Human interleukin 6 (IL-6) quantitative detection kit (ELISA) instruction manual

Example: Human Interleukin-6 (IL-6) Quantitative Assay Kit (ELISA)

【product name】
Generic Name: Human Interleukin 6 (IL-6) Quantitative Assay Kit (ELISA)
English name: Huamn Interleukin-6 (IL-6) ELISA KIT

[Package Specifications]
48 servings/box, 96 servings/box

【expected usage】
For scientific research only, quantitatively measure the concentration of human interleukin-6 (IL-6) in serum, plasma and cell culture supernatants.

[Test principle]
The kit uses a double antibody sandwich enzyme-linked immunosorbent assay (ELISA). In the microporous plate pre-coated with anti-human interleukin-6 (IL-6) antibody (solid phase antibody), add human interleukin 6 (IL-6) calibrator and sample to be tested, and then add another An HRP-labeled anti-human interleukin-6 (IL-6) antibody (enzyme-labeled antibody) is incubated and washed thoroughly to remove unbound components and form a solid phase antibody-antigen on the surface of the microplate solid phase. - a sandwich complex of enzyme-labeled antibodies. Substrate A and B, the substrate is catalyzed by HRP to produce a blue product, which is finally converted to yellow under the action of a stop solution (2M sulfuric acid). The absorbance (OD value) and absorbance (OD value) are measured on a microplate reader. ) is positively correlated with the concentration of human interleukin 6 (IL-6) in the sample to be tested. By fitting the calibrator curve, the concentration of human interleukin-6 (IL-6) in the sample can be calculated.

[main components]
Main component quantity Quantity Main component calibrator 0.5 ml/tube*6 tube Six standard preparations for antigen preparation coated microplate 96T pre-coated solid phase antibody
HRP labeled antibody 6 mL HRP labeled detection antibody substrate A 6 mL carbamide peroxide working solution substrate B 6 mL TMB working solution
20× concentrated washing solution 30 mL PBS containing 0.15% Tween20
Instructions 1 copy --
Ziplock bag 1 --
Sticker 2 pieces --
The concentration of the calibrator is: 320, 160, 80, 40, 20, 0 pg/ml. The calibrator has been tested and the results indicate that the HBs antigen is negative and HIV1, HIV2 and HCV antibodies are negative. Since there is no test method that can completely guarantee the absence of these substances, the product must be treated according to the potential infectivity, and the treatment should be followed. General security measures.

Materials and consumables that are required but not provided
1, microplate reader
2, precision pipette and disposable tips
3, distilled water
4, washing bottles or automatic washing machine
5, 37 ° C water bath or incubator
6, 500 ml measuring cylinder
7, powder-free disposable latex gloves
8, quality control products

[Storage conditions and expiration date]
1, 2-8 ° C preservation, do not freeze, valid for 6 months.
2. After opening the package, the coated microplate is placed in a ziplock bag with a desiccant, the ziplock bag is sealed, and all reagents are returned to the 2-8 °C refrigerator.
3. After opening, preserve according to the recommended conditions, calibrator, coated microplate and HRP labeled antibody, valid for 14 days, other components are stable within the validity period indicated by the label.

[Applicable instrument]
Semi-automatic microplate reader, such as Thermo MK3, or domestic microplate reader.

[sample requirements]
Sample Types and Acquisitions The following are just general guidelines for sample collection. Sodium azide should not be used as a preservative during all sample collection.
1. Cell culture supernatant: Centrifuge for 20 min at 4000 rpm to remove cell pellets and polymer. The supernatant is stored at - 20 °C to avoid repeated freezing and thawing.
2. Serum: Use a tube containing no pyrogen and endotoxin. Avoid any cell stimulation during the operation. Centrifuge at 4000 rpm for 20 min. Separate the serum carefully and store at -20 °C to avoid repeated freezing and thawing.
3. Plasma: Heparin, EDTA, or sodium citrate as an anticoagulant. The supernatant was centrifuged at 4000 rpm for 20 minutes, and the plasma was stored at -20 ° C or less to avoid repeated freezing and thawing.

Sample storage and stability samples can be stored at 2-8 ° C for 72 h or at - 20 ° C for 6 months. After the sample is collected, it is not once tested. Please freeze it by using the dosage once, avoid repeated freezing and thawing, and thaw at room temperature during use to ensure that the sample is fully thawed.

【Testing method】
Reagent preparation
1. All components should be rewarmed for at least 30 minutes before use to ensure adequate rewarming to room temperature.
2. Concentrated washing solution: The concentrated washing liquid taken out from the refrigerator will be crystallized, which is a normal phenomenon, and the water bath is heated to completely dissolve the crystal. Concentrate the washing solution and distilled water, dilute 1:20, that is, 1 part of the concentrated washing solution, and add 19 parts of distilled water.
3. Substrate: Substrate liquids A and B, thoroughly mixed in a 1:1 volume before use, and used within 15 minutes after mixing.

