Rat glucose-dependent insulinotropic hormone EIA kit instructions

Rat glucose-dependent insulinotropic hormone EIA kit instructions

principle

This experiment used a double antibody sandwich ELISA method. The anti-rat GIP monoclonal antibody is coated on the microplate, the standard and the GIP in the sample are combined with the monoclonal antibody, and the enzyme-labeled antibody is added to form an immune complex, which is attached to the plate, and the enzyme substrate TMB is added to appear blue. Add stop solution sulfuric acid, the color turns yellow, measure the OD value at 450nm, the GIP concentration is directly proportional to the OD value, and the GIP concentration in the sample can be obtained by drawing a standard curve.

Kit composition ( 2-8 ° C preservation)

Prepare reagents and collect blood samples

1. Collection of specimens: serum, plasma (EDTA, citrate, heparin anticoagulation), cell culture supernatant, tissue homogenate, etc., as early as possible, stored at 2-8 ° C for 48 hours; longer time must be frozen (-20 ° C Or -70 °C) to avoid repeated freezing and thawing.

2. When the standard product is used for the first time, dissolve it with 0.5ml of the sample diluent, place it for half an hour, mix it, use it, and store it at -20oC. The concentrations after dissolution were 14, 8, 3.2, 1.6, 0.8, and 0 ng/ml, respectively.

3. Washing solution: diluted 1:20 with re-distilled water (example: 1 ml concentrated washing solution added to 19 ml of re-distilled water)

Test procedure

1. Establish a standard curve: set 6 holes of standard holes, and add 50ul of standard to each hole.

2. Adding sample: 50 ul of the sample to be tested is added to each hole of the sample to be tested.

3. Add 50 ul of enzyme-labeled antibody working solution per well. The reaction plate was placed at 37 ° C for 120 minutes.

4. Wash the board: the same as before.

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

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

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

Result calculation and judgment

1. All OD values ​​should be subtracted from the blank value before calculation.

2. Take the standard products 14, 8, 3.2, 1.6, 0.8, 0ng/ml as the abscissa and OD as the ordinate. Draw on the coordinate paper and draw the standard curve.

3. Find the corresponding GIP content on the graph based on the OD value of the sample.

Kit performance

1. Sensitivity: The minimum GIP detection concentration is less than 0.4 ng/ml.

2. Specificity: Recombinant or natural rat GIP can be detected simultaneously. Does not cross-react with other cytokines in rats.

3. Repeatability: The coefficient of variation in both the plate and the plate is less than 10%.

Precautions

1. It is recommended to make double holes for the above standard holes and samples to be tested. The standard curve should be made at the same time for each measurement.

2. The washing process is critical. Insufficient washing will result in an accuracy error and an erroneous rise in the OD value.

3. After the slats are opened, 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 and cannot be used for clinical diagnosis!