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Human (Human) Hepatitis B Surface Antigen (HBsAg) ELISA Kit Instructions
Human Hepatitis B Surface Antigen (HBsAg) ELISA Test Kit
Test principle :
The HBSAG kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA). Standards of known HBSAG concentrations and samples of unknown concentration are added to the microplates for detection. HBSAG and biotinylated antibodies were first incubated simultaneously. After washing, avidin-labeled HRP was added. After incubation and washing, the unbound enzyme conjugate is removed, then the substrates A, B, and the enzyme conjugate are added simultaneously. Produce color. The depth of the color is proportional to the concentration of HBSAG in the sample.
Kit contents and their preparation :
Kit ingredients
96-well configuration
48 hole configuration
96/48 ELISA plate
1 board (96T)
Half board (48T)
Plastic diaphragm cover
1 block
Half block
Standard: 500pg/ml
1 bottle (1.0ml)
1 bottle (0.5ml)
Blank control
1 bottle (1.0ml)
1 bottle (0.5ml)
Standard dilution buffer
1 bottle (8.0ml)
1 bottle (4.0ml)
Biotinylated anti-HBSAG antibody
1 bottle (8.0ml)
1 bottle (4.0ml)
Affinity chain enzyme-HRP
1 bottle (12ml)
1 bottle (5ml)
Washing buffer
1 bottle (20ml)
1 bottle (10ml)
Substrate A
1 bottle (6.0ml)
1 bottle (3.0ml)
Substrate B
1 bottle (6.0ml)
1 bottle (3.0ml)
Stop solution
1 bottle (6.0ml)
1 bottle (3.0ml)
Bring your own materials :
1. Distilled water.
2. Sampler: 5ul, 10ul, 50ul, 100ul, 200ul, 500ul, 1000ul.
3. Oscillators and magnetic stirrers.
4.
Sample collection, processing and storage methods :
1. Serum... Avoid any cell irritation during the procedure. Use tubes without pyrogens and endotoxins. After collecting blood, centrifuge at 1000 × g for 10 minutes to red blood
The cells are quickly and carefully separated.
2, plasma ... EDTA, citrate, heparin plasma can be used for testing. The pellet was removed by centrifugation at 1000 x g for 30 minutes.
3. Cell supernatant. Centrifuge at 1000 xg for 10 minutes to remove particles and polymer.
4. Tissue homogenization... Add the appropriate amount of normal saline to the tissue. Centrifuge at 1000 xg for 10 minutes and take the supernatant.
5. Save... If the sample is not used immediately, it should be divided into small parts - 70 ° C to avoid repeated freezing. Do not use hemolysis or hyperlipemia as much as possible. If there are large amounts of particles in the serum, centrifuge or filter before testing. Do not heat thaw at 37 ° C or higher. It should be thawed at room temperature and ensure that the sample is fully thawed evenly.
Operational notes :
â— Reagents should be stored according to the label instructions and returned to room temperature before use. Standards after dilution should be discarded and cannot be stored.
â— The slats not used in the experiment should be immediately put back into the bag and sealed to prevent deterioration.
â— Other reagents that are not used should be packaged or covered. Do not mix reagents of different batches. Use before warranty.
â— Use disposable tips to avoid cross-contamination. Avoid pipettes with metal parts when drawing stop solution and substrate A and B.
â— Use a clean plastic container to configure the wash solution. Mix all the ingredients and samples in the kit thoroughly before use.
â— Substrate A should be volatilized to avoid opening the lid for a long time. Substrate B is sensitive to light and avoids prolonged exposure to light. Avoid contact with hands and be toxic. The OD value should be read immediately after the experiment is completed.
â— The order of adding reagents should be the same to ensure that all wells are incubated for the same time.
â— Carry out the incubation according to the time indicated in the instruction manual, the amount of liquid addition and the order.
Security :
1. Avoid direct contact with the stop solution and substrate A, B. Once exposed to these liquids, rinse with water as soon as possible.
2. Do not eat, drink, smoke or use cosmetics.
3. Do not use the mouth to absorb any ingredients in the kit.
Preparation of reagents :
1. Standard: The serial dilution of the standard should be prepared during the experiment and cannot be stored. Mix the standard shakes before dilution. The dilution ratio is as follows:
500
Pg/ml
(No. 6 standard)
The original concentration is directly added to 50ul without dilution.
250
Pg/ml
(No. 5 standard)
Add 100 ul of standard dilution to 100 ul of the original standard
125
Pg/ml
(No. 4 standard)
100ul of standard 5 is added to 100ul of standard dilution
62.5
Pg/ml
(No. 3 standard)
100ul of standard 4 is added to 100ul of standard dilution
31.2
Pg/ml
(No. 2 standard)
100ul of standard 3 is added to 100ul of standard dilution
15.6
Pg/ml
(No. 1 standard)
100ul of standard 2 is added to 100ul of standard dilution
0
Pg/ml
(blank control)
The original concentration is directly added to 50ul without dilution.
2. Dilution of wash buffer (50×): dilute with distilled water 50 times.
Kit performance :
1. Sensitivity: The minimum detection concentration is less than the No. 1 standard. The linearity of the dilution. The linear regression coefficient of the sample and the expected concentration correlation coefficient R value was 0.990.
2. Specificity: Does not react with other cytokines.
3. Repeatability: The coefficient of variation between the plate and the plate is less than 10%.
Operation steps :
1. Mix all reagents thoroughly before use. Do not allow the liquid to generate a large amount of foam, so as to avoid adding a large amount of air bubbles during the loading, resulting in errors in the loading.
2. Determine the number of slats required based on the number of samples to be tested plus the number of standards. Multiple holes are recommended for each standard and blank hole. Each sample is determined according to its own quantity, and it is possible to use the double hole as much as possible to make a double hole.
3. Add 50 ul of the diluted standard to the reaction well and add 50 ul of the sample to be tested to the reaction well. Immediately add 50 ul of biotinylated antibody. Cover the membrane, mix gently by shaking, and incubate for 45 minutes at 37 °C.
4. Remove the liquid from the wells, fill each well with the washing solution, shake for 30 seconds, remove the washing solution, and pat dry with absorbent paper. Repeat this operation 4 times. If washing with a washer, the number of washings is increased once.
5. Add 100 ul of affinity streptavidin-HRP to each well, mix gently by shaking, and incubate for 30 minutes at 37 °C.
6. Remove the liquid from the wells, fill each well with the washing solution, shake for 30 seconds, remove the washing solution, and pat dry with absorbent paper. Repeat this operation 4 times. If washing with a washer, the number of washings is increased once.
7. Add 50 ul of substrate A and B to each well, mix gently by shaking, and incubate for 5 minutes at 37 °C. Avoid lighting.
8. Remove the microplate and quickly add 50 ul of stop solution. Immediately after adding the stop solution, the results should be determined.
9. Determine the OD value of each well at a wavelength of 450 nm.
Result judgment and analysis :
1. Instrument value: read the OD value of each hole on a microplate reader with a wavelength of 450 nm.
2. The absorbance OD value is the ordinate (Y), and the corresponding HBSAG standard concentration is the abscissa (X), and the corresponding curve is obtained. The HBSAG content of the sample can be converted into the corresponding concentration according to the OD value from the standard curve. Multiply by the dilution factor; or calculate the regression equation of the standard curve using the concentration of the standard and the OD value, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply by the dilution factor, which is the actual concentration of the sample.
3. Detection range: 0-500pg/ml
4. Sensitivity: 1.95pg/ml