Human rotavirus IgG (RV-IgG) Elisa kit

Instructions for use of human rotavirus IgG (RV-IgG) Elisa kit

The human rotavirus IgG (RV-IgG) Elisa kit is for research use only.

Detection range: 48T

Drug generic name: Human rotavirus IgG (RV-IgG) ELISA kit

Purpose of use: This kit qualitatively determines rotavirus IgG (RV-IgG) in human blood or other related tissues.

Experimental principle: This kit uses double antibody sandwich enzyme-linked immunoassay (ELISA) to determine human rotavirus IgG (RV-IgG) in the specimen. Coated microtiter plates with purified rotavirus IgG (RV-IgG) antibodies to make solid phase antibodies, which can be combined with rotavirus IgG (RV-IgG) antigens in samples. After washing to remove unbound antigen and Other components are combined with HRP-labeled rotavirus IgG (RV-IgG) antibody to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and compared with the CUTOFF value to determine the presence or absence of human rotavirus IgG (RV-IgG) in the specimen.

Human rotavirus IgG (RV-IgG) Elisa kit composition:

1 20 times concentrated washing solution 20ml × 1 bottle 7 Stop solution 3ml × 1 bottle

2 Enzyme label reagent 3ml × 1 / bottle 8 Positive control 0.5ml × 1 vial

3 Enzyme label coated plate 12 well × 4 strips 9 negative control 0.5ml × 1 bottle

4 Sample diluent 3ml × 1 bottle 10 instructions 1 copy

5 Developer A solution 3ml × 1 bottle 11 2 sealing film

6 Developer B solution 3ml × 1 bottle 12 sealed bag 1

Specimen requirements:

1. Perform the experiment as soon as possible after the specimen is prepared as required. If it can not be detected in time, the specimen can be stored at -20 ℃ for one month, but repeated freezing and thawing should be avoided.

2. Specimen processing: serum and plasma specimens can be directly detected

3. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.

Steps:

1. Numbering: Number the corresponding microwells in sequence, each plate should be set with 2 wells for the negative control, 2 wells for the positive control, and 1 well for the blank control (the blank control well does not add the sample and enzyme labeling reagent, the rest of the steps are the same)

2. Add sample: Add 50μl of negative control and positive control (standard) to the negative and positive control wells respectively. Then add 40μl of sample diluent to the sample well, and then add 10μl of the sample to be tested. Add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, gently shake to mix,

3. Incubation: seal the plate with the sealing film and incubate at 37 ° C for 30 minutes.

4. Mixing solution: add 30 times concentrated washing liquid to distilled water to 600ml and reserve

5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing liquid, let it stand for 30 seconds, then discard, repeat 5 times and pat dry.

6. Add enzyme: add 50μl of enzyme label reagent to each well, except blank well.

7. Incubation: The operation is the same as 3.

8. Washing: The operation is the same as 5.

9. Color development: add 50μl of developer A to each well, then add 50μl of developer B, mix gently, and develop at 37 ℃ for 15 minutes in the dark

10. Termination: Add 50μl of stop solution to each well to terminate the reaction (at this time, the blue color turns to yellow).

11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution.

Result judgment:

Test effectiveness: the average of positive control wells ≥1.00; the average of negative control wells ≤0.15

Calculation of cut-off value (CUT OFF): cut-off value = average value of negative control well + 0.15

Positive judgment: the sample with OD value ≥ critical value (CUT OFF) is positive for rotavirus IgG (RV-IgG)

Negative judgment: samples with OD value <cut-off value (CUT OFF) are negative for rotavirus IgG (RV-IgG)

Precautions

1. The operation is strictly in accordance with the instructions. The components of different batches of this reagent must not be mixed.

2. The kit should be taken out of the refrigerated environment and equilibrated at room temperature for 15-30 minutes before use. If the enzyme-coated plate is unsealed after opening, the strip should be stored in a sealed bag.

3. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in a water bath during dilution, and the results will not be affected during washing.

4. The sealing film is limited to one-time use to avoid cross-contamination.

5. Please keep the substrate away from light.

6. The test results must be determined based on the reading of the microplate reader. When using dual wavelength detection, the reference wavelength is 630nm

7. All samples, washing liquids and various wastes should be treated as infectious agents. The stop solution is 2M sulfuric acid, you must pay attention to safety when using it.

8. Kit storage: 2-8 ° C.

9. Validity: 6 months