Digital method for evaluating nonspecific adsorption of bioactive substance diluent

Abstract

The invention discloses a digital method for evaluating non-specific adsorption of a bioactive substance diluent, belongs to the technical field of immunodetection, and relates to a digital method for evaluating non-specific adsorption of a bioactive substance diluent by establishing a digital fluorescence immunoadsorption analysis system. The influence of a certain step or a certain material on non-specific adsorption in the experiment process is evaluated by replacing a bioactive substance sample diluent, so that the non-specific adsorption in the experiment process is evaluated, the experiment method is perfected, and the detection accuracy is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of immunoassay, and in particular relates to a bioactive substance diluent, and specifically is a scheme for evaluating the effect of the bioactive substance diluent on nonspecific adsorption. Background Art

[0002] The study of bioactive substances is of great significance in the fields of medicine and biotechnology, especially in disease diagnosis, prognosis assessment, treatment effect monitoring and drug development. Among them, bioactive substance dilution, as an indispensable part of biological activity research, plays a decisive role in the identification sensitivity and accuracy of related bioactive substances.

[0003] Nonspecific adsorption refers to the nonspecific binding phenomenon that occurs between the molecule to be tested and the solid phase carrier in the experimental reaction. This phenomenon is usually caused by factors such as the physicochemical properties of the solid phase carrier, the structural complexity of the molecule to be tested, and inappropriate reaction conditions. The polarity of the molecule to be tested, the hydrophobicity and charge distribution on the surface of the solid phase carrier, the attachment state of the molecule to be tested on the solid phase carrier, etc. will affect the degree of nonspecific adsorption. This adsorption phenomenon is a common problem in biomedical experiments, which may cause deviations in experimental results, thereby affecting the accuracy and reliability of the experiment. Therefore, understanding and controlling nonspecific adsorption is crucial to improving the quality of experiments, but in the current prior art, no perfect scheme for evaluating nonspecific adsorption has been found. Summary of the invention

[0004] In order to address the deficiencies of the prior art, the present invention proposes a digital method for evaluating the nonspecific adsorption of a dilution of a bioactive substance, which can more accurately evaluate the experimental results, thereby improving the accuracy and reliability of the experiment.

[0005] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0006] A digital method for evaluating nonspecific adsorption of a dilution of a bioactive substance comprises the following steps:

[0007] Determine the experimental detection process based on the biological detection method of digital fluorescent immunoadsorption;

[0008] Replace the diluent of bioactive substances in the detection process;

[0009] According to the test results, the effects of different diluent formulations on nonspecific adsorption during the experiment were evaluated.

[0010] The diluents used include: PBS (phosphate buffered saline) solution containing 0.1% BSA (bovine serum albumin), or PBS solution containing 0.1% BSA and 0.1% casein, or PBS solution containing 0.1% BSA and 0.01% Tween 20.

[0011] In the present invention, PBS is the basic buffer, and its preparation method is: weigh 3.40g Na2HPO4 and 6.32g NaH2PO4 in a biscuit, then add ultrapure water to 1L, stir and dissolve, and the pH is 7.2-7.4. The present invention uses a PBS solution containing 0.1% BSA as the optimized basic diluent, and adds 0.1% volume mass ratio (m / v) casein or 0.01% Tween 20 as the preferred diluent.

[0012] In the present invention, the test detection process includes coating of base antibody, sample incubation, detection antibody incubation, and machine reading.

[0013] In the present invention, the bioactive substance is interleukin (IL)-6, the antibody used is an interleukin-6 antibody pair, and the base plate of the solid phase carrier is a 96-well ELISA plate made of polystyrene (PS).

[0014] In the present invention, the coating step of the substrate antibody comprises:

[0015] S1-1: Add 100 μL of IL-6 antibody diluted with pH 9.6 carbonate buffer to the wells of the substrate plate, with an antibody concentration of 2 μg / mL, and place in a 37°C incubator for reaction for 2 h. After the reaction, wash the plate with PBST (phosphate buffered saline with Tween-20) solution;

[0016] S1-2: Add 150 μL of PBS solution containing 1% BSA and place at 37°C for 2 h for blocking. After blocking, wash the plate with PBST solution.

[0017] In the present invention, the sample incubation step comprises:

[0018] S2-1: Select IL-6 antigen standard and add it to different diluents to prepare different dilution samples of the antigen standard;

[0019] S2-2: Take the treated substrate plate, add 50 μL of each antigen standard diluted sample, place at 37°C for 1 hour, and wash the plate with PSBT solution;

[0020] S2-3: Add 150 μL of PBS solution containing 1% BSA and place at 37°C for 2 h for blocking. After blocking, wash the plate with PBST solution.

[0021] In the present invention, the detection antibody incubation step comprises:

[0022] S3-1: Add 100 μL of fluorescent microspheres containing detection antibodies and place at 37°C for 1 hour. After completion, wash the plate with PBST solution.

