Intended use

This HAV-IgG ELISA kit is intended Laboratory for Research use only ａnd is not for use in diagnostic or therapeutic procedures.The S Solution changes the color from blue to yellow ａnd the intensity of the color is measured at 450 nm using a spectrophotometer. In order to measure the concentration of HAV-IgG in the sample, this HAV-IgG ELISA Kit includes a set of calibration standards. The calibration standards are assayed at the same time as the samples ａnd allow the operator to produce a standard curve of Optical Density versus HAV-IgG concentration. The concentration of HAV-IgG in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Sample collection ａnd storages

Serum - Use a serum separator tube ａnd allow samples to clot for 30 minutes before centrifugation for 10 minutes at approximately 3000×g. Remove serum ａnd assay immediately or aliquot ａnd store samples at -20℃ or -80℃.Avoid repeated freeze-thaw cycles

Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 30 minutes at 3000×g at 2-8℃ within 30 minutes of collection. Store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.

Cell culture supernates ａnd other biological fluids - Remove particulates by centrifugation ａnd assay immediately or aliquot ａnd store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.

Note:  The samples shoule be centrifugated dequately ａnd no hemolysis or granule was allowed.

Materials required but not supplied

1.  Standard microplate reader(450nm)

2.  Precision pipettes ａnd Disposable pipette tips.

3.  37 ℃ incubator

Precautions

1.  Do not substitute reagents from one kit to another. Standard, conjugate and microplates are matched for optimal performance. Use only the reagents supplied by manufacturer.

2.  Do not remove microplate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.

3.  Mix all reagents before using.

Remove all kit reagents from refrigerator ａnd allow them to reach room temperature ( 20-25°C)

Materials supplied

Reagent preparation

20×wash solution:Dilute with Distilled or deionized water 1:20.

Assay procedure

1.  Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the Microelisa Stripplate.

2.  Separately add Positive control ａnd Negative control 50μl to the Positive ａnd Negative well; Add testing sample 10μl then add Sample Diluent 40μl to testing sample well.

3.  Add 100μl of HRP-conjugate reagent to each well, cover with an adhesive strip and incubate for 60 minutes at 37°C. 

4.  Aspirate each well ａnd wash, repeating the process four times for a total of five washes. Wash by filling each well with Wash Solution (400μl) using a squirt bottle, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting. Invert the plate ａnd blot it against clean paper towels.

5.  Add chromogen solution A 50μl ａnd chromogen solution B 50μl to each well. Gently mix and incubate for 15 minutes at 37°C. Protect from light.

6.  Add 50μl S Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not

appear uniform, gently tap the plate to ensure thorough mixing. 

7.  Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.

Determine the result

1.  Test validity: the average of Positive control well≥1.00; the average of Negative control well ≤0.15.

2.  Calculate Critical (CUT OFF): Critical= the average of Negative control well + 0.15.

    Negative Result: sample OD< Calculate Critical (CUT OFF) is Negative.

    Positive Result: sample OD≥ Calculate Critical (CUT OFF) is Positive.

Storage ａnd validity

Storage：  2-8℃.

validity： six months.

FOR RESEARCH USE ONLY;

NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! 

PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!