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Kit instruction manual
This kit is for research use only.
purpose of usage:
This kit is used to determine the content of Brucella IgG in human serum, plasma and related liquid samples.
Experimental principle
This kit uses an indirect method to determine the levels of Brucella IgG in the specimen. A microplate is coated with purified antigen to prepare a solid-phase antigen, and Brucella IgG standard (known concentration) and Brucella (unknown concentration) are added to the monoclonal antibody-coated microwells in sequence. IgG antibody (Brucella IgG) sample to be tested, after incubation, add biotin-labeled anti-IgG antibody, and then combine with streptavidin-HRP to form an immune complex, 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 color depth is positively correlated with Brucella IgG in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of human Brucella IgG antibody (Brucella IgG) in the sample was calculated by a standard curve.
Kit composition
1
30 times concentrated washing solution
20ml × 1 bottle
8
Standard product S1 (90 ng / L)
0.5ml × 1 bottle
2
Streptavidin-HRP
6ml × 1 bottle
Standard product S2 (60ng / L)
0.5ml × 1 bottle
3
Enzyme coated plate
12 holes × 8
Standard product S3 (30ng / L)
0.5ml × 1 bottle
4
Biotin-labeled anti-IgG antibody
6ml × 1 bottle
Standard product S4 (15ng / L)
0.5ml × 1 bottle
5
Developer A liquid
6ml × 1 bottle
Standard product S5 (7.5ng / L)
0.5ml × 1 bottle
6
Developer B liquid
6ml × 1 / bottle
9
Instructions
1 serving
7
Stop solution
6ml × 1 bottle
10
Sealing film
2 sheets
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. Determine the number of slats required based on the number of samples to be tested plus the number of standard products. It is recommended to make multiple holes for each standard and blank hole. Each sample is determined according to its own quantity, and those that can use multiple holes can be used as much as possible.
2. Adding samples: set up blank wells (the blank control wells do not add samples, the rest of the operations are the same), standard wells, and sample wells to be tested. Then add 50 μl of the standard product to the standard well, add 50 μl of the sample to the sample reaction well, cover with the sealing film, shake gently to mix, and incubate at 37 ° C for 45 minutes.
3. Mixing solution: Dilute 30 times concentrated washing liquid with distilled water 30 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. 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.
Calculation
Taking the concentration of the standard as the abscissa and the OD value as the ordinate, draw a standard curve on the coordinate paper, and find the corresponding concentration from the standard curve according to the OD value of the sample; then multiply it by the dilution factor; Calculate the linear regression equation of the standard curve with the OD value, put the OD value of the sample into the equation, calculate the sample concentration, and then multiply it by the dilution factor, which is the actual concentration of the sample.
Precautions
1. 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.
2. 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.
3. The sampler should be used at each step of sample addition, and the accuracy should be regularly checked to avoid test errors. It is best to control the sampling time within 5 minutes. If there are many specimens, it is recommended to use a volley gun to add samples.
4. If the content of the substance to be tested in the specimen is too high (the OD value of the sample is greater than the OD value of the first well of the standard well), please dilute the sample by a certain multiple (n times) and then determine it. When calculating, please multiply the dilution factor (× 5 × n).
5. The sealing film is limited to one-time use to avoid cross-contamination.
6. The components of different batches of this reagent shall not be mixed. Store developer B in dark place.
7. Strictly follow the instructions, and the test results must be determined by the microplate reader.
8. All samples, washing liquids and various wastes should be treated as infectious agents.
9. If there is any difference with the English manual, the English manual shall prevail.
Linear range:
5ng / L-120ng / L
specification:
96 servings / box
Storage conditions and validity period
1. Kit storage :; 2-8 ℃.
2. Validity: 6 months
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