Method for making anti trypsin assay kit

The anti trypsin assay kit is usually made based on the principle of enzyme-linked immunosorbent assay (ELISA). The following are the core steps and key points of its production method:

1. Core principle

The anti trypsin assay kit uses a double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). By pre coating micro pores with anti trypsin capture antibodies, samples, standards, and HRP labeled detection antibodies are sequentially added. After incubation and thorough washing, the substrate TMB is used for color development. TMB is converted to blue under the catalysis of peroxidase and to the final yellow under the action of acid. The depth of color is positively correlated with the anti trypsin content in the sample. The absorbance (OD value) can be measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450nm to calculate the sample concentration.

2. Preparation steps

Preparation of reagents and materials:

Microporous enzyme-linked immunosorbent assay (ELISA) plate: used to carry solid-phase antibodies, usually microporous plates, with each well pre coated with anti trypsin antibodies.

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Standard: Used to draw a standard curve and help calculate the concentration of trypsin in the sample.

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Enzyme labeled reagent: an HRP labeled antibody that binds to anti trypsin in the sample to form a complex.

. Washing solution: used for washing enzyme-linked immunosorbent assay plates to remove unbound components.

Chromogenic reagent: It produces a color change after reacting with enzyme-linked antibodies and is used for quantitative detection of the concentration of anti trypsin.

. Termination solution: used to stop the color reaction for OD value determination.

Other materials: such as enzyme-linked immunosorbent assay (ELISA) reader, high-precision sampler and nozzle, 37 ℃ constant temperature box, distilled water or deionized water, etc.

Coating antibody:

Dilute the specific antibody globulin with coating buffer to the optimal concentration (such as 1-10 μ g/ml).

. Add an appropriate amount of diluted antibody (such as 0.3ml) to each well, overnight at 4 ℃ or in a 37 ℃ water bath for 3 hours, and then store in the refrigerator.

Washing:

Remove the coating and wash the concave holes three times with washing buffer (containing 0.05% Tween-20) for 5 minutes each time.

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Adding samples and standards:

Add an appropriate amount of antigen containing test sample (such as 0.2ml) diluted with dilution buffer to each well, and let it sit at 37 ℃ for 1-2 hours.

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Simultaneously set up standard wells and add standard samples of different concentrations to each.

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Add enzyme labeled specific antibody:

Add an appropriate amount of enzyme labeled specific antibody solution (such as 0.2ml) diluted with dilution buffer to each well, and incubate at 37 ℃ for 1-2 hours or determine the incubation time through preliminary experiments.

. Wash: Wash the wells again with washing buffer to remove unbound enzyme labeled antibodies.

Color development:

Add an appropriate amount of substrate solution (such as OPD or OT) to each well and let it sit at room temperature for 30 minutes (as a blank control, add 0.1ml of termination agent to 0.4ml of substrate).

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Termination reaction:

Add an appropriate amount of termination agent (such as 2M H2SO4 or 2M citric acid 0.05ml) to each well to stop the color reaction.

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Determination of OD value:

Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the OD value of each well at a wavelength of 450nm.

. Draw a standard curve with the OD value of the measured standard as the horizontal axis and the concentration value of the standard as the vertical axis, and obtain a linear regression equation. Substitute the OD value of the sample into the equation and calculate the concentration of the sample.

III. Precautions

Reagent Storage: All reagents must reach room temperature (20-25 ℃) before use and be immediately refrigerated after use.

. Washing operation: Incorrect washing can lead to inaccurate results. Make sure to absorb as much liquid as possible from the pores before adding the substrate, and do not let the micropores dry out during the incubation process.

Avoid contamination: Eliminate residual liquid and fingerprints at the bottom of the board, otherwise it will affect the OD value.

. The substrate color solution should be colorless or very light in color, and the substrate solution that has turned blue cannot be used. Avoid cross contamination between reagents and specimens. Light intensity control: Avoid direct exposure to strong light during storage and incubation.

Reagent processing: Any reaction reagent should not come into contact with bleach solvents or the strong gases emitted by bleach solvents, otherwise it will damage the biological activity of the reaction reagents in the reagent kit.

. Expired products cannot be used. Sample management: If there is a possibility of disease transmission, all samples should be managed and processed according to the prescribed procedures and testing devices.