Product number: YP-W11077

Specification:96T

Detection range: 50 μg/mL – 1600 μg/mL.

Sensitivity: Minimum detected dose is less than 10 μg/mL.

Precision: In-batch variation coefficient CV% is less than 10%; interbatch variation coefficient CV% is less than 15%.

Recovery rate: The recovery rate is between 85% and 115%.

Specificity: This kit recognizes natural and recombinant HumanImmunoglobulin M（IgM） without crossing with structural analogs. Stability: Storage at 2℃-8℃, valid for 6 months.

Usage: Used to detect the concentration of  HumanImmunoglobulin M（IgM） in samples such as serum, plasma, cell culture supernatant and tissue.

Experimental principle

This kit uses double antibody sandwich enzyme-linked immunosorbent assay (ELISA). To the microporous enzyme plate precoated with anti-HumanImmunoglobulin M（IgM） antibody (solid phase antibody), HumanImmunoglobulin M（IgM） calibrator and sample to be tested were added, and then HRP-labeled anti-HumanImmunoglobulin M（IgM） antibody (enzyme-labeled antibody) was added. After incubation and thorough washing, the unbound components were removed, and a sandwich complex of solid-phase antibody-antigen-enzyme-labeled antibody was formed on the solid phase surface of the microplate. Substrates A and B are added, and the substrate is catalyzed by HRP, and the substrate is produced, which is finally converted to yellow under the action of the stop solution (acid solution). The absorbance (OD value) was measured at a wavelength of 450 nm on the microplate reader, and the absorbance (OD value) was positively correlated with the concentration of Immunoglobulin M（IgM） in the sample to be tested. Fitting the calibrator curve, the concentration of Immunoglobulin M（IgM） in the sample can be calculated.

Kit components and storage Unopened kits are kept at 2-8 degrees and expired kits must not be used.

The concentrations of calibrators are:  1600、800、400、200、100、50 μg/mL  in turn.

Note:

1: Before use, please check whether the label and quantity of the reagent in the kit are consistent with the table.

2: If the components of the kit need to be used again, please make sure that they are not contaminated after the last use. 3: The enzyme label plate has not been used for a single time. Remember to seal it and store it at 2-8℃.

Self-provided testing equipment required for testing

 (not provided, but can be assisted in purchasing)

1) Microplate reader that can detect absorbance of 450 nm

2) Pipet and gun tip, sample filling tank

3) 37℃ constant temperature box or water bath pot

4) Test tubes, centrifuge tubes, measuring cylinders, etc. for preparing reagents

5) Distilled water or deionized water

6) Vortex oscillator, microplate oscillator

Notes

1) It is for scientific research only and shall not be used for clinical diagnosis.
2) Used within the validity period marked by the kit, expired products shall not be used.

3) Do not mix with other manufacturers' kits or components, use the sample diluent that is equipped with the kit.

4) If the sample value is higher than the highest standard concentration value, please dilute the sample appropriately before re-measurement.

5) The heterophilic antibodies such as human anti-mouse in the sample to be tested will interfere with the detection results. Please excrete this factor before testing.

6) The test results obtained by other methods are not directly comparable to the test results of this kit.

7) Please wear experimental clothes and latex gloves to protect yourself during the test. Especially when testing blood or other body fluid samples, please follow the National Biological Laboratory Safety Protection Regulations.

8) Incubate strictly in accordance with the prescribed time and temperature to ensure accurate results. All reagents must reach room temperature 20-25°C before use. Refrigerate immediately after use.

9) Incorrect washing of the board can lead to inaccurate results. Make sure to suck the liquid in the pores as much as possible before adding the substrate. Do not let the micropores dry out during incubation.

10) Eliminate residual liquid and finger prints on the bottom of the plate, otherwise it will affect the OD value.

11) The substrate color-producing liquid should be colorless or very light.

12) Avoid cross-contamination of reagents and specimens to avoid causing erroneous results.

13) Avoid direct exposure of strong light during storage and incubation.

14) The microplate reader used for detection needs to be installed with a filter that can detect wavelengths of 450±10nm, with an optical density range between 0-3.5. It is recommended to warm up 15 minutes in advance when using it.

15) The EP tube and suction head used in the test are both used once and are strictly prohibited from being mixed.

Sample preparation and preservation

            The following are just general guidelines for sample collection and preservation. During the collection and storage of all samples, sodium azide must not be used as a preservative. If the sample is not analyzed immediately, it should be aliquoted and frozen and avoid repeated freeze-thawing.

Cell culture supernatant - centrifuge to remove the precipitate, analyze immediately or aliquot it and store it frozen at -20°C.

Serum - Collect blood with a clean test tube, coagulate at room temperature for 30 minutes, centrifuge 2000×g for 20 minutes, and collect serum. Immediately analyze or aliquot it and store it frozen at -20°C.

