Manual of human anti-platelet antibody IgM (PA-IgM) ELISA kit

Manual of human anti-platelet antibody IgM (PA-IgM) ELISA kit

This kit is for research use only

Product Numbers: CSB-E05177h

Minimum detection limit: 1 ng / ml

Specificity: This kit can detect human PA-IgM, and does not cross-react with other related antibodies.

Validity: 6 months

Intended application: ELISA method for semi-quantitative determination of PA-IgM content in human serum, plasma or other related biological fluids.

Explanation

1. Kit storage: -20 ℃ (when not in use for a long time); 2-8 ℃ (when used frequently).

2. The concentrated washing liquid will be salted out at low temperature, and it can be heated and dissolved in the water bath when diluted.

3. There may be inconsistencies between the Chinese and English instructions. Please refer to the English instructions.

4. The well of the ELISA plate just opened may contain a little water-like substance. This is normal and will not have any impact on the experimental results.

Experimental principle

The enzyme-labeled plate is coated with purified platelet lysis antigen to make a solid-phase carrier. The sample to be tested is added to the microwells for reaction, and then horseradish peroxidase-labeled anti-human IgM is added for reaction. After thorough washing, the color is developed with the substrate TMB. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The color depth is positively correlated with PA-IgM in the sample. Measure the absorbance (OD value) at 450nm wavelength with a microplate reader, and calculate the titer of the antibody in the sample (the highest dilution factor that the antiserum can eventually develop as the titer).

Kit composition and reagent preparation

1. Assay plate: one piece (96 wells).

2. Sample Diluent: 1 × 20ml / bottle.

3. Horseradish peroxidase labeled anti-human IgM Diluent (HRP-anti-human IgM Diluent): 1 × 10ml / bottle.

4. Horseradish peroxidase labeled anti-human IgM (HRP-anti-human IgM): 1 × 120μl / vial (1: 100).

5. Substrate solution (TMB Substrate): 1 × 10ml / bottle.

6. Wash Buffer: 1 × 20ml / bottle, each bottle is diluted 25 times with distilled water.

7. Stop Solution (Stop Solution): 1 × 10ml / bottle (2N H2SO4).

Reagents and equipment needed but not provided

1. Standard Specification Microplate Reader

2. High-speed centrifuge

3. Electric heating thermostat incubator

4. Clean test tubes and Eppendof tubes

5. Series adjustable pipettes and tips. When testing more samples at one time, it is best to use multi-channel pipettes

6. Distilled water, volumetric flask, etc.

Collection and preservation of specimens

1. Serum: Whole blood samples should be left at room temperature for 2 hours or overnight at room temperature and centrifuged at 1000 xg for 20 minutes. Take the supernatant for detection, or store the samples at -20 ℃ or -80 ℃, but avoid repeated freezing. melt.

2. Plasma: EDTA or heparin can be used as anticoagulant. Centrifuge the sample at 2-8 ° C 1000 xg for 15 minutes within 30 minutes after collection, or store the specimen at -20 ° C or -80 ° C, but avoid repeated freezing melt.

3. Cell culture supernatant or other biological specimens: centrifuge at 1000 xg for 20 minutes, take the supernatant for detection, or store the specimen at -20 ℃ or -80 ℃, but avoid repeated freezing and thawing.

Note: Hemolysis of specimens will affect the final test results, so hemolysis specimens should not be tested.

Dilution principle of specimens:

First of all, we should know the approximate content of the sample to be tested through literature search, and determine the appropriate dilution factor. Detailed records should be made during the dilution process. When the antibody titer was finally calculated, it was diluted "N" times, and the antibody titer in the specimen was "N".

Dilution principle of horseradish peroxidase labeled anti-human IgM:

Before use, dilute with horseradish peroxidase-labeled anti-human IgM dilution. Before dilution, prepare according to the pre-calculated total amount required for each experiment (100μl per well). 0.1-0.2ml should be prepared during actual preparation . For example, 10 μl horseradish peroxidase-labeled anti-human IgM is added in a ratio of 990 μl horseradish peroxidase-labeled anti-human IgM dilution, mixed gently, and prepared within one hour before use.

