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Human Granzyme B ELISA kit
$295/ 48T
QuantityTotal: $295
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Product Information
NameSpecificationsPreservation conditionsAntibody precoated plate8 × 12 or 8 × 6*4℃Lyophilized standard3 / 2 vials * 2ng/vial4℃Standard & sample universal diluent1 Bottle 20ml / 12ml*4℃Concentrated biotinylated antibody1 vial*4℃Biotinylated antibody diluent1 Bottle 16ml / 10ml*4℃Concentrated enzyme conjugates1 vial*4 ℃ (dark)Enzyme conjugate diluent1 Bottle 16ml / 10ml*4℃Concentrated detergent 20 ×1 Bottle 50ml / 25ml*4℃Chromogenic substrate (TMB)1 Bottle 12ml / 6ml*4 ℃ (dark)Reaction termination solution1 Bottle 12ml / 6ml*4℃Sealing tape6/3 pieces* Product datashete1 manual *For 48 tests.
Detection principleDouble antibody sandwich ELISA was used in this study.Anti human granzyme B monoclonal antibody is coated on the enzyme plate. Granzyme B in the sample and standard sample will combine with the monoclonal antibody, and the free components will be washed away. Biotinylated anti human granzyme B antibody and horseradish peroxidase labeled avidin were added. Biotin specifically binds to avidin; anti human granzyme B antibody binds to human granzyme B on monoclonal antibody to form immune complex, and the free components are washed away.If there is granzyme B in the reaction well, horseradish peroxidase will make the colorless reagent turn blue and the stop solution turn yellow. The concentration of granzyme B was directly proportional to the OD450 value measured at 450 nm. The concentration of granzyme B in the sample could be calculated by drawing a standard curve.
Specimen collection1.The test tube for collecting blood should be disposable pyrogen free and endotoxin free.
2. EDTA is recommended as plasma anticoagulant.Avoid using hemolytic and hyperlipidemic samples.
3.The samples should be clear and transparent, and the suspended matter should be removed by centrifugation.
4.If the samples are not detected in time after collection, they should be packed according to the amount of one-time use and stored in refrigerators at - 20 ℃ and - 70 ℃ to avoid repeated freezing and thawing, and detected within 3-6 months.
5.If the concentration of the test substance in your sample is higher than the maximum value of the standard substance, please dilute it in an appropriate multiple according to the actual situation (it is recommended to do a pre experiment to determine the dilution multiple).
Self prepared equipment required for the test (not provided)
1. Microplate reader (450nm wavelength filter)
2. Imported brand high precision dispenser and disposable suction head: 0.5-10ul, 2-20ul, 20-200ul, 200-1000ul
3. 37Thermostat, double distilled water or deionized water, coordinate paper.
2. EDTA is recommended as plasma anticoagulant.Avoid using hemolytic and hyperlipidemic samples.
3.The samples should be clear and transparent, and the suspended matter should be removed by centrifugation.
4.If the samples are not detected in time after collection, they should be packed according to the amount of one-time use and stored in refrigerators at - 20 ℃ and - 70 ℃ to avoid repeated freezing and thawing, and detected within 3-6 months.
5.If the concentration of the test substance in your sample is higher than the maximum value of the standard substance, please dilute it in an appropriate multiple according to the actual situation (it is recommended to do a pre experiment to determine the dilution multiple).
Self prepared equipment required for the test (not provided)
1. Microplate reader (450nm wavelength filter)
2. Imported brand high precision dispenser and disposable suction head: 0.5-10ul, 2-20ul, 20-200ul, 200-1000ul
3. 37Thermostat, double distilled water or deionized water, coordinate paper.
Matters needing attention1. Please keep the kit at 2-8 ℃ before use.If the standard sample after re dissolving is not used up, please discard it.
2.Concentration of biotinylated antibody, concentration of enzyme conjugates small volume, transportation bumps and possible inversion, will make the liquid stick to the wall or cap.Therefore, before use, please shake a few times by hand or separate the heart by 1000 rpm for 1 minute, so as to deposit the liquid attached to the tube wall or bottle cap to the bottom of the tube.Before using, please use pipette to blow 4-5 times carefully to mix the solution.
3. The concentrated washing solution taken out from the refrigerator may crystallize, which is a normal phenomenon. Reheat to 40 ℃ to completely dissolve the crystals before preparing the washing solution.(heating temperature should not exceed 50 ℃) the detergent should be at room temperature.
4. The kit components of different batch numbers should not be mixed (except washing solution and reaction termination solution).Please consult EnoGene Biotechnology Co., Ltd. in advance.
5. It's very important to mix well. It's better to use a micro oscillator (the lowest frequency). If there is no micro oscillator, you can tap the enzyme plate and frame by hand before the reaction.
