Horse coccidiosis enzyme-linked immunoassay kit instruction manual - Database & Sql Blog Articles

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Horse coccidiosis enzyme-linked immunoassay kit instruction manual

This kit is for research use only.

Experimental principle

Horse coccidiosis enzyme-linked immunosorbent assay kit

The expression of horse coccidiosis in the specimens was determined by double antibody sandwich method. The microporous plate is coated with the purified horse phobia antibody to prepare a solid phase antibody, which can be combined with the coccidiosis in the sample, and the unbound antigen and other components are washed to remove the HRP-labeled coccidiosis antibody. The antibody-antigen-enzyme-labeled antibody complex is formed by binding, and after thorough washing, the substrate TMB is added for color development. TMB is converted to blue under the catalysis of HRP enzyme and converted to the final yellow color by the action of an acid. The absorbance (OD value) was measured at 450 nm using a microplate reader, and compared with the CUTOFF value to determine the presence or absence of horse coccidiosis in the specimen.

Horse coccidiosis enzyme-linked immunosorbent assay kit

1

,

30 times concentrated washing solution 20ml × 1 bottle 7 stop solution 6ml × 1 bottle

2

,

Enzyme standard reagent 6ml × 1 bottle 8 positive control 0.5ml × 1 bottle

3

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Enzyme label coated plate 12 holes × 8 strips 9 negative control 0.5 ml × 1 bottle

4

,

Sample dilution 6ml × 1 bottle 10 instructions 1 copy

5

,

Reagent A liquid 6ml × 1 bottle 11 sealing film 2 sheets

6

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Color developer B liquid 6ml × 1 bottle 12 sealed bag 1

Horse coccidiosis enzyme-linked immunosorbent assay kit specimen requirements

1. The specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to the relevant literature. The experiment should be carried out as soon as possible after extraction. If not

Immediately test, the specimen can be stored at -20 ° C, but should avoid repeated freezing and thawing

2. Samples containing NaN3 could not be detected because NaN3 inhibited horseradish peroxidase (HRP) activity.

Horse coccidiosis enzyme-linked immunoassay kit procedure

1. No.: The corresponding micropores of the sample are numbered sequentially. Each plate should have 2 holes for negative control, 2 holes for positive control, and 1 for blank control.

Hole (the blank control well is not added with the sample and the enzyme standard reagent, the other steps are the same)

2. Loading: 50 μl of negative control and positive control were added to the negative and positive control wells, respectively. Then in the sample hole to be tested first

Add 40 μl of the sample dilution, and then add 10 μl of the sample to be tested. Add the sample to the bottom of the well of the ELISA plate, try not to

Touch the hole wall and gently shake it to mix.

3. Incubation: The plate was sealed with a sealing film and incubated at 37 ° C for 30 minutes.

4. Dosing: 30 times concentrated washing solution diluted with distilled water 30 times and used

5. Washing: Carefully remove the sealing film, discard the liquid, dry it, fill each well with the washing liquid, let stand for 30 seconds, then discard it.

Repeat 5 times and pat dry.

6. Add enzyme: 50 μl of enzyme labeling reagent was added to each well, except for blank wells.

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

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

9. Color development: add 50μl of color developer A, then add 50μl of color developer B, gently shake and mix, avoid light and develop color at 37 °C

15 minutes.

10. Termination: 50 μl of stop solution was added to each well to terminate the reaction (when the blue color turned yellow).

11. Measurement: The absorbance (OD value) of each well was measured sequentially with a blank air conditioner of zero and a wavelength of 450 nm. Determination should be terminated

It is carried out within 15 minutes after the liquid.

Calculation and result determination:

Test validity: mean value of positive control well ≥ 1.00; mean value of negative control ≤ 0.10

CUTOFF calculation: critical value = negative control hole average + 0.15

Negative judgment: sample OD value <critical value (CUTOFF) is negative for coccidiosis

Positive judgment: The sample OD value ≥ critical value (CUTOFF) is positive for coccidiosis.

Horse coccidiosis enzyme-linked immunosorbent assay kit considerations

1. The operation is carried out in strict accordance with the instructions. The components of the different batches of this reagent shall not be mixed.

2. The kit should be taken out from the refrigerated environment and should be used at room temperature for 15-30 minutes before use. If the enzyme label is unsealed,

When used up, the slats should be stored in a sealed bag.

3. Concentrated washing liquid may crystallize out. When diluted, it can be heated and dissolved in a water bath. The washing will not affect the result.

4. The sealing film is intended for single use only to avoid cross-contamination.

5. Please keep the substrate away from light.

6. The test results must be determined by the microplate reader. When using dual-wavelength detection, the reference wavelength is 630nm.

7. All samples, washings and various wastes should be treated as infectious materials. The stop solution is 2M sulfuric acid, which must be used

be careful.

Storage conditions and expiration date

1. Kit storage: 2-8 ° C.

2. Validity: 6 months