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Application of pepsin Kit

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Pepsin kit is used to quantitatively detect the content of human pepsin in serum, plasma, tissue homogenate and related liquid samples in vitro.

Experimental principle

The kit adopts double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). Add the sample, standard and HRP labeled detection antibody into the coated micropores pre coated with pepsin capture antibody, and then incubate and wash them thoroughly. With the substrate TMB for color development, TMB is converted into blue under the catalysis of peroxidase and into the final yellow under the action of acid. The depth of color is positively correlated with pepsin in the sample. Determine the absorbance (OD value) with a microplate reader at the wavelength of 450nm and calculate the sample concentration.

Sample handling and requirements

1. Serum: place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or 4 ℃ overnight, and then 1000 × G centrifuge for 20 minutes, take the supernatant, or store the supernatant at -20 ℃ or -80 ℃, but avoid repeated freezing and thawing.

2. Plasma: use EDTA or heparin as anticoagulant to collect samples, and the samples will be stored at 2-8 ℃ 1000 within 30 minutes after collection × G centrifuge for 15 minutes, take the supernatant for detection, or store the supernatant at -20 ℃ or -80 ℃, but avoid repeated freezing and thawing.

Operation steps:

1. Take out the required strips from the aluminum foil bag after the room temperature is balanced for 20min, and seal the remaining strips with a self sealing bag and put them back at 4 ℃.

2. Set the standard hole and sample hole, and add standard 50 of different concentrations to the standard hole μ L；

3. Add 50 samples to be tested into the sample hole μ L； Blank hole is not added.

4. In addition to the blank hole, add horseradish peroxidase (HRP) labeled detection antibody 100 to each hole in the standard hole and sample hole μ 50. Seal the reaction hole with a sealing film, and incubate in a 37 ℃ water bath or incubator for 60min.

5. Discard the liquid, pat it dry on the absorbent paper, and fill each hole with detergent (350 μ 50) Let it stand for 1min, throw away the washing liquid, pat it dry on the absorbent paper, and repeat washing the plate for 5 times (you can also use the plate washing machine to wash the plate).

6. Add 50 substrates A and B to each hole μ 50. Incubate at 37 ℃ away from light for 15min.

7. Add termination liquid 50 to each hole μ 50. Measure the OD value of each hole at the wavelength of 450nm within 15min.

Conclusion: the detection of salivary pepsin is of great significance in the diagnosis of gastroesophageal reflux disease, and has the advantages of noninvasive, efficient, simple and easy to accept. It is expected to become an important diagnostic tool for gastroesophageal reflux disease. The sampling time for salivary pepsin detection in the early morning is more appropriate than 1 hour after three meals.

Classification and efficacy of protease

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Classification and efficacy of protease

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