Mouse HbA1c ELISA Kit
Mouse HbA1c ELISA Kit
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Product Specification
| Usage |
I. Self-provided test equipment required for the experiment: 1. Plate reader ( 450nm ） 2. High-precision sampler and gun tip: 0.5-10μL 、 5-50μL 、 20-200μL 、 200-1000μL 3.37℃ thermostatic box 4. Distilled water or deionized water II. Sample Handling and Requirements: 1. The detection range of the kit is not equivalent to the concentration range of the substance to be tested in the sample. It is recommended to estimate the concentration of the substance to be tested in the sample through relevant literature before the experiment and determine the actual concentration of the sample through pre-experiments. If the concentration of the test substance in the sample is too high or too low, dilute or concentrate the sample appropriately. 2. If the tested sample is not among the sample types listed in the instructions, it is recommended to conduct a preliminary experiment to verify its testing effectiveness. 3. Serum: Whole blood samples collected in serum separation tubes were placed at room temperature 2 Hours or 2-8℃ Stay overnight, then 1000×g centrifuge 20 Simply remove the supernatant for minutes, or place the supernatant in... -20℃ or -80℃ Store, but avoid repeated freezing and thawing. (Limited to in vivo related hemolytic diseases or in vitro destructive hemolytic samples, target produced only within red blood cells.) 4. Plasma: use EDTA Alternatively, heparin can be used as an anticoagulant to collect samples, and the samples can be collected after collection. 30 Within minutes 2-8℃ 1000×g centrifuge 15 The supernatant can be taken for testing within minutes, or the supernatant can be placed in... -20℃ or -80℃ Store, but avoid repeated freezing and thawing. (Limited to in vivo related hemolytic diseases or in vitro destructive hemolytic samples, target produced only within red blood cells.) 5. Erythrocyte lysate: Collect a volume of anticoagulant blood (e.g. 1mL ), at 4°C ， 10000rpm centrifuge 10min , collecting red blood cells. Discard the supernatant, add pre-cooled pure water and resuspend to the original volume (e.g. 1mL ), placed in -20°C freezing 30-60 Minutes, after 3 Repeated freezing and thawing damages cell membranes. The cells were then suspended in 4°C ， 10000rpm centrifuge 10 Minutes, take the supernatant, and the supernatant can be detected immediately; Or repackage, and place the repackaged sample in -20°C Store, but avoid repeated freezing and thawing. The thawed sample should be centrifuged again before testing. 6. Sample appearance: The sample should be clear and transparent, and suspended matter should be removed by centrifugation. 7. Sample preservation: After sample collection, if... 1 Tests conducted within the week can be stored in 4℃ If testing is not possible in time, please repackage according to the single-use amount and freeze it. -20℃ （ 1 Testing within a month), or -80℃ （ 6 (Tested within one month), avoid repeated freezing and thawing, as sample hemolysis will affect the final test results, so hemolyzed samples should not be tested. III. Sample dilution protocol: Please estimate the concentration range of your sample in advance. If your test sample needs to be diluted, the reference dilution scheme is as follows: dilution 100 Double: Dilute in one step. take 5μL Sample to 495μL In the general diluent, make 100 fold dilution; dilution 1000 Double: Two-step dilution. take 5μL Sample to 95μL In the general diluent, make 20 Dilute twice, then take 5μL20 Fold the diluted sample to 245μL In the general diluent, make 50 Fold dilution, total dilution 1000 times; dilution 100000 Double: Three-step dilution. take 5μL Sample to 195μL In the general diluent, make 40 Dilute twice, then take 5μL40 Fold the diluted sample to 245μL In the general diluent, make 50 Dilute twice, and finally take... 5μL 2000 Fold the diluted sample to 245μL In the general diluent, make 50 Fold dilution, total dilution 100000 times; The amount of liquid taken during each dilution step shall not be less than 3μL Dilution factor not exceeding 100 Double. Each dilution step should be mixed evenly to avoid foaming. IV. Pre-testing preparations: 1. Please advance 10 Remove the kit from the refrigerator and allow to equilibrate to room temperature. 2. Preparation of standard gradient working solution: Add 1mL Add the general diluent to the lyophilized standard and let stand. 15 After it has been completely dissolved for minutes, mix gently (concentration is 5000ng/mL ), and then at the following concentrations: 5000ng/mL 、 2500ng/mL 、 1250ng/mL 、 625ng/mL 、 312.5ng/mL 、 156.25ng/mL 、 78.12ng/mL 、 0ng/mL Dilute. Double dilution method: Take 7 branch EP Tubes, add to each tube 500μL Universal diluents, 5000ng/mL Absorb from the standard working solution 500μL To the first one EP Mix well in the tube 2500ng/mL The standard working solution is then sucked and mixed sequentially according to this step. The last tube is directly used as a blank hole, so there is no need to draw liquid from the penultimate tube, as shown in the figure below.  4. Preparation of enzyme conjugate working solution: Before use 15 minutes will 100× Concentrated enzyme conjugate 1000×g centrifuge 1 Minutes, with universal diluent 100× The concentrated enzyme conjugate was diluted into 1× working concentration ( Example: 10μL concentrate +990μL Universal diluent ) Ready to use. 5. 1× Washing liquid preparation: Take 10mL 20× washing liquid to 190mL In distilled water (the concentrated washing liquid taken out of the refrigerator may have crystals, which is a normal phenomenon. It can be placed at room temperature and prepared after the crystals have completely dissolved). ) 。 V. Operating Steps: 1. From room temperature equilibrium 10 After a few minutes, remove the required strips from the aluminum foil bag, and seal the remaining strips back in a self-sealing bag. 4℃ . 2. Sampling: Samples or standards of different concentrations were added according to 100μL Each well is added to the corresponding well, and the blank well is added. 