Usage of Total Bile Acid Test Kit

The usage method of total bile acid (TBA) determination kit varies depending on the specific type of kit (such as ELISA method, circulating enzyme method, etc.) and the manufacturer. The following is an overview of the usage of an ELISA based total bile acid assay kit for your reference:

1、 Preparation work

Sample preparation: Prepare the test sample according to the requirements of the reagent kit, such as serum, plasma, etc. Ensure timely processing of samples after collection to avoid contamination and repeated freeze-thaw cycles.

Reagent preparation: Remove the reagent kit from the refrigerator and let it equilibrate at room temperature for 20 minutes. Check if the reagents in the kit are complete, including standard samples, detection antibodies, washing solutions, etc.

Instrument preparation: Prepare the enzyme-linked immunosorbent assay (ELISA) reader (wavelength set to 450nm), high-precision sampler, and gun head.

2、 Operation steps

Set up standard and sample wells: Set up standard and sample wells in the microplate, add 50 μ L of different concentrations of standard to each standard well, first add 10 μ L of the test sample to the sample well, and then add 40 μ L of sample diluent (note: the specific operating amount may vary depending on the kit, please refer to the instructions).

Add detection antibody: In addition to blank wells, add 100 μ L of horseradish peroxidase (HRP) labeled detection antibody (or other specified amount of detection antibody) to each well of the standard and sample wells, and seal the reaction well with a sealing membrane.

Incubation: Place the microplate in a 37 ℃ water bath or constant temperature box and incubate for 60 minutes (specific time according to the instructions).

Washing: After incubation, discard the liquid in the well and wash 5 times with washing solution. After each wash, pat dry on absorbent paper.

Color development: Add 50 μ L of substrate A and 50 μ L of substrate B (or other specified amount of substrate) to each well, and incubate at 37 ℃ in the dark for 15 minutes.

Termination reaction: Add 50 μ L of termination solution to each well to terminate the color reaction.

Measure absorbance: Measure the absorbance (OD value) of each well using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450nm.

3、 Result calculation

Draw a standard curve: Using the concentration of the standard substance as the horizontal axis and the corresponding OD value as the vertical axis, draw a linear regression curve of the standard substance in an Excel worksheet.

Calculate sample concentration: Based on the standard curve equation, calculate the concentration values of each sample. Note that if the sample has been diluted before testing, the final result needs to be multiplied by the corresponding dilution factor.

4、 Precautions

Operating standards: Strictly follow the operating steps in the manual to avoid cross contamination.

Sample preservation: After sample collection, it should be processed in a timely manner. If preservation is required, please pack according to the instructions and freeze at -20 ℃ to avoid repeated freeze-thaw cycles.

Reagent storage: The reagent kit should be stored at 2-8 ℃, avoiding direct sunlight and high temperatures.

Instrument calibration: The enzyme-linked immunosorbent assay (ELISA) reader should be calibrated before use to ensure the accuracy of the measurement results.

Please note that the above is only an overview of the usage method of a total bile acid determination kit based on ELISA method. Different brands and models of reagent kits may vary. Please carefully read the product manual and follow its instructions before use.