Human hepatitis B surface antigen (HBsAg) ELISA
Kit Instruction Manual This kit is for research use only.
Drug Name:
Generic name: Human hepatitis B surface antigen (HBsAg) enzyme-linked immunoassay kit Purpose of use:
This kit is used to determine the content of hepatitis B surface antigen (HBsAg) in human serum, plasma and related liquid samples.
Experimental principle The kit uses an indirect method to determine the level of human hepatitis B surface antigen (HBsAg) in the specimen. A microplate was coated with purified human hepatitis B surface antigen (HBsAg) antibody to prepare a solid-phase antibody, and a known concentration of hepatitis B surface antigen (HBsAg) standard and an unknown concentration of HBsAg were sequentially added to the monoclonal antibody-coated microwells. 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 the hepatitis B surface antigen (HBsAg) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of human hepatitis B surface antigen (HBsAg) in the sample was calculated by a standard curve.
Kit composition
1
20 times concentrated washing liquid 50ml × 1 bottle
9
Standard product S1 (90IU / L)
0.5ml × 1 bottle
2
Streptavidin-HRP
6ml × 1 bottle of standard product S2 (60IU / L)
0.5ml × 1 bottle
3
Enzyme label coating plate 12 well × 8 strips Standard S3 (30IU / L)
0.5ml × 1 bottle
4
Biotin-labeled anti-IgG antibody 6ml × 1 bottle Standard S4 (15IU / L)
0.5ml × 1 bottle
5
Developer A liquid 6ml × 1 bottle Standard product S5 (7.5IU / L)
0.5ml × 1 bottle
6
Developer B liquid 6ml × 1 / bottle 10
1 sealed bag
7
Stop solution 6ml × 1 bottle 11
3 sheets of sealing film
8
Sample diluent 6ml × 1 bottle 12
Instructions 1
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. Add samples: set up blank wells (no sample is added to the blank control wells, biotin-labeled anti-IgG antibody, streptavidin-HRP, the rest of the steps are the same), standard wells, and test wells Then add 50μl of the standard to the standard well, add 10μl of the sample to be tested to the sample reaction well and then add 40μl of the sample diluent (the final dilution of the sample is 5 times), cover with the sealing film, shake gently and mix well, 37 ℃ Incubate 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 50 μl of streptavidin-HRP to each well (except the blank 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.
Calculate the standard concentration 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; multiply by the dilution factor; or use the standard The linear regression equation of the standard curve is calculated from the concentration and the OD value. The OD value of the sample is entered into the equation to calculate the sample concentration and then multiplied 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:
5IU / L -100IU / L
specification:
96 servings / box
Storage conditions and validity period
1. Kit storage :; 2-8 ℃.
2. Validity: 6 months
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