Instructions for use of Trypanosoma mayis ELISA kit
This kit is for research use only.
Drug Name:
Common name: ELISA Kit for Trypanosoma mayi
purpose of usage:
This kit is used for the qualitative determination of Trypanosoma cruzi in horse serum, plasma and related liquid samples.
Experimental principle
This kit uses an indirect method to determine Trypanosoma marieii in specimens. The microplate was coated with purified trypanosoma eosinii antibody to make a solid phase antibody. After incubating with the trypanosoma eosinophilus in the sample, biotin-labeled anti-IgG antibody was added and then combined with streptavidin-HRP , To form immune complexes, after thorough washing, add substrate TMB to develop color. 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 Trypanosoma maritima in the specimen. ,
Kit composition
120 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 wells × 8 strips 10 positive controls 0.5ml × 1 bottle
4 Biotin-labeled anti-IgG antibody 6ml × 1 bottle 11 sealed bags 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 sample containing NaN3 cannot be detected because NaN3 inhibits the activity of horseradish peroxidase (HRP).
2. The specimens should be extracted as soon as possible after collection. The extraction should be carried out according to relevant literature. 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, biotinylated 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 it stand for 30 seconds and then discard, repeat this 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 50μl streptavidin-HRP (except the blank well) to each well, gently shake and mix, and incubate at 37 ℃ 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 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 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 cut-off value (CUT OFF): cut-off value = average value of negative control well + 0.15
Negative judgment: samples with OD value <cut-off value (CUT OFF) are negative for Trypanosoma mayi
Positive judgment: the sample whose OD value ≥ critical value (CUT OFF) is positive for Trypanosoma mayi
Precautions
1. The operation is carried out in strict 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 slats should be stored in sealed bags.
3. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in the 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 result 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 liquid 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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