AIV-ab exists in the sample or not.

Storage Conditions

• The kit shall be stored at [2-8 ℃].

• The opened Microelisa Stripplate can be stored at [2-8 ℃] and avoid damp. Use for at least 2 months.

Instrument

Mircoplate reader (contain: 450nm, 630nm wavelength), thermostatic equipment (37 degrees Celsius), adjustable Pipette

 

Composition of the Kit

Reagent	Quantity
Microelisa Stripplate	8well×12strips（1）
Positive control	1×1.0ml
Negative control	1×1.0ml
HRP-Conjugate Reagent	1×11ml
Sample Diluent	1×50ml
Substrate A solution	1×6ml
Substrate B solution	1×6ml
Stop Solution	1×6ml
Concentrated Wash Solution 20×	1×40ml
User manual	1
Adhesive Membrane	1
Sealed bag	1

Washing Method

• Manually washing method:

Shake away the remained liquid in the enzyme plates; place some bibulous papers on the test-bed, and flap the plates on the upside down strongly. Inject at least 0.3ml after-dilution washing solution into the well, and marinate 30 second. Repeat this process at 5 times.

• Automatic washing method:

If there is automatic washing machine, it should only be used in the test when you are quite familiar with its function and performance.

Sample Preparation

• serum:

Coagulation at room temperature for 10-20 min, centrifuge at the speed of 2000-3000 rpm for 20min. Remove supernatant, if precipitation appeared, centrifuge again.

Extract as soon as possible after samples collection, and should be tested as soon as possible after the extraction. If be used recently, kept the samples in 2- 8℃.If not, samples can be kept in -20℃. Avoid repeated freeze-thaw cycles.

• Don’t detect the samples which contain NaN3, because NaN3 inhibits HRP activity.

• sample dilution：

40 times dilution（add 5ul sample into 195ul sample diluent, mix）

• 20× Concentrated wash buffer should be restored to room temperature to dissolve the precipitate before use, then dilute the 20× concentrated washing buffer at a ratio of 1:20. If using the entire bottle (40ml), add to 760 ml of the distilled or deionized water.

Assay Procedure

Step 1: Number: determine the number of well to be used and store unused wells in 4 ℃. Set a blank well without any solution.

Step 2: Prepare sample: pipette Positive control and Negative control 100μl to the well respectively. Controls need test in duplicate. Pipette testing sample 100μl to testing sample well. Pipette sample to the bottom of well, don’t touch the wall as far as possible, and mix gently.

Step 3: Incubate: Cover with the adhesive Membrane provided, incubate for 30 min at 37℃(recommend water bath) .

Step 4: Configurate liquid: Dilute 20fold concentrated wash solution with distilled water.

Step 5: Washing: Uncover the adhesive Membrane, discard liquid, pipette washing buffer to every well, still for 30s then drain, repeat 5 times.

Step 6: Add enzyme: Pipette HRP-Conjugate reagent 100μl to each well, except blank well.

Step 7: Incubate: Operation with 3.

Step 8: Washing: Operation with 5.

Step 9: Color: Pipette 50ul Substrate A solution, and then pipette 50ul Substrate B solution to each well, avoid the light preservation for 10 min at 37℃

Step 10: Stop the reaction: Pipette Stop Solution 50μl to each well, stop the reaction (the blue change to yellow).

Step 11: Calculate: take blank well as zero. Read absorbance at 450nm after pipette Stop Solution within 10min.

Calculation of Result

• Test validity: the average of Positive control well≥0.6; the average of Negative control well ≤0.10.
• A sample value ≥0.38 is positive;

• A sample value between 0.2 and 0.38 for suspicious;

A sample values < 0.2 is negative.

Test Method Limitation

This test is used only for the qualitative detection of Avian Influenza Virus Antibody in serum, plasma. Evaluate the level of antibody according to the A value with three classes: strong, middle and weak.

Expiration

12 months [see label on the outer box for the specific date].

Attention

• The kit takes out from the refrigeration should be balanced 15-30 minutes in the room temperature, if the coated ELISA plates have not been used up after opening, the plate should be stored in sealed bag.
• Washing buffer will Crystallization separation, it can be heated in water to dissolve.
• Pipette sample with pipettors each step, and proofread its accuracy frequently to avoid the experimental error. Pipette sample within 5 min, if the number of sample is big, recommend using multichannel pipettor.
• The test sample should be kept fresh.
• Adhesive Strip only limits the disposable use to avoid cross-contamination.
• The substrate should evade the light to be preserved.
• Please refer to the user instruction strictly, the test result determination must take the microtiter plate reader as a standard.
• The preparation of samples and all the reagents should refer to infective material process.
• Do not mix reagents with those from other lots.