6. In the test, the standard and sample should be tested by double well.
7. The test tube and suction head used in the test are disposable. It is strictly forbidden to mix different liquids!Otherwise, the test results will be affected.
8. During the test, please wear test clothes and latex gloves for protection.Especially when testing blood or other body fluid samples, please follow the safety protection regulations of national biological laboratory.
2.Concentration of biotinylated antibody, concentration of enzyme conjugates small volume, transportation bumps and possible inversion, will make the liquid stick to the wall or cap.Therefore, before use, please shake a few times by hand or separate the heart by 1000 rpm for 1 minute, so as to deposit the liquid attached to the tube wall or bottle cap to the bottom of the tube.Before using, please use pipette to blow 4-5 times carefully to mix the solution.
3. The concentrated washing solution taken out from the refrigerator may crystallize, which is a normal phenomenon. Reheat to 40 ℃ to completely dissolve the crystals before preparing the washing solution.(heating temperature should not exceed 50 ℃) the detergent should be at room temperature.
4. The kit components of different batch numbers should not be mixed (except washing solution and reaction termination solution).Please consult EnoGene Biotechnology Co., Ltd. in advance.
5. It's very important to mix well. It's better to use a micro oscillator (the lowest frequency). If there is no micro oscillator, you can tap the enzyme plate and frame by hand before the reaction.
6. In the test, the standard and sample should be tested by double well.
7. The test tube and suction head used in the test are disposable. It is strictly forbidden to mix different liquids!Otherwise, the test results will be affected.
8. During the test, please wear test clothes and latex gloves for protection.Especially when testing blood or other body fluid samples, please follow the safety protection regulations of national biological laboratory.
Preparationbefore testing:1. Please remove the kit from the refrigerator 20 minutes in advance to balance to room temperature.
2. Dilute the concentrated washing solution with double distilled water.The unused ones should be put back to 4 ℃.(1:20)
3. Standard: add the standard & sample general diluent into the freeze-dried standard, and let it stand for 15 minutes. After it is fully dissolved, mix it gently (the concentration is).Then dilute as needed.(the following concentrations are recommended for the standard curve:).0 pg / ml was the blank well.If the original solution of re dissolving standard is not used up, please discard it.0.5ml
4. Biotinylated antibody working solution: according to the amount required in the current test, dilute and concentrate biotinylated antibody into working concentration 20 minutes before use.Used on the same day.If the amount of biotinylated antibody is insufficient due to too many experiments, the biotinylated antibody can be purchased from EnoGene company alone.(antibody batch number must be provided)(1:30)
5. Enzyme conjugate working solution: according to the amount required in the current test, dilute the human granzyme B conjugate with enzyme conjugate diluent to the working concentration 20 minutes before use.Used on the same day.If too many experiments lead to insufficient amount of concentrated enzyme conjugate, you can purchase concentrated enzyme conjugate from EnoGene company.(batch number of enzyme conjugates must be provided)(1:30)
Legend of standard dilution method:(take 500ul / tube as an example, it can also be diluted according to the actual dosage, such as 200ul / tube)
2. Dilute the concentrated washing solution with double distilled water.The unused ones should be put back to 4 ℃.(1:20)
3. Standard: add the standard & sample general diluent into the freeze-dried standard, and let it stand for 15 minutes. After it is fully dissolved, mix it gently (the concentration is).Then dilute as needed.(the following concentrations are recommended for the standard curve:).0 pg / ml was the blank well.If the original solution of re dissolving standard is not used up, please discard it.0.5ml
4. Biotinylated antibody working solution: according to the amount required in the current test, dilute and concentrate biotinylated antibody into working concentration 20 minutes before use.Used on the same day.If the amount of biotinylated antibody is insufficient due to too many experiments, the biotinylated antibody can be purchased from EnoGene company alone.(antibody batch number must be provided)(1:30)
5. Enzyme conjugate working solution: according to the amount required in the current test, dilute the human granzyme B conjugate with enzyme conjugate diluent to the working concentration 20 minutes before use.Used on the same day.If too many experiments lead to insufficient amount of concentrated enzyme conjugate, you can purchase concentrated enzyme conjugate from EnoGene company.(batch number of enzyme conjugates must be provided)(1:30)
Legend of standard dilution method:(take 500ul / tube as an example, it can also be diluted according to the actual dosage, such as 200ul / tube)
Washing method1. Automatic washing machine: 350 uL washing liquid is required to be injected, and the interval between injection and suction is 30-60 seconds.Wash the plate 5 times.
2. Manual plate washing: shake all the liquid in the hole, dry it on the clean absorbent paper, add 350ul of washing liquid to each hole, leave it standing for 30 seconds, shake all the liquid, and dry it on the thick laminated absorbent paper.Wash the plate 5 times.
