May 27, 2020

Human anti-filamentin antibody (AFA) ELISA kit

Human anti-filamentin antibody (AFA) ELISA kit
This kit is for research use only.
Drug Name:
Generic name: Human anti-filamentin antibody (AFA) ELISA kit
purpose of usage:
This kit is used for qualitative determination of anti-filaggrin antibodies (AFA) in human serum, plasma and related liquid samples.
Experimental principle
This kit uses an indirect method to determine the human anti-filaggrin antibody (AFA) in the specimen. After coating the microtiter plate with purified silk fibroin (AFA) antigen to prepare a solid-phase antigen, after incubating with the anti-filaggrin antibody (AFA) in the sample, biotin-labeled anti-IgG antibody was added, followed by streptomyces The avidin-HRP combines to form an immune complex, 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 human anti-filaggrin antibody (AFA) 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 label coating plate 12 well × 8 strips 10 positive control 0.5ml × 1 bottle
4 Biotin-labeled anti-IgG antibody 6ml × 1 bottle 11 sealed bag 1
5 Developer A solution 6ml × 1 bottle 12 sealing film 3 sheets
6 Developer B liquid 6ml × 1 / bottle 13 Instructions 1 copy
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: The microwells corresponding to the samples are numbered in order. Each plate should be provided with 2 wells for negative control, 2 wells for positive control, and 1 well for blank control
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. Incubate at 37 ° C for 30 minutes
6. Washing: The operation is the same as 4.
7. Add streptavidin-HRP: add 50μl streptavidin-HRP to each 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 human filaggrin (AFA)
Positive judgment: the sample with OD value ≥ critical value (CUT OFF) is positive for human filaggrin antibody (AFA)

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

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Human anti-filamentin antibody (AFA) ELISA kit

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