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(US DRG imported original, article number: EIA 4138 )
Kit Composition <br>Sufficient for 96-person reagent kits stored at 2-8 ° C
The expiration date of each reagent can be found in the reagent bottle label kit. Once opened, it can be stored for 2 months at 2-8 °C.
Materials required for the experiment but not provided in the kit
This experiment can also be operated with an automatic microplate reader
Sample Collection and Preparation <br>Tests can detect serum or plasma samples. The appropriate amount of lipemia samples will not affect the results. High levels of lipemia or hemolysis may affect the test. Fibrin will interfere with the test; ensure that the sample is clear before testing. The sample can be stored at 2-8 ° C for 1 week. If you want to keep it for a longer period of time, it is recommended to freeze at -20 ° C to avoid repeated freeze-thaw. Before use: Use a previously diluted sample dilution at a ratio of 1:300. Sample dilution (eg 10 ul sample + 2990 ul dilution)
Experimental Procedures <br>Reagents and samples are equilibrated to room temperature.
Experimental operation diagram
The results were calculated on the linear coordinate paper with the standard concentration as the X-axis and the absorption value as the Y-axis, and the standard curve was drawn; the corresponding anti-CagA IgG antibody concentration (RU/mL) was obtained from the standard curve based on the absorbance of each sample.
Calculation example <br>The following values ​​are for demonstration purposes only and should not be used in place of the experimental values. The sample anti-CagA IgG antibody titer is 70 RU/mL from the standard curve.
Before verifying the standard <br> results, first ensure that the QC absorption value is within the following expected range
This translation is for reference only, please refer to the original manual.
Human cytotoxin associated protein A ( CagA ) detection kit
Experimental principle <br>This kit is based on enzyme-linked immunosorbent assay using HRP conjugate; in the first incubation, the anti-CagA IgG antibody in the sample binds to the coated CagA antigen, washes the plate to remove unbound, and incubates again The secondary antibody (peroxidase-labeled anti-human IgG) is combined with the CagA-antigen-antibody complex, and after washing again, the colorless developer (TMB) substrate solution is added to the micropores, and the peroxide The enzymatic reaction produces a colored complex, which is terminated by the addition of sulfuric acid. The color intensity is measured at 450nm and 405nm. The concentration of anti-CagA IgG antibody in the standard and sample is proportional to the color intensity.
1. [ MTP ] coated plate, 96 wells, coated with CagA antigen (recombinant DNA), unused slats were stored in sealed bags at 2-8 °C
2. [ CAL ] standard, 6 bottles, 1.5ml/bottle, anti-CagA IgG serum matrix, concentration 0; 15; 30; 60; 120; 240 RU/ml, ready to use, red, sodium azide (< 0.1%) as a preservative
3. [ CONJ ] Enzyme conjugate, 1 bottle, 14ml, HRPO-labeled mouse monoclonal antibody anti-human IgG, TRIS buffer, stabilizer, ready-to-use, pink, neomycin as a preservative
4. [ WASH ] Washing buffer (concentrated), 1 bottle, 50ml, PBS-Tween 20, thimerosal (<0.05%) as preservative, diluted 1:20 in distilled water before use; if insoluble crystals appear , need to be incubated at 37 ° C for several minutes; diluted washing solution can be stored at 2-8 ° C for 30 days
5. [ DIL ] Sample diluent (concentrated,) 1 bottle, 20ml, serum matrix, stabilizer, red, sodium azide (<0.1%) as a preservative, diluted 1:20 in diluted detergent solution before use. Diluted sample dilution can be stored for 30 days at 2-8 °C
6. [ TMB ] developer, 2 bottles, 15ml, TMB containing citric acid-phosphate buffer, DMSO and H 2 O 2 , ready to use
7. [ STOP ] blocking solution, 1 bottle, 14ml, 1N sulfuric acid, ready to use
8. [ CPA ] cover
9. Plastic bags
1. Adjustable, automatic micropipette with disposable tip
2. Drying oven, adjusted to 37±2°C
3. Scale container for reagent dilution
4. Suction pump or automatic washing equipment
5. Microplate reader (450nm and 405nm)
6. Coordinate paper
7. Distilled water
1. Prepared microwells: blanks, standards and samples
2. Add 100 ul of standard and previously diluted sample to the corresponding microwell
Note: Standards are not diluted
1. Add 100 ul of sample diluent to the blank well
2. Cover the coated plate with the adhesive paper provided in the kit and incubate at 37±2°C for 60±5 min.
3. 350 ul of diluted washing solution, wash the plate 4 times, and absorb all the liquid in the micropore
4. Add 100 ul of enzyme conjugate per well
5. Cover the coated plate with the adhesive paper provided in the kit and incubate at 37±2°C for 30±2 min.
6. Follow step 5) to wash the plate.
7. Add 100 ul of developer to each well
8. Incubate for 15 min at 37 ± 2 °C to avoid direct sunlight
9. Add 100 ul of blocking solution per well
10. Read the absorbance at 50 nm using a suitable microplate reader at a reference wavelength of 620 nm (zero with a blank control well). In the event of an absorption overflow, select a reading at 405 nm; the test must be completed within 15 minutes of the end of the test. reading
(If using automatic ELISA equipment, follow the relevant manual)
Coated hole
blank
Standard
sample
Reagent
Standard
-
100 ul
-
Dilute sample
-
-
100 ul
Sample diluent
100 ul
-
-
Incubation: 37 ± 2 ° C, 60 ± 5 min
Wash board: 4 x 350 ul
Enzyme complex
100 ul
100 ul
100 ul
Incubation: 37 ± 2 ° C, 30 ± 2 min
Wash board: 4 x 350 ul
Reagent
100 ul
100 ul
100 ul
Incubation: 37 ° C, 15 min
Blocking solution
100 ul
100 ul
100 ul
Reading: 450 nm
1. If the sample IgG value is less than 10 RU/mL, it is judged that the anti-CagA IgG antibody is not reactive.
2. If the sample IgG value is 10 -15 RU/mL, it is judged to be weak reaction.
3. If the sample IgG value is higher than 15 RU/mL, it is judged that the anti-CagA IgG antibody is reactive.
description
Expected value
Negative control
<0.100
Ratio of OD values ​​(standard 240 RU/mL/standard 15 RU/mL)
>2.5
Ratio of OD values ​​(standard 15 RU/mL/standard 0 RU/mL)
>3.5
If the value is not within the expected range, you need to re-experiment