| Usage | Specimen requirements:
1. Extract specimens as soon as possible after collection, and the extraction should be carried out according to relevant literature. Experiments should be carried out as soon as possible after extraction. If the test cannot be carried out immediately, the specimen can be stored at-20 ℃, but repeated freezing and thawing should be avoided
2. Samples containing NaN3 cannot be detected because NaN3 inhibits the activity of horseradish peroxidase (HRP).
Operation steps:
1. Dilution of standard product: This kit provides one original standard product. Users can dilute it in a small test tube according to the following chart.
| 8umol/L | Standard No. 5 | 150uL of original standard was added to 150uL of standard dilution | | 4umol/L | Standard No. 4 | 150uL of standard No. 5 was added to 150uL of standard dilution | | 2umol/L | Standard No.3 | 150uL of standard No. 4 was added to 150uL of standard dilution | | 1umol/L | Standard No.2 | 150uL of standard No. 3 was added to 150uL of standard dilution | | 0.5 umol/L | Standard No. 1 | 150uL of Standard No. 2 was added to 150uL of Standard Diluent |
2. Sample addition: Set up blank wells (the blank control wells do not add samples and enzyme-labeled reagents, and the other steps are the same), standard wells, and sample wells to be tested respectively. Accurately add 50uL of the standard on the enzyme-labeled coated plate, first add 40uL of sample diluent to the sample well to be tested, and then add 10uL of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the enzyme labeled plate, try not to touch the well wall, and gently shake and mix well.
3. Incubation: Incubate at 37 °C for 30 minutes after sealing with a plate sealing film.
4. Liquid preparation: 30 times concentrated washing solution is diluted 30 times with distilled water and used for later use
5. Washing: Carefully remove the sealing film, discard the liquid, spin dry, fill each hole with washing liquid, let it stand for 30 seconds and then discard it. Repeat this 5 times and pat dry.
6. Enzyme addition: 50uL of enzyme-labeled reagent is added to each well, except for blank wells.
7. Incubation: The procedure is the same as 3.
8. Washing: The operation is the same as 5.
9. Color development: First add 50uL of color developer A to each well, then add 50uL of color developer B, gently shake and mix well, and develop color at 37 °C in the dark for 10 minutes.
10. Termination: Add 50uL of stop solution to each well to terminate the reaction (blue immediately turns yellow at this time).
11. Measurement: Zero the blank well, and measure the absorbance (OD value) of each well sequentially at a wavelength of 450nm. The determination should be performed within 15 minutes after the addition of the stop solution.
Calculation:
Taking the concentration of the standard substance as the abscissa and the OD value as the ordinate, draw a standard curve on the coordinate paper, and find out the corresponding concentration from the standard curve according to the OD value of the sample; Multiply by the dilution factor; Or use the concentration of the standard substance and the OD value to calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and then multiply it by the dilution factor, which is the actual concentration of the sample. |
| Species Reactivity | Human |
| Theory | This kit uses double antibody sandwich method to determine the level of human homocysteine in specimens. Coating the microplate with purified human homocysteine antibody to make solid phase antibody, adding homocysteine sequentially to the microwells coated with monoclonal antibody, and then combining with HRP-labeled homocysteine antibody to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB is added to develop color. TMB is converted to blue under the catalysis of HRP enzyme and to the final yellow under the action of acid. There was a positive correlation between the depth of color and the homocysteine in the sample. The absorbance (OD value) was measured with a microplate reader at 450nm wavelength, and the concentration of human homocysteine in the sample was calculated by the standard curve. |
| Synonym | Hcy ELISA Kit |
| Composition | Kit composition:
| Components | Specifications | | 30 times concentrated wash | 20mL × 1 vial | | Enzyme labeled reagent | 6mL × 1 vial | | Enzyme-labeled coated plate | 12 holes × 8 strips | | Sample dilution | 6mL × 1 vial | | Developer A liquid | 6mL × 1 vial | | Developer B liquid | 6mL × 1 vial | | Stop liquid | 6mL × 1 vial | | Standard (16umol/L) | 0.5 mL × 1 vial | | Standard dilution | 1.5 mL × 1 vial | | Sealing film | 2 sheets | | Sealed bag | 1 |
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| General Notes | 1. The kit should be taken out of the refrigerated environment and balanced at room temperature for 1 hour before it can be used. If the enzyme-labeled coated plate is not used up after opening, the strips should be stored in a sealed bag.
2. Crystals may precipitate in the concentrated washing liquid. When diluting, it can be heated in a water bath to help dissolve, and the results will not be affected during washing.
3. A sampler should be used in each step of sampling, and its accuracy should be checked frequently to avoid test errors. It is best to control the sample addition time within 5 minutes. If the number of specimens is large, it is recommended to use a row gun to add samples.
4. Please make a standard curve at the same time of each measurement, preferably a double 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 it with the sample diluent for a certain multiple (n times) before measuring, and please finally multiply it by the total dilution multiple (× n × 5) when calculating.
5. The sealing film is only for one-time use to avoid cross-contamination.
6. Please keep the substrate away from light.
7. Strictly follow the instructions, and the test results must be determined based on the reading of the microplate reader.
8. All samples, washing solutions and various wastes should be treated as infectious agents.
9. Components of different batch numbers of this reagent shall not be mixed. |
| Storage Temp. | Store at 2-8 °C, shelf life 6 months. |
| Test Range | 0.2umol/L-8umol/L |
| Applications | It is used to determine homocysteine content in human serum, plasma and related fluid samples. |
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