Plasma - Use heparin, citrate or EDTA to anticoagulate, and centrifuge at 2-8°C for 2000×g for 20 minutes within 30 minutes after blood drawing. To eliminate the effects of platelets, it is recommended to further centrifuge at 2-8°C for 10,000×g for 10 minutes. Immediately analyze or aliquot it and cryopreserve at -20°C.

Cell Lysate - For adherent cells, remove the culture medium and wash it with PBS, saline or serum-free culture medium. Add an appropriate amount of lysate and blow it with a gun for a few times to make the lysate and the cells fully contact. Usually after 10 seconds, the cells are lysed. For suspended cells, collect the cells by centrifugation and wash them with PBS, saline or serum-free culture medium. Add an appropriate amount of lysate, blow the cells with a gun, and flick them with your fingers to fully lyse the cells. After sufficient cleavage, centrifuge at 10000-14000×g for 3-5 minutes and remove the supernatant. Immediately analyze or aliquot it and cryopreserve at -20°C.

Tissue homogenate - Rinse the tissue with pre-cooled PBS (0.01M, pH=7.4) to remove residual blood (the lysed red blood cells in the homogenate will affect the measurement results), and cut the tissue after weighing. The sheared tissue and the corresponding volume of PBS (usually at a weight-volume ratio of 1:9, for example, 1g of tissue sample corresponds to 9mL of PBS. The specific volume can be adjusted appropriately according to experimental needs and recorded. It is recommended to add protease inhibitors to PBS) to a glass homogenizer and grind them thoroughly on ice. To further lyse tissue cells, the homogenate can be sonicated or repeatedly freeze-thawed. Finally, the homogenate was centrifuged at 5000×g for 5~10 minutes, and the supernatant was taken for testing.

Urine – collect with a sterile tube and centrifuge at 2000×g for 20 minutes. Collect the supernatant carefully. If precipitation forms, centrifuge again.

Reagent preparation

1. Before use, all components must be re-warmed for at least 60 minutes to ensure full re-warming to room temperature.
2. Concentrated washing liquid: The concentrated washing liquid taken out of the refrigerator will produce crystallization, which is a normal phenomenon. The crystallization is completely dissolved by heating in a water bath. The concentrated washing liquid and distilled water are diluted at 1:20, that is, 1 part of the concentrated washing liquid, and 19 parts of the distilled water are added. 3. Substrate: substrate liquids A and B, mix thoroughly at 1:1 volume before use, and use within 15 minutes after mixing.

Operating procedures:

All reagents and components are first restored to room temperature, and are recommended to make compound holes.

1. Prepare the working liquid of various components of the kit according to the method described in the previous instructions.

2. Remove the required slats from the aluminum foil bag, and seal the remaining slats with a self-sealing bag and put them back into the refrigerator.

3. Set up standard product wells, 0-value wells, blank wells and sample wells. Add 50μL of standard products of different concentrations to each standard product wells, add 50μL of sample dilution for 0-value wells, and do not add blank wells, add 50μL of sample to be tested.

4. In addition to the blank wells, standard wells, 0 value wells and sample wells, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody.

5. Cover the reaction plate with a sealing film, incubate at 37℃ water bath or constant temperature box away from light for 60 minutes.

6. Remove the sealing membrane, discard the liquid, pat dry on the absorbent paper, fill each hole with washing liquid, let stand for 20S, shake off the washing liquid, pat dry on the absorbent paper, and repeat this 5 times. If you use an automatic plate washing machine, please follow the plate washing machine operating procedure and add a program of soaking for 30 seconds to improve the detection accuracy. After washing the plate, pat the reaction plate thoroughly on clean paper that does not remove shaved content before adding substrate. (Tip: To obtain ideal experimental results, the residual liquid must be completely removed. After washing the plate, please perform the next step immediately and do not let the microplate dry.) 7. Mix substrate A and B well in a 1:1 volume, and add 100μL of substrate mixture to all wells. Cover the reaction plate with a sealing film, incubate at 37°C water bath or a constant temperature box for 15 minutes away from light.

8. Add 50μL of the stop solution to all wells and read the absorbance (OD value) of each well on a 450nm wavelength microplate reader.

Result calculation

1.        Use the concentration of the standard substance as the abscissa and the corresponding absorbance (OD value) as the ordinate. Use computer software and four-parameter Logistic curve fitting (4-pl) to create a standard curve equation. Through the absorbance (OD value) of the sample value), use the equation to calculate the concentration value of the sample. [Calculate using ELISA Calc software. It is recommended to use four-parameter fitting for the standard curve, but it is not the only fitting method]

2.        If the sample is diluted, the concentration value measured by the above method must be multiplied by the dilution factor to determine the final value of the sample. concentration. Note: Experimenters need to establish a standard curve based on their own experiments. For each test, a standard curve must be established for each enzyme plate.