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Human Epidemic Hemorrhagic Fever Antibody (EHF-Ab) Enzyme-Linked Immunoassay Kit Instruction Manual
This kit is for research use only.
                                                                   48T
purpose of usage:
This kit is used to determine the expression of epidemic hemorrhagic fever antibody (EHF-Ab) in human serum, plasma and related fluid samples.
Experimental principle
The kit uses a double antigen sandwich method to determine the expression of human epidemic hemorrhagic fever antibody (EHF-Ab) in the specimen. The microporous plate is coated with purified human epidemic hemorrhagic fever antigen to prepare a solid phase antigen, which can be combined with the epidemic hemorrhagic fever antibody in the sample, and the unbound antigen and other components are washed to remove the epidemic with HRP. The hemorrhagic fever antigen binds to form an antigen-antibody-enzyme-labeled antigen complex, which is thoroughly washed and then added with the substrate TMB. 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 a wavelength of 450 nm using a microplate reader, and compared with the CUTOFF value to determine the presence or absence of a human epidemic hemorrhagic fever antibody (EHF-Ab) in the specimen.
Kit composition
1
20 times concentrated washing solution
20ml × 1 bottle
7
Stop solution
3ml × 1 bottle
2
Enzyme standard reagent
3ml × 1 bottle
8
Positive control
0.5ml × 1 bottle
3
Enzyme label coated plate
12 holes × 4
9
Negative control
0.5ml × 1 bottle
4
Sample diluent
3ml × 1 bottle
10
Instruction manual
1 copy
5
Developer A solution
3ml × 1 bottle
11
Sealing film
2 sheets
6
Developer B solution
3ml×1/bottle
12
sealed bag
1
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 the test cannot be performed immediately, the specimen can be stored at -20 °C, but repeated freezing and thawing should be avoided.
2. Samples containing NaN3 could not be detected because NaN3 inhibited horseradish peroxidase (HRP) activity.
Steps
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 well for blank control (no blank sample and enzyme standard reagent for blank control well, 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, add 40 μl of the sample dilution to the sample well to be tested, and then add 10 μl 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, and shake it gently.
3. Incubation: The plate was sealed with a sealing film and incubated at 37 ° C for 30 minutes.
4. Liquor: 20 times concentrated washing solution diluted with distilled water 20 times and used
5. Washing: Carefully remove the sealing film, discard the liquid, dry it, fill each well with the washing solution, let stand for 30 seconds, then discard it, repeat 5 times, and pat dry.
6. Add enzyme: Add 50 μl of enzyme labeling reagent to each well, except for blank wells.
7. Incubation: The operation is the same as 3.
8. Wash: Operate the same as 5.
9. Color development: add 50 μl of color developer A, and then add 50 μl of color developer B, gently shake and mix, and color for 15 minutes at 37 °C.
10. Termination: 50 μl of stop solution was added to each well to stop the reaction (the blue color turned yellow).
11. Measurement: The absorbance (OD value) of each well was measured in sequence with a blank air conditioner of zero and a wavelength of 450 nm. The measurement should be carried out within 15 minutes after the addition of the stop solution.
Calculation and result determination:
Test validity: mean value of positive control well ≥ 1.00; mean value of negative control ≤ 0.10
CUT OFF calculation: critical value = negative control well average + 0.15
Negative judgment: sample OD value < critical value (CUT OFF) is negative for human epidemic hemorrhagic fever antibody (EHF-Ab)
Positive judgment: sample OD value ≥ critical value (CUT OFF) is human epidemic hemorrhagic fever antibody (EHF-Ab) positive
.
Precautions
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 after opening, 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 and must be used safely.
Storage conditions and expiration date
1. The kit is stored at: 2-8 °C.
2. Validity: 6 months