Hemin Assay Kit

This reagent kit is used to determine the heme content in samples.Detection range:96Ttwo point five μ G/ml -85 μ G/mlExperimental principle:This reagent kit uses the double antibody sandwich method to determine the level of heme in the sample. Purified heme antibodies are coated on microplates to produce solid-phase antibodies. Hemae is sequentially added to the microplates of the coated monoclonal antibody, and then bound to HRP labeled heme antibodies to form an antibody antigen enzyme antibody complex. After thorough washing, TMB substrate is added for color development. TMB is converted to blue under the catalysis of HRP enzyme, and finally to yellow under the action of acid. The depth of color is positively correlated with the heme in the sample. Measure the absorbance (OD value) at a wavelength of 450nm using an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the concentration of heme in the sample through a standard curve.Kit composition:130倍浓缩洗涤液20ml×1瓶7终止液6ml×1瓶2酶标试剂6ml×1瓶8标准品（160μg/ml）0.5ml×1瓶3酶标包被板12孔×8条9标准品稀释液1.5ml×1瓶4样品稀释液6ml×1瓶10说明书1份5显色剂A液6ml×1瓶11封板膜2张6显色剂B液6ml×1/瓶12密封袋1个Specimen requirements:1. Extract the specimens as soon as possible after collection, following relevant literature, and conduct experiments as soon as possible after extraction. If the experiment cannot be conducted immediately, the specimen can be stored at -20 ℃, but repeated freeze-thaw should be avoided2. Samples containing NaN3 cannot be detected as NaN3 inhibits the activity of horseradish peroxidase (HRP).Operation steps:1. Dilution of standard substance: This reagent kit provides one original standard substance, and users can dilute it in a small test tube according to the following chart.80μg/ml5号标准品150μl的原倍标准品加入150μl标准品稀释液40μg/ml4号标准品150μl的5号标准品加入150μl标准品稀释液20μg/ml3号标准品150μl的4号标准品加入150μl标准品稀释液10μg/ml2号标准品150μl的3号标准品加入150μl标准品稀释液5μg/ml1号标准品150μl的2号标准品加入150μl标准品稀释液2. Sample addition: Set up blank wells (blank control wells do not include samples and enzyme-linked immunosorbent assay reagents, the rest of the steps are the same), standard wells, and sample wells for testing. Accurately add 50 samples of standard sample on the enzyme-linked immunosorbent assay (ELISA) coated plate μ l. Add sample diluent 40 to the test sample well first μ l. Then add 10 more samples to be tested μ L (The final dilution of the sample is 5 times). Add the sample to the bottom of the enzyme-linked immunosorbent assay (ELISA) plate well, avoiding touching the well wall as much as possible. Gently shake and mix well.3. Warm incubation: Seal the plate with a sealing film and incubate at 37 ℃ for 30 minutes.4. Solution preparation: Dilute 30 times the concentrated washing solution with distilled water and set aside for later use5. Washing: Carefully remove the sealing film, discard the liquid, shake dry, fill each well with washing solution, let it stand for 30 seconds, then discard. Repeat this process 5 times and pat dry.6. Enzyme addition: Add 50 enzyme labeled reagents to each well μ l. Excluding blank holes.7. Warm education: The operation is the same as 3.8. Washing: The operation is the same as 5.9. Color development: Add color development agent A50 to each well first μ l. Add color developer B50 again μ l. Gently shake and mix well, and develop color in the dark at 37 ℃ for 10 minutes10. Termination: Add 50% termination fluid to each hole μ l. Terminate the reaction (at this point, the blue color immediately turns yellow).Measurement: Measure the absorbance (OD value) of each well in sequence with a blank air conditioner at a wavelength of 450nm. The measurement should be conducted within 15 minutes after adding the termination solution.Summary of operating procedures:calculateDraw a standard curve on a coordinate paper with the concentration of the standard substance as the x-axis and the OD value as the y-axis. Based on the OD value of the sample, determine the corresponding concentration from the standard curve; Multiply it by the dilution factor; Alternatively, a linear regression equation can be used to calculate the standard curve using the concentration and OD value of the standard substance. The OD value of the sample can be substituted into the equation to calculate the sample concentration, which is then multiplied by the dilution factor to obtain the actual concentration of the sample.Notes:1. The kit should be balanced at room temperature for 15-30 minutes before use when taken out from the cold storage environment. If the enzyme coated plate is not used up after opening, the Flat noodles should be stored in a sealed bag.2. Concentrated detergent may precipitate crystals. When diluted, it can be heated in a water bath to aid in dissolution. Washing does not affect the results.3. A sampler should be used for each step of sample addition, and its accuracy should be regularly calibrated to avoid experimental errors. It is best to control the sample addition time within 5 minutes. If there are a large number of specimens, it is recommended to use a firing gun for sample addition.4. Please make a standard curve at the same time as each measurement, preferably with a re hole. If the content of the substance to be tested in the sample 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 diluent by a certain multiple (n times) before measurement. When calculating, please multiply the total dilution multiple by the final factor（ × N × 5) .5. The sealing film is only for one-time use to avoid cross contamination.6. Please store the substrate in dark.7. Strictly follow the instructions and determine the test results based on the reading of the enzyme-linked immunosorbent assay (ELISA) reader8. All samples, washing liquids, and various waste should be treated as infectious substances.9. The components of this reagent with different batch numbers shall not be mixed.Storage conditions and validity period:1. Kit storage:; 2-8 ℃.2. Validity period: 6 months. Specification: 96T Related Documents: https://ald-pub-files.oss-cn-shanghai.aliyuncs.com/aladdinsci/pdp/sds/1/H486178-SCI_1dae97059a067ec1821a35a1e2a47df0.pdf