Rat connective tissue growth factor (CTGF) ELISA kit instruction manual

Shanghai Xitang Biological Technology Co., Ltd.   021-55229872,   65333639    

Rat connective tissue growth factor (CTGF) ELISA kit instruction manual

  ( used in serum, plasma, cell culture supernatants and other biological fluids )

principle

This experiment used double antibody sandwich ABC-ELISA . The anti-rat CTGF mAb was coated on the microtiter plate, the CTGF of the standard and the sample was combined with the monoclonal antibody, and biotinylated anti-rat CTGF was added to form an immune complex attached to the plate, horseradish peroxidation. enzyme-labeled Streptavidin and biotin binding, substrate working VIS blue was added, the final sulfuric acid stop solution, the OD value measured at 450nm, and OD is proportional to the value of the concentration of CTGF, CTGF samples can be determined by the standard curve concentration.

Kit composition ( 2 -8 °C save)

Prepare reagents and collect blood samples

1.           Collect specimens: serum, plasma ( EDTA , citrate, heparin anticoagulation), cell culture supernatant, tissue homogenate, etc., as early as possible, 2 -8 °C Store for 48 hours; freeze for a longer time ( -20 °C or -70 °C ) Save to avoid repeated freezing and thawing.

2.           Standard product preparation: Add 1ml of distilled water before use and mix well to form a solution of 8000pg/ml . Set 8 tubes of standard tube , add 300ul of sample dilution to each tube . Add 300 ul of the standard solution of 8000 pg/ml to the first tube, mix and aspirate 300 ul with the sampler , and transfer to the second tube. Repeat the dilution in this way, and remove 300 ul from the seventh tube and discard it. The eighth tube is a blank control.

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

4.          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.           Adding sample: Add 100 ul of standard or sample to be tested in each well , and mix the reaction plate thoroughly. 37 °C 120 minutes.

2.           Washing the plate: The reaction plate was thoroughly washed 4-6 times with a washing solution , and dried on a filter paper.

3.           Add 1 ul of the first antibody working solution to each well . Mix the reaction plate thoroughly 37 °C 60 minutes.

4.           Wash the board: the same as before.

5.           Add 100 ul of enzyme-labeled antibody working solution per well . Reaction plate 37 °C 30 minutes.

6.           Wash the board: the same as before.

7.           Add 100 ul of substrate working solution per well , set 37 °C The reaction was carried out in the dark for 15 minutes.

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

9.           The absorbance was measured at 45 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.           The standard products 4000 , 2000 , 1000 , 500 , 250 , 125 , 62.5 , 0 pg/ml are plotted on the abscissa, and the OD is plotted on the coordinate paper. The standard curve is drawn on the coordinate paper.

3.           The corresponding CTGF content was found on the graph based on the sample OD value .

Kit performance

              1.    Sensitivity : The minimum CTGF detection concentration is less than 30pg/ml .

2.         Specificity: Recombinant or natural rat CTGF 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, and 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 4 ^o C refrigerator.

  5.    This kit is for scientific research only and cannot be used for clinical diagnosis!

2.         Specificity: Recombinant or natural rat CTGF 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, and 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 4 ^o C refrigerator.

  5.    This kit is for scientific research only and cannot be used for clinical diagnosis!

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