Instructions for use of rat IgM quantitative EIA kit

Instructions for use of rat IgM quantitative EIA kit

principle

This experiment uses the double antibody sandwich ELISA method. Coated with anti-rat IgM monoclonal antibody on the enzyme-labeled plate, the IgM in the standard and the sample is combined with the monoclonal antibody, enzyme-labeled antibody is added to form an immune complex and connected to the plate, and the enzyme substrate TMB is added, blue , Add stop solution sulfuric acid, the color turns yellow, measure the OD value at 450nm, the IgM concentration is proportional to the OD value, the IgM concentration in the specimen can be obtained by drawing a standard curve.

Kit composition (stored at 2-8 ° C)

Coated Wells

96 wells

Enzyme Conjugate

12ml

10 × Sample Buffer

12ml

20 × concentrated washing solution (Wash Buffer)

50ml

Standards (Standards): 5ug / bottle

2 bottles

Substrate working solution (TMB Solution)

12ml

Cover paper

One

Stop Solution (Stop Solution)

12ml

Prepare reagents and collect blood samples

1. Collect specimens: serum, plasma (EDTA, heparin anticoagulation), cell culture supernatant, urine, etc. as soon as possible, stored at 2-8 ℃ for 48 hours; longer time must be frozen (-20 ℃ or -70 ℃) Store to avoid repeated freezing and thawing. Before the urine measurement, make a 1:10 dilution with the specimen diluent (take 20ul, add the specimen diluent 180ul, and dilute 10 times). Serum or plasma is diluted at least 10,000 times.

2. Preparation of standard solution: add 0.5ml of distilled water to mix well before use to make a solution of 10000ng / ml. Set 8 standard tubes, add 900ul of sample dilution in the first tube, and 500ul of sample dilution in the second to eighth tubes. Add 100ul of the standard solution of 10000ng / ml to the first tube and mix it. Then aspirate 500ul with the pipette and move to the second tube. Repeat the dilution in this manner repeatedly, draw 500ul from the seventh tube and discard it. The eighth tube is a blank control.

3. The 10 × specimen dilution is diluted 1:10 with distilled water (example: 1ml concentrated dilution + 9ml distilled water).

4. Washing solution: 1:20 dilution with redistilled water (example: 1ml concentrated washing solution is added with 19ml of redistilled water)

Testing procedures

1. Add sample: add 100ul of standard or sample to be tested to each well, mix the reaction plate thoroughly and place at 37 ℃ for 120 minutes.

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

3. Add 100ul of enzyme-labeled antibody working solution to each well. Place the reaction plate at 37 ° C for 60 minutes.

4. Wash board: same as above.

5. Add 100ul of substrate working solution to each well, and let it react in the dark at 37 ℃ for 15 minutes.

6. Add 100ul of stop solution to each well and mix well.

7. Measure absorbance at 450nm with a microplate reader within 30 minutes.

Result calculation and judgment

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

2. With the standard products 1000, 500, 250, 125, 62, 31, 15.6, 0 ng / ml as the abscissa and the OD value as the ordinate, draw a graph on the graph paper and draw a standard curve.

3. Find the corresponding IgM content on the graph according to the OD value of the sample, and then multiply it by the dilution factor.

Kit performance

1. Sensitivity: The smallest IgM detection concentration is less than 8ng / ml.

2. Specificity: simultaneous detection of recombinant or natural rat IgM. Does not cross-react with other cytokines in rats.

3. Repeatability: The coefficient of variation within the board and the board is less than 10%.

Precautions

1. The above standard holes and samples to be tested are recommended to be re-holes, and the standard curve should be made at the same time for each measurement.

2. The washing process is critical. Insufficient washing will lead to an increase in accuracy error and OD value.

3. After opening the slats, the remaining slats should be sealed again to keep the slats dry.

4. This kit should be stored in a 4oC refrigerator.

5. This kit is for scientific research only, not for clinical diagnosis!