Manual of ELISA Kit for Equine Encephalitis Virus

2024-05-18 09:06:52

Horse encephalitis virus enzyme-linked immunoassay (ELISA)

Kit instruction manual

This kit is for research use only.

Drug Name:

Generic name: Horse Blue Ear Virus ELISA Kit

purpose of usage:

This kit is used for qualitative determination of Japanese encephalitis virus in horse serum, plasma and related liquid samples.

Experimental principle

This kit uses an indirect method to determine equine encephalitis virus in specimens. Coated microtiter plates with purified Japanese encephalitis virus antibodies to make solid phase antibodies. After incubation with Japanese encephalitis virus in samples, biotin-labeled anti-IgG antibodies were added, followed by streptavidin- HRP binds to form immune complexes, and 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 equine encephalitis virus in the specimen. ,

Kit composition

1

20 times concentrated washing liquid

50ml × 1 bottle

8

Sample diluent

6ml × 1 bottle

2

Streptavidin-HRP

6ml × 1 bottle

9

Negative control

0.5ml × 1 bottle

3

Enzyme coated plate

12 holes × 8

10

Positive control

0.5ml × 1 bottle

4

Biotin-labeled anti-IgG antibody

6ml × 1 bottle

11

sealed bag

1

5

Developer A liquid

6ml × 1 bottle

12

Sealing film

3 sheets

6

Developer B liquid

6ml × 1 / bottle

13

Instructions

1 serving

7

Stop solution

6ml × 1 bottle

Specimen requirements

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

2. Specimens are extracted as soon as possible after collection, and extraction is performed according to relevant literature, and experiments should be conducted as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided

Steps

1. Numbering: Number the samples corresponding to the microwells in sequence, each plate should be set with 2 wells for negative control, 2 wells for positive control, blank control (no sample added, biotin-labeled anti-IgG antibody, streptavidin-HRP , The rest are the same) 1 hole

2. Add sample: add negative control and positive control 50μl 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 and add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, shake gently to mix, and incubate at 37 ℃ for 45 minutes.

3. Mixing solution: dilute 20 times concentrated washing solution with distilled water 20 times and reserve

4. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing solution, let stand for 30 seconds and then discard, repeat 4 times, pat dry.

5. Add biotin-labeled anti-IgG antibody: Add 50 μl of biotin-labeled anti-IgG antibody to each well (except blank wells). Incubate at 37 ° C for 30 minutes

6. Washing: The operation is the same as 4.

7. Add streptavidin-HRP: add 50 μl of streptavidin-HRP to each well (except the blank well), gently shake and mix, and incubate at 37 ° C for 30 minutes.

8. Washing: The operation is the same as 4.

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

10. Termination: Add 50μl of stop solution to each well to stop the reaction (at this time the blue will turn to yellow).

11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450 nm 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 ≤0.10

Calculation of critical value (CUT OFF): critical value = average value of negative control wells + 0.15

Negative judgment: the sample with OD value <cut-off value (CUT OFF) is negative for equine encephalitis virus

Positive judgment: the sample whose OD value ≥ critical value (CUT OFF) is positive for equine encephalitis virus

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 equilibrated at room temperature for 15-30 minutes before being taken out of the refrigerated environment. If the enzyme label coated plate is unopened, 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, and you must pay attention to safety when using it.

specification:

96 servings / box

Storage conditions and validity period

1. Kit storage :; 2-8 ℃.

2. Validity: 6 months

Shanghai Yifeng Biotechnology Co., Ltd. acts as an agent for ELISA kits of different brands and price grades, such as the original R & D RB BD in the United States. Tens of thousands of antibody products, etc., with many varieties, good quality, high sensitivity, affordable, and also provide free testing services

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