Operating procedure
1. All reagents and components are first restored to room temperature, standard products, quality control products and samples. It is recommended to make double holes.
2. Prepare the working fluids of the various components of the kit according to the method described in the previous reagent preparation.
3. Remove the required slats from the aluminum foil bag and seal the remaining slats back to the refrigerator with a ziplock bag.
4. Set the standard product hole, sample hole and blank hole. Add 50μL standard sample to each standard hole, add 50μL sample hole to the sample, and add blank hole.
5. In addition to the blank wells, standard wells and sample wells were added with horseradish peroxidase-labeled detection antibody 100 μL.
6. Cover the reaction plate with a sealing film and incubate for 60 min at 37 °C in a water bath or incubator.
7. Uncover the sealing film, discard the liquid, pat dry on the absorbent paper, fill each well with the washing liquid, let stand for 20s, remove the washing liquid, and pat dry on the absorbent paper.
8. Repeat this 4 times (to wash the plate 5 times). If you use the automatic washer, please wash the board according to the washing machine operation program, add the program soaked for 20s, can improve the detection accuracy. At the end of the washing process, before adding the substrate, fully dry the reaction plate on the clean and non-chip paper.
9. Mix the substrates A and B in a 1:1 volume and add 100 μL of the substrate mixture to all wells. The reaction plate was covered with a sealing film and incubated for 15 min in a 37 ° C water bath or incubator.
10. Add 50 μL of stop solution to all wells and read the absorbance (OD value) of each well on the microplate reader.

[Explanation of test results]
After the test is completed, the standard concentration is used as the ordinate, and the corresponding absorbance (OD value) is taken as the abscissa. Using the computer software, a four-parameter Logistic curve fitting (4-pl) is used to create a standard curve equation, and the absorbance of the sample is passed. (OD value), using the equation to calculate the concentration value of the sample.
If the sample is diluted, the concentration value measured by the above method is multiplied by the dilution factor to determine the final concentration of the sample.

[Limitations of inspection methods]
1. It can only be used for scientific research and should not be used for clinical diagnosis.
2. It should be used within the validity period marked by the kit, and expired products should not be used.
3, can not be mixed with other manufacturers' kits or components.
4. Use the sample dilution solution supplied with the kit.
5. If the sample value is higher than the highest standard concentration value, please dilute the sample properly and then re-measure.
6. Human anti-mouse and other heterologous antibodies present in the sample to be tested may interfere with the test results. Please exclude this factor before testing.
7. The test results obtained by other methods are not directly comparable with the test results of the kit.

[product performance indicators]
1. Physical properties The liquid components of the kit should be clear, free of precipitates or flocs. The microplate aluminum foil bag should be vacuum packed without damage and leakage.
2. Dose response curve Linear calibrator dose response curve correlation coefficient r value, greater than or equal to 0.9900.
3. Precision intra-assay precision: Three sets of known high, medium and low concentration samples were evaluated in the same plate for 20 times. The intra-assay coefficient of variation CV% is less than 10%.
Inter-assay precision: Three sets of known high, medium and low concentration samples were evaluated for accuracy within 20 different plates. The inter-assay coefficient of variation CV% is less than 15%.
4. The minimum sensitivity of the detected dose is less than 0.7pg/mL.
5. Recovery rate Three sets of known high, medium and low concentration samples were evaluated for five recovery rates with recoveries ranging from 85% to 115%.
6. Specificity This kit recognizes native and recombinant human interleukin 6 (IL-6) and does not cross the structural analog.
7. Stability 2 °C-8 °C preservation, valid for 6 months.

【Precautions】
Biosecurity
1. The test must comply with the regulations of the laboratory management regulations and strictly prevent cross-contamination. All samples, washing liquids and various wastes should be disposed of according to the infectious materials.
2. The liquid component of the kit contains proclin-300 preservative, which may cause skin allergic reactions and avoid inhalation of fumes and skin contact.
3, the substrate liquid has a stimulating effect on the skin, eyes and upper respiratory tract, to avoid inhaling smoke. Put on protective gloves and wash your hands thoroughly after the experiment is completed.

Technical tips
1. Avoid foaming when mixing protein solutions.
2. When adding calibrators and samples, the pipette tip should be replaced for each calibrator concentration and sample. The common components should be cantilevered to avoid cross-contamination.
3. Appropriate incubation time and sufficient washing steps are necessary conditions to ensure the accuracy of the experimental results.
4. The substrate solution is a colorless liquid, which turns blue during storage, indicating that the substrate solution has failed and should not be used.
5. The order of loading the stop liquid is the same as the order of loading the substrate solution. After the stop solution is added, the blue substrate product will turn yellow instantly.
6. In the experiment, the remaining slats should be immediately put back into the ziplock bag and sealed (low temperature dry) for storage.
7. All liquid components should be shaken well before use, and the incubation operation should be carried out in strict accordance with the time indicated in the manual, the amount of sample added and the order of loading.

Waste disposal of all used or unused reagents, all contaminating disposable materials should follow the handling procedures for infectious or potentially infectious products, and each laboratory is responsible for carrying out waste according to the type and level of risk of the experiment. And the disposal of dirt, while treating all waste and dirt in strict accordance with relevant regulations.


Boao Joint Medical Experiment Center <br>Operating Headquarters: 1302, No. 833, Hongmei South Road, Minhang District, Shanghai
Experimental Center: Joint Experiment of Boao Joint Medical Experiment Center, No. 85, Shashan Road, Jiangyin City, Wuxi City:
Reagent supplies:
Platform website:
Sales mailbox:
After-sales technology:
Toll-free number: 400-775-1808
Switchboard number: 86-021-54800626
Fax number: 86-021-54800626 ext 8006

Tips: Not for clinical treatment.