Steps

Before starting the experiment, please configure all reagents in advance. When the reagents or samples are diluted, they should be mixed well. Try to avoid foaming when mixing. If the sample concentration is too high, dilute with sample diluent to make the sample meet the detection range of the kit.

1. Add sample: set blank hole and sample hole to be tested respectively. Add 100μl of sample diluent to the blank well and 100μl of the sample to be tested in the remaining well. Be careful not to have air bubbles. Add the sample to the bottom of the well of the microtiter plate. Do not touch the wall of the well as much as possible. Cover or cover the film and react at 37 ° C for 120 minutes.

2. Discard the liquid and spin dry without washing. Add 100μl of anti-human IgM working solution labeled with horseradish peroxidase to each well (the ratio of 1μl biotin-labeled antibody plus 99μl biotin-labeled antibody dilution is prepared, mix gently, and prepare within one hour before use), 37 ℃, 60 minutes.

3. After incubating for 60 minutes, discard the liquid in the wells, spin dry, wash the plate 5 times, soak for 1-2 minutes each time, 350μl / per well, spin dry.

4. Add 90μl of substrate solution to each well in sequence, and develop color at 37 ℃ in the dark.

5. Add 50μl of stop solution to each well in sequence to stop the reaction (at this time, the blue color turns to yellow). The order of adding the stop solution should be the same as that of the substrate solution. In order to ensure the accuracy of the experimental results, the termination solution should be added as soon as possible after the substrate reaction time expires.

6. Measure the optical density (OD value) of each well in sequence using an enzyme-linked instrument at a wavelength of 450 nm. Test within 15 minutes after adding stop solution.

Note:

1. When using the reagent kit for the first time, the user should centrifuge various reagent tubes for several minutes so that the reagents are concentrated to the bottom of the tube.

2. Leave one well for each experiment as a blank zero-adjusting well. No reagents are added to this well, only the substrate solution and 2N H2SO4 are added at the end. Use this hole to adjust the OD value to zero when measuring.

3. To prevent the sample from evaporating, place the reaction plate in a closed box covered with a damp cloth during the test, and add a cover or film to the enzyme label plate.

4. Store unused microplates or reagents at 2-8 ° C. Horseradish peroxidase labeled anti-human IgM working solution should be configured according to the required amount. Do not reuse diluted horseradish peroxidase labeled anti-human IgM working solution.

5. It is recommended to set double-hole measurement when testing samples to ensure the accuracy of the test results.

Plate washing method Manual plate washing method: suck (do not touch the wall) or shake off the liquid in the microplate; place a few layers of absorbent paper on the experimental table, and force the microplate down several times; pat the recommended wash buffer Inject at least 0.3ml of solution into the hole and soak for 1-2 minutes. Repeat this process several times as needed.
Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment process after being used skillfully.

Calculation

In order to detect the PA-IgM titer in the sample, the customer can take a 2-fold dilution of the sample to calculate the antibody titer. Generally, the starting dilution factor is 1:40 (use the sample diluent for dilution). The maximum dilution factor is determined by the customer's own test requirements. The final color development is compared with the negative control well. The maximum dilution with obvious difference is determined as the final titer of the antibody.

Precautions

1. When mixing protein solutions, try to be as gentle as possible to avoid foaming.

2. The washing process is very important. Insufficient washing can easily cause false positives.

3. It is best to control the sampling time within 5 minutes. If there are many specimens, it is recommended to use a volley gun to add samples.

4. If the content of the substance to be tested in the specimen is too high, please dilute it and then determine it. When calculating, please multiply by the dilution factor.

5. When preparing the working solution of the test solution, please prepare it with the corresponding diluent, not to be confused.

6. Please keep the substrate away from light.

7. Do not replace the reagents in the kit with reagents from other manufacturers.