| Usage | Sample Processing and Requirements: Serum: Place whole blood specimens collected in serum separator tubes at room temperature for 2 hours or at 4°C overnight, then centrifuge at 1000×g for 20 minutes. Remove the supernatant, or store at -20°C or -80°C, but avoid repeated freezing and thawing. Plasma: Collect specimens using EDTA or heparin as an anticoagulant and centrifuge at 1000×g for 15 minutes at 2-8°C within 30 minutes of collection. Remove the supernatant for testing, or store at -20°C or -80°C, but avoid repeated freezing and thawing. Tissue Homogenate: Rinse tissue with pre-chilled PBS (0.01M, pH=7.4) to remove residual blood (lysed red blood cells in the homogenate will affect the measurement results). Weigh and mince the tissue. Mix the minced tissue with the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1g of tissue sample to 9mL of PBS. The specific volume can be adjusted according to experimental needs and recorded. It is recommended to add protease inhibitors to the PBS) in a glass homogenizer and grind thoroughly on ice. To further lyse the tissue cells, the homogenate can be sonicated or repeatedly freeze-thawed. Finally, centrifuge the homogenate at 5000×g for 5-10 minutes, and collect the supernatant for analysis.
Cell Lysis Buffer: Adherent cells are gently washed with ice-cold PBS, then trypsinized and harvested by centrifugation at 1000×g for 5 minutes. Suspension cells can be harvested directly by centrifugation. Wash the harvested cells three times with ice-cold PBS and resuspend in 150-200µL of PBS per 1×10^6 cells (it is recommended to add protease inhibitors to the PBS; if the cell count is very low, reduce the PBS volume appropriately). Disrupt the cells by repeated freeze-thaw cycles or sonication. Centrifuge the extract at 1500×g for 10 minutes at 2-8°C and remove the supernatant for testing.
Cell culture supernatant: Centrifuge at 1000×g for 20 minutes and remove the supernatant for testing, or store at -20°C or -80°C, but avoid repeated freezing and thawing.Other biological fluids: Centrifuge at 1000xg for 20 minutes, remove the supernatant, and test. Pre-test preparation: 1. Remove the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Preparation of standard gradient working solution: Add 1mL of universal diluent to the lyophilized standard, let it stand for 15 minutes to completely dissolve, then gently mix (concentration is 50ng/mL). Then dilute to the following concentrations: 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL, 3.125ng/mL, 1.5625ng/mL, 0.78125ng/mL, and 0ng/mL. Serial dilution method: Take 7 EP tubes and add 500μL of universal diluent to each tube. Pipette 500μL of the 50ng/mL standard working solution into the first EP tube and mix thoroughly to make a 25ng/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube directly serves as a blank well; there is no need to pipette liquid from the penultimate tube.
3. Preparation of Biotin-Antibody Working Solution: 15 minutes before use, centrifuge the concentrated Biotin-Antibody at 1000×g for 1 minute. Dilute the 100× concentrated Biotin-Antibody to a 1× working concentration with universal diluent (e.g., 10μL concentrate + 990μL universal diluent). Use the same day. 4. Preparation of Enzyme Conjugate Working Solution: 15 minutes before use, centrifuge the 100x concentrated enzyme conjugate at 1000×g for 1 minute. Dilute the 100x concentrated HRP enzyme conjugate to a 1x working concentration with universal diluent (e.g., 10μL concentrate + 990μL universal diluent). Use the same day. 5. Preparation of 1x Wash Solution: Dispense 10mL of 20x Wash Solution into 190mL of distilled water (Concentrated Wash Solution removed from the refrigerator may crystallize; this is normal. Allow to stand at room temperature until the crystals have completely dissolved before preparing). Procedure: 1. Remove the desired strips from the aluminum foil bag after equilibration at room temperature for 10 minutes. Seal the remaining strips in a ziplock bag and return to 4°C. 2. Sample Addition: Add 50μL of sample or standard of varying concentrations to the corresponding wells. Add 50μL of universal diluent to the blank wells. Then, add 50μL of Biotin-Antibody Working Solution to each well. Cover with a sealing film and incubate at 37°C for 1 hour. (Recommendation: Dilute the sample to be tested at least 1-fold with universal diluent before adding it to the ELISA plate for testing to reduce the influence of matrix effects on the test results. The final calculation of sample concentration should be multiplied by the corresponding dilution factor. It is recommended to set up duplicate wells for all samples to be tested and standards.) 3. Wash: Discard the liquid, add 300uL of 1x wash buffer to each well, let it stand for 1 minute, shake off the wash buffer, pat dry on absorbent paper, and repeat this process three times (a microplate washer can also be used for washing). 4. Add enzyme conjugate working solution: Add 100μL of enzyme conjugate working solution to each well, cover with a sealing film, and incubate at 37°C for 30 minutes. 5. Wash: Discard the liquid and wash the plate five times according to the washing method in step 3.
