Instructions for using the B-factor assay kit

The usage method of the B-factor assay kit (taking ELISA kit as an example) includes steps such as reagent preparation, sample collection and processing, standard dilution, sample addition, incubation, washing, color development, reaction termination, measurement and result calculation. The specific steps are as follows:

1. Reagent preparation

Reagent reheating: Remove the kit from the refrigerated environment, equilibrate at room temperature for 15-30 minutes, and then use it again.

Preparation of washing solution: Dilute the concentrated washing solution with distilled water or deionized water in a certain proportion to form a working solution. The unused concentrated washing solution should be stored in a refrigerator at 4 ℃.

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Other reagent preparation: Prepare other required reagents according to the instructions, such as enzyme-linked immunosorbent assay (ELISA) reagents, color reagents A and B, stop buffer, etc.

II. Sample Collection and Processing

Serum samples: Whole blood samples are left at room temperature for 2 hours or overnight at 4 ℃, centrifuged at 1000g for 20 minutes, and the supernatant is taken for detection.

. Alternatively, the specimen can be stored at -20 ℃ or -80 ℃, but repeated freezing and thawing should be avoided.

Plasma samples: EDTA or heparin can be used as anticoagulants. Within 30 minutes after specimen collection, centrifuge at 1000g at 2-8 ℃ for 20 minutes, or store the specimen at -20 ℃ or -80 ℃, but avoid repeated freezing and thawing.

. Other samples, such as cell culture supernatant and tissue homogenate, should also be processed according to the instructions.

III. Standard dilution

According to the concentration and dilution method provided in the instruction manual, perform gradient dilution of the standard.

. For example, some reagent kits may require diluting the standard into multiple different concentrations for plotting the standard curve. During the dilution process, attention should be paid to aseptic operation to avoid contamination.

4. Sample addition

Set up standard wells and sample wells: Set up standard wells and sample wells respectively on the enzyme-linked immunosorbent assay (ELISA) coated plate, add different concentrations of standard to each standard well, and add the test sample to the sample well.

. Sample amount: According to the instructions, add an appropriate amount of sample or standard to each well. For example, some test kits may require the addition of 50 μ L of sample or standard per well.

Sample addition sequence: The standard should be added first, followed by the sample. When adding the sample, the sample or standard should be added to the bottom of the enzyme-linked immunosorbent assay plate well, avoiding touching the well wall as much as possible. Gently shake and mix well.

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Fifth, Incubation

Seal the plate with a sealing film and incubate at 37 ℃ for a certain period of time, such as 30 minutes or 60 minutes.

. During the incubation process, temperature stability should be maintained to avoid temperature fluctuations affecting the experimental results. After incubation, remove the sealing film, discard the liquid, and shake dry.

Sixth, Wash

Fill each well with detergent, let it stand for a certain period of time, and then discard it. Repeat the washing process several times.

. During the washing process, it should be ensured that the washing solution fully covers the bottom of the hole, and the liquid inside the hole should be drained after each washing. The purpose of washing is to remove unbound enzyme-linked immunosorbent assay reagents and other impurities, improving the accuracy and sensitivity of the experiment.

7. Color development

Add color developer A to each well first, then add color developer B, gently shake and mix well, and develop color at 37 ℃ in the dark for a certain period of time, such as 10 or 15 minutes.

. During the color development process, light and vibration should be avoided to avoid affecting the color rendering effect.

Eighth, Termination Reaction

Add termination solution to each well to terminate the reaction, and the blue color will immediately turn yellow.

. The amount of termination solution added should be determined according to the instructions. After terminating the reaction, measurements should be taken immediately to avoid color changes affecting the experimental results.

9. Determination and Result Calculation

Determination of absorbance: Measure the absorbance (OD value) of each well in sequence using blank air conditioning zero and 450nm wavelength.

. The measurement should be conducted within 15 minutes after adding the termination solution. Draw a standard curve: Plot the standard curve on a coordinate paper with the concentration of the standard substance as the horizontal axis and the OD value as the vertical axis. Find the corresponding concentration based on the OD value of the sample from the standard curve. Calculate sample concentration: Multiply the obtained sample concentration by the dilution factor to obtain the actual concentration of the sample. If the sample has been diluted multiple times, it should be multiplied by the total dilution factor.