100μL Universal diluent. After covering the sealing film 37℃ incubation 60 minutes. (Recommendation) Minimally dilute the sample to be tested with a general-purpose diluent. 1 After doubling, it was added to the enzyme-labeled plate for testing. Thereby reducing the influence of matrix effect on the test results. Finally, when calculating the sample concentration, the corresponding dilution factor needs to be multiplied. Duplicate wells are recommended for all samples and standards to be tested. 3. Add biotinylated antibody: remove the enzyme label plate, discard the liquid, and do not need to wash. Add biotinylated antibody working solution directly to each well 100μL After covering with a sealing film 37℃ incubation 60 minutes. 4. Washing plate: Discard the liquid and add it to each well. 300μL 1x Washing liquid, let stand 1 After 10 minutes, remove the detergent, pat dry on absorbent paper, and repeat the washing process. 3 (You can also wash the board using a board washing machine). 5. Add enzyme conjugate working solution: Add enzyme conjugate working solution to each well. 100μL After covering with a sealing film 37℃ incubation 30 minutes. 6. Washing plate: Discard the liquid according to the steps. 4 Washing method, washing plate 5 Next. 7. Add substrate: Add substrate to each well ( TMB ） 90μL Cover with a sealing film, 37℃ Incubation in light 15 minutes. 8 Add stop solution: Remove the enzyme-labeled plate and add stop solution directly to each well. 50μL Immediately 450nm Measure each hole at the wavelength OD Value. VI. Calculation of Experimental Result Judgment: 1. Calculate the average of standard and sample replicates OD Value and subtract the blank hole OD Value as the correction value. With concentration as the horizontal axis, OD Value is ordinate Draw the standard curve of the four-parameter logic function on double-logarithmic coordinate paper. 2. If the sample OD Values above the upper limit of the standard curve should be diluted appropriately and retested and multiplied by the appropriate dilution factor when calculating the sample concentration.  VII. Kit performance: 1. Repeatability: The coefficient of variation within the plate is less than 10% The coefficient of variation between plates is less than 10% 。 2. Recovery: Add to selected healthy mouse erythrocyte lysate 3 Mice at different concentration levels HbA1c Calculate the recovery rate.
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| Sensitivity | 38.96ng/mL | |||||||||||||||||||||||||||||||||
| Theory | This kit uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Into microwells previously coated with mouse glycated hemoglobin A1c (HbA1c) capture antibodies, a sample, a standard, a biotin-labeled detection antibody, and an HRP enzyme conjugate were sequentially added, followed by incubation and washing, and developed with a substrate TMB. TMB was converted into blue under the catalysis of peroxidase (HRP) and finally into yellow under the action of acid. The depth of color was positively correlated with the mouse glycated hemoglobin A1c (HbA1c) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the sample concentration was calculated. | |||||||||||||||||||||||||||||||||
| Source | Mouse | |||||||||||||||||||||||||||||||||
| Synonym | Mouse Glycated Hemoglobin A1c (HbA1c) ELISA Detection Kit | |||||||||||||||||||||||||||||||||
| Detection Type | double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Glycosylated hemoglobin (glycohemoglobin, hemoglobin A1c, HbA1c, less commonly HbA1c, HgbA1c, Hb1c, etc., and also informal A1c in patients) is a form of hemoglobin (Hb) chemically linked to glycochemistry. It is the product of the combination of hemoglobin and glucose in red blood cells. It is usually used as a reference standard for the average plasma glucose concentration over a period of time. Generally speaking, the proportion of hemoglobin glycosylated is directly proportional to the level of plasma glucose concentration over a period of time. The measurement of glycated hemoglobin is mainly used to determine the average blood glucose level over three months. It is used as a diagnostic test for diabetes and can also be used as an evaluation test for glycemic control in diabetic patients. | |||||||||||||||||||||||||||||||||
| General Notes | 1. Incubate strictly according to the specified time and temperature to ensure accurate results. All reagents must reach room temperature of 20-25℃ before use. Refrigerate and store reagents immediately after use. 2. Incorrect washing of the board may lead to inaccurate results. Ensure that the fluid in the wells is sucked dry as much as possible before adding the substrate. Do not allow the microwells to dry out during incubation. 3. Eliminate residual liquid and fingerprints from the bottom of the plate, otherwise it will affect the OD value. 4. The substrate developing solution should be colorless or very light in color. Substrate solutions that have turned blue should not be used. 5. Avoid cross-contamination of reagents and specimens to prevent erroneous results. 6. Avoid direct exposure to strong light during storage and incubation. 7. No reactant should come into contact with the bleaching solvent or the strong gases emitted by the bleaching solvent. Any bleaching ingredient will destroy the biological activity of the reaction reagents in the kit. 8. Expired products must not be used, and ingredients with different product numbers and batch numbers must not be mixed. 9. Recombinant proteins from sources other than the kit may not match the antibodies in this kit and may not be recognized. 10. If there is a possibility of disease transmission, all samples should be managed properly, and the samples and testing devices should be handled according to the prescribed procedures. |
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| Instructions | Serum, plasma and erythrocyte lysates | |||||||||||||||||||||||||||||||||
| Storage Temp. | Unopened kit, stored at 4°C with a shelf life of 6 months. | |||||||||||||||||||||||||||||||||
| Test Range | 78.12-5000ng/mL |