6. Add substrate: Add 90uL of substrate (TMB) to each well, cover with sealing film, and incubate at 37℃ in the dark for 15 minutes.
7. Add stop solution: Remove the ELISA plate and add 50uL of stop solution directly to each well. Immediately measure the OD value of each well at a wavelength of 450nm.
Calculation of experimental results:
Result evaluation:
1. Calculate the average OD value of the standard and sample replicates and subtract the OD value of the blank well as the correction value. With concentration as the horizontal axis and OD value as the vertical axis, draw a standard curve of the four-parameter logistic function on double logarithmic coordinate paper.
2. If the sample OD value is higher than the upper limit of the standard curve, it should be appropriately diluted and re-measured and the corresponding dilution factor should be multiplied when calculating the sample concentration. |
| Sensitivity | 0.35 ng/mL |
| Theory | This kit uses a competitive enzyme-linked immunosorbent assay (ELISA). Samples, standards, biotin-labeled antibodies, and HRP enzyme conjugates are sequentially added to microwells pre-coated with universal histamine (HIS) antigens. After incubation and washing, the cells are developed using the substrate TMB. TMB is converted to blue under the catalysis of peroxidase (HRP) and to the final yellow under the action of acid. The color depth is negatively correlated with the universal histamine (HIS) content in the sample. The absorbance (OD value) is measured at a wavelength of 450 nm using a microplate reader to calculate the sample concentration. |
| Source | All |
| Synonym | General Histamine ELISA Kit |
| Detection Type | Competition Law |
| Composition | | Name | 9 6 T Configuration | Remarks | | Pre-coated 96-well enzyme plate | 8 holes×12 strips | None | | Standard | 2 bottles | Dilution according to the instructions | Universal diluent | 2×20mL | None | Concentrated Biotin-Antibody (100×) | 60uL | Dilution according to the instructions | Concentrated enzyme conjugate (100×) | 120uL | Dilution according to the instructions | 20× Wash solution | 2×10mL | Dilute according to instructions | Substrate (TMB) | 10mL | None | Stop solution | 6mL | none | Sealing film | 4 sheets | None | Instructions | 1 serving | None |
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| Background | Histamine is an organic nitrogenous compound involved in local immune responses, regulating intestinal physiology, and acting as a neurotransmitter in the brain, spinal cord, and uterus. Since its discovery in 1910, histamine has been considered a local hormone (autologous) because it lacks a typical endocrine gland to secrete it. However, in recent years, histamine has been recognized as a central neurotransmitter. It participates in inflammatory responses and plays a central role as a mediator of itch. Histamine is produced by basophils and mast cells in nearby connective tissue as part of the immune response to foreign pathogens. It increases capillary permeability to white blood cells and some proteins, allowing them to come into contact with pathogens in infected tissues. It consists of an imidazole ring attached to an ethylamine chain. Under physiological conditions, the amino group in the side chain is protonated. |
| General Notes | 1. Strictly adhere to the specified incubation time and temperature to ensure accurate results. All reagents must be at room temperature (20-25°C) before use. Refrigerate reagents immediately after use.
2. Improper plate washing may lead to inaccurate results. Ensure that the wells are completely aspirated before adding substrate. Do not allow the wells to dry out during incubation.
3. Remove any residual liquid and fingerprints from the bottom of the plate, as this will affect the OD value.
4. The substrate developer solution should be colorless or very light in color. Do not use substrate solution that has turned blue.
5. Avoid cross-contamination of reagents and specimens to prevent erroneous results.
6. Avoid direct exposure to strong light during storage and incubation.
7. Do not expose any reagents to bleaching solvents or the strong fumes emitted by bleaching solvents. Any bleaching component will destroy the biological activity of the reagents in the kit.
8. Do not use expired products, and do not mix components from different product numbers and batches. 9. Recombinant proteins from sources other than the test 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 detection devices should be handled according to the prescribed procedures. |
| Storage Temp. | Store at 2-8°C. Valid for 6 months. |
| Test Range | 0.78-50 ng/mL |
| Applications | Serum, plasma, tissue homogenate, cell lysate, cell culture supernatant and other biological fluids |
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