2. Manual plate washing: shake all the liquid in the hole, dry it on the clean absorbent paper, add 350ul of washing liquid to each hole, leave it standing for 30 seconds, shake all the liquid, and dry it on the thick laminated absorbent paper.Wash the plate 5 times.
Operation steps1.Take out the strips needed for the test from the sealed bag which has been balanced to room temperature. Put the unused strips and desiccant back into the aluminum foil bag, compact the self sealing strip, seal the bag, and put them back to 4 ℃.
2.The blank hole was added with standard, and the other corresponding holes were added with samples or different concentrations of standard (100ul / hole). The reaction hole was sealed with sealing tape, and incubated in 37 ℃ incubator in dark for 90 minutes.
3.Prepare biotinylated antibody working solution 20 minutes in advance.
4. Wash the plate 5 times.
5.The blank well was added with biotinylated antibody diluent, and the remaining wells were added with biotinylated antibody working solution (100ul / well).The reaction holes were sealed with new sealing tape and incubated in 37 ℃ incubator for 60 minutes.
6.Prepare the enzyme conjugate working solution 20 minutes in advance.Keep away from light and room temperature (22-25 ℃).
7. Wash the plate 5 times.
8.The blank well was added with enzyme conjugate diluent, and the other wells were added with enzyme conjugate working solution (100ul / well).The reaction holes were sealed with new sealing tape and incubated in 37 ℃ incubator for 30 minutes.
9.Turn on the power of the microplate reader, preheat the instrument, and set up the detection program.
10. Wash the plate 5 times.
11.Add color substrate (TMB) 100ul / well, incubate in 37 ℃ incubator and keep away from light for 15 minutes.
12.Add 100ul / well of reaction termination solution and measure OD450 immediately after mixing (within 3 minutes).Save the reading result in the instrument and print a paper result.
13.After the experiment, put the unused reagents back to the refrigerator according to the specified storage temperature and keep them until the end of the validity period.
It is suggested that the enzyme labeled plate and frame should be preserved for the next or future test.
The results were determined as following：
1. The OD value of the blank hole should be subtracted from the OD value of each standard and
specimen.
2. Using standard concentration as abscissa and OD value as ordinate, the best fitting curve was drawn by hand or by software, and quadratic polynomial equation was recommended.Through the OD value of the sample, its concentration can be found on the standard curve.
3. If the OD value of the sample is higher than the upper limit of the standard curve, it should be diluted properly and retested, and the concentration should be multiplied by the dilution multiple.
2.The blank hole was added with standard, and the other corresponding holes were added with samples or different concentrations of standard (100ul / hole). The reaction hole was sealed with sealing tape, and incubated in 37 ℃ incubator in dark for 90 minutes.
3.Prepare biotinylated antibody working solution 20 minutes in advance.
4. Wash the plate 5 times.
5.The blank well was added with biotinylated antibody diluent, and the remaining wells were added with biotinylated antibody working solution (100ul / well).The reaction holes were sealed with new sealing tape and incubated in 37 ℃ incubator for 60 minutes.
6.Prepare the enzyme conjugate working solution 20 minutes in advance.Keep away from light and room temperature (22-25 ℃).
7. Wash the plate 5 times.
8.The blank well was added with enzyme conjugate diluent, and the other wells were added with enzyme conjugate working solution (100ul / well).The reaction holes were sealed with new sealing tape and incubated in 37 ℃ incubator for 30 minutes.
9.Turn on the power of the microplate reader, preheat the instrument, and set up the detection program.
10. Wash the plate 5 times.
11.Add color substrate (TMB) 100ul / well, incubate in 37 ℃ incubator and keep away from light for 15 minutes.
12.Add 100ul / well of reaction termination solution and measure OD450 immediately after mixing (within 3 minutes).Save the reading result in the instrument and print a paper result.
13.After the experiment, put the unused reagents back to the refrigerator according to the specified storage temperature and keep them until the end of the validity period.
It is suggested that the enzyme labeled plate and frame should be preserved for the next or future test.
The results were determined as following：
1. The OD value of the blank hole should be subtracted from the OD value of each standard and
specimen.
2. Using standard concentration as abscissa and OD value as ordinate, the best fitting curve was drawn by hand or by software, and quadratic polynomial equation was recommended.Through the OD value of the sample, its concentration can be found on the standard curve.
3. If the OD value of the sample is higher than the upper limit of the standard curve, it should be diluted properly and retested, and the concentration should be multiplied by the dilution multiple.
Legend of standard dilution method:(take 500ul / tube as an example, it can also be diluted according to the actual dosage, such as 200ul / tube)
Note: This figure is for reference only. The sample content should be standard curve drawn by the same test standard.
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