Rat Neurotensin ELISA Kit Instructions for Use

Rat Neurotensin ELISA Kit Instructions for Use

This experiment used double antibody sandwich ABC-ELISA . The anti-rat Neurotensin monoclonal antibody was coated on the plate, the Neurotensin in the standard and the sample was combined with the monoclonal antibody, and biotinylated anti-rat Neurotensin was added to form an immune complex attached to the plate, horseradish peroxidation. The enzyme-labeled Streptavidin is combined with biotin, the substrate working solution is blue, and finally the stop solution sulfuric acid is added . The OD value is measured at 450 nm . The concentration of Neurotensin is directly proportional to the OD value. The standard curve can be used to find the Neurotensin in the specimen. concentration.

1.           Collect specimens: serum, plasma ( EDTA ), cell culture supernatant, tissue homogenate, urine, 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 solution preparation: Add 0.5 ml of distilled water before use and mix well to prepare a solution of 4000 pg/ml . Set 8 tubes of standard tube , add 200ul of specimen dilution solution to each tube . Add 200 ul of the standard solution of 4000 pg/ml to the first tube, mix and aspirate 200 ul with the sampler, and transfer to the second tube. Repeat the dilution as described above, and aspirate 200 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)

Standard

              1.    Add 450 ul of the sample dilution to a 1.5 ml imported polypropylene tube and add 10 ul of serum or plasma samples.

  2.    Add 20ul 1 N HCI, cover tightly and mix up and down. 2 - 8 °C Leave for 60 ± 2 minutes.

     3.    Add 20ul of 1 N NaOH, cover tightly, and mix up and down.

     4.    Ready to use, or put -20/ -70 °C Save for 3 days. When the calculation result is multiplied by 50 dilution. ( Note: The level of Neurotensin may vary greatly depending on the actual situation. Please flexibly grasp the dilution according to the actual situation)

     5.    Cell culture supernatant was diluted 10-fold or tissue homogenates (410ul of sample diluent + 50ul sample + 20ul of 1N HCI + 20ul of 1N NaOH).

Test procedure

1.           Adding: Add 100ul of standard or test sample (activated) to 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 2000 , 1000 , 500 , 250 , 125 , 62.5 , 31.2 , 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.           According to the OD value of the sample , the corresponding Neurotensin content is found on the graph , and then multiplied by the dilution factor .

Kit performance

              1.    Sensitivity : The minimum concentration of Neurotensin is less than 15 pg/ml .

2.         Specificity: Recombinant or natural rat Neurotensin 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 was less than 8.9 %.

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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