ELISA determination operating procedures

ELISA determination operating procedures (taking mouse TNF-a as an example)

1. Principle and significance

Through various phenomena after the specific binding of antigen and antibody in vitro, the antigen or antibody in the sample is qualitatively and quantitatively detected. In this experiment, the double antibody sandwich ABC-ELISA method was used. The anti-mouse TNF-a monoclonal antibody was coated on the enzyme labeling plate. TNF-a antibody forms an immune complex and is connected to the plate. Horseradish peroxidase-labeled streptavidin (Streptavidin) is combined with biotin. The enzyme substrate o-phenylenediamine (OPD) is added and yellow appears Add stop solution concentrated sulfuric acid, the color becomes darker, and the OD value is measured at 492nm. The TNF-a concentration is proportional to the OD value. The TNF-a concentration in the specimen can be obtained by drawing a standard curve. It is a qualitative and quantitative method for detecting protein content in samples.

Second, the composition of the kit

96/48 microplate (1 piece, coated with anti-mouse TNF-a monoclonal antibody), sample diluent (1 bottle), first antibody working solution (1 bottle), enzyme-labeled antibody working solution (1 bottle) , Substrate dilution (1 bottle), stop solution (1 bottle), washing solution (20 ×, 1 bottle), standard product (2000 pg / ml, 2 tubes, lyophilized powder), OPD tablets (3 tablets), Coordinate paper (1 sheet).

3. Experiment preparation

1. Reagent preparation: The washing solution is diluted with 1:20 three-distilled water; the standard product can be dissolved by adding 200 μl of distilled water to each tube before use (see the instructions for details); the substrate working solution should be 5-10 min before color development Dissolve the OPD tablets in the substrate diluent and add 5 ml of solution to each tablet.

2. Specimen collection: collect serum or body fluid for early detection, and store at 2 ~ 8 ℃ for a longer period of time to be frozen (-20 ℃) ​​to avoid repeated freeze-thaw; tissues (fetal liver, fetal brain, etc.) are well organized the day before Slurry, tissue homogenate with 90% volume RIPA lysate (PH 8.0, 50 mM Tris HCl, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA) and 10% volume 10 mM PMSF (10%), cracked on ice for 30 min, take the supernatant after 12000 × 20min, and use at 4 ℃.

3. Equipment preparation: microplate reader, 37 ℃ constant temperature oven, filter paper, tray, measuring cylinder, beaker, pipettes of various specifications, centrifuge tubes, tube racks and waste tanks, etc. Bring your own distilled water.

4. Operation steps

1.Establish 8 wells (can be adjusted to 6 wells according to the protein concentration of the sample), add 100 ul of sample diluent to each well, and add 100 ul of the standard product to the first well. Move to the second well, dilute it repeatedly to the seventh well in this way, and finally, aspirate 100 ul from the seventh well and discard it, so that the volume is 100 ul. Well 8 is a blank control.

2. Sample addition: add 100ul of the sample to be tested to each well of the sample to be tested.

3. Place the reaction plate at 37 ℃ × 120 min.

4. Wash the plate: wash the reaction plate 4-6 times with the washing solution, and buckle it on the filter paper.

5. Add 50ul of the first antibody working solution to each well.

6. Mix the reaction plate thoroughly and place at 37 ℃ × 60 min.

7. Plate washing: Wash the reaction plate 4-6 times with the washing solution, and buckle it on the filter paper.

8. Add 100ul of enzyme-labeled antibody working solution to each well.

9. Place the reaction plate at 37 ℃ for 120 min.

10. Washing the plate: Wash the reaction plate 4-6 times with the washing solution, and buckle it on the filter paper.

11. Add 100ul of substrate working solution to each well and place in a dark place at 37 ℃ for 5-10 minutes.

12. Add 50ul of stop solution to each well and mix well.

13. Measure the absorbance at 492 nm with a microplate reader.

V. Calculation results and judgment

1. Draw the standard curve with the OD values ​​of standard products 1000, 500, 250, 125, 62.5, 31.25, 15.625, 0 pg / ml on semi-logarithmic paper.

2. All OD values ​​should be calculated after subtracting the blank tube value.

According to the OD value of the sample, the content of TNF-a in the corresponding sample is found on the standard curve diagram or converted according to the standard curve formula.

Six, matters needing attention

1. The above standard holes and the samples to be checked are recommended to be re-holes, and the standard holes should be used for each measurement.

2. This kit should be stored at -20 ℃ for refrigeration.

3. The reagents taken out of this kit are used at room temperature (20 ~ 25 ℃). The solution should be shaken gently before use.

4. This kit contains 2M sulfuric acid. Do not mix it with waste containing sodium azide.

5. After opening the lath, the remaining lath should be sealed again to keep the lath dry.

6. Avoid cross contamination. For example, when washing, pour to the side where the protein content of the sample may be high.

7. Turn on the oven 24 hours in advance to preheat.

8. Substrate working solution should be prepared 5 ~ 10 minutes before color development. If the preparation time is too long, it will deteriorate.

9. Drain all the liquid in the hole, add about 350 ul of washing liquid to each hole, and shake off after 30 seconds of rest. Especially when adding antibody working solution or substrate, try to dry the liquid in the well. And during the incubation process, the reaction wells should be covered to prevent the liquid from recovering.

10. Try to prevent the concentration of protein to be measured in the sample from being too high and the microplate reader display out. When the measured value of the microplate reader appears a lot of out, it can be measured by a visible spectrometer after dilution by a certain multiple, and all wells are measured at the same time.