[0023] In the present invention, the computer reading step includes:

[0024] S4-1: Place the processed substrate in a digital fluorescence immunoassay analyzer to read and analyze the fluorescence signal to obtain digital data of the experimental results;

[0025] S4-2: Perform statistical analysis on the obtained data to determine the effects of different dilutions on nonspecific adsorption.

[0026] The present invention provides a digital method to evaluate nonspecific adsorption during the experiment and determine the effects of different dilutions on nonspecific adsorption. Through this method, researchers can more accurately evaluate the experimental results, thereby improving the accuracy and reliability of the experiment, which not only helps to improve the diagnosis and treatment of diseases, but also provides new tools and methods for biomedical research. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is an appearance diagram of the base plate used in the present invention. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with the accompanying drawings and specific preferred embodiments, but the protection scope of the present invention is not limited thereby.

[0029] Example 1

[0030] A digital method for evaluating the nonspecific adsorption of bioactive substance diluents, according to the biological detection method of digital fluorescent immunoadsorption, determine the experimental detection process; replace the bioactive substance diluent in the detection process; according to the detection effect, evaluate the effect of different formulations of dissolving solutions on nonspecific adsorption during the experiment. In this embodiment, taking interleukin-6 factor as an example, the experimental detection process is as follows, wherein interleukin-6 antibodies and antigens are sourced from Hangzhou Yibaixin Biotechnology Co., Ltd., fluorescent microspheres are purchased from Suzhou Weidu Biotechnology Co., Ltd., the labeling method of microspheres refers to the manufacturer's instructions, and the digital fluorescent immunoassay analyzer is a self-developed device of the Institute of Physics, Henan Academy of Sciences. The specific steps include:

[0031] (1) Pre-preparation

[0032] According to the experimental requirements and expected results, select the appropriate diluent: Solution 1: PBS solution containing 0.1% BSA; Solution 2: PBS solution containing 0.1% BSA and 0.1% casein; Solution 3: PBS solution containing 0.1% BSA and 0.01% Tween 20. Select a suitable substrate plate, such as a 96-well ELISA plate made of PS material, such as Figure 1 shown.

[0033] (2) Base antibody coating

[0034] ① Antibody dilution: Use carbonate buffer at pH 9.6 to dilute the substrate antibody interleukin-6 antibody to the desired concentration, such as 2 μg / mL;

[0035] ② Coating: Add 100 μL of the diluted substrate antibody solution to the wells of the substrate plate and place in a 37°C incubator for 2 hours to fix the antibody on the surface of the substrate plate;

[0036] ③ Washing the plate: After the reaction is completed, wash the plate with PBST solution to remove unbound antibodies.

[0037] (3) Blocking redundant sites

[0038] ① Preparation of blocking solution: Use PBS solution containing 1% BSA as blocking solution;

[0039] ② Blocking: Add 150 μL of blocking solution to each well of the substrate plate and place in a 37°C incubator for 2 hours to block sites on the substrate plate that are not occupied by antibodies to prevent nonspecific adsorption;

[0040] ③ Washing the plate: After blocking, wash the plate again with PBST solution to remove excess blocking solution.

[0041] (4) Sample incubation

[0042] ① Sample preparation: add the antigen standard containing interleukin-6 into different diluents to prepare different dilution samples;

[0043] ② Incubation: Add 50 μL of different sample solutions to each well of the substrate plate and incubate in a 37°C incubator for 1 hour to allow the detection antibody to bind to the bioactive substances bound to the substrate plate;

[0044] ③ Washing the plate: After incubation, wash the plate with PBST solution to remove unbound sample components.

[0045] (5) Detection antibody incubation

[0046] ① Preparation of detection antibodies: dilute the fluorescently labeled interleukin-6 detection antibody to an appropriate concentration;

[0047] ② Incubation: Add 100 μL of the diluted detection antibody solution to each well of the substrate plate and incubate in a 37°C incubator for 1 hour to allow the detection antibody to bind to the bioactive substance bound to the substrate plate;

[0048] ③ Washing: After incubation, wash the plate with PBST solution to remove unbound detection antibodies.

[0049] (6) Reading on the computer

[0050] ① Instrument preparation: Ensure that the digital fluorescence immunoassay analyzer is in normal working condition and set the instrument parameters according to experimental requirements;

[0051] ② Reading: Place the processed substrate plate in the digital fluorescence immunoassay analyzer to read and analyze the fluorescence signal to obtain digital data of the experimental results.

[0052] Based on the above dilution and test process, the experimental data were recorded, and the sample addition layout was shown in Table 1, where T1-T10 were samples diluted with solution 1 of interleukin-6, T11-T20 were samples diluted with solution 2 of interleukin-6, and T21-T30 were samples diluted with solution 3 of interleukin-6, with 10 tests for each solution.

[0053] Table 1: Sample layout of substrate board

[0054] 1 2 3 4 5 6 7 8 9 10 11 12 A T1 T9 T17 T25 B T2 T10 T18 T26 C T3 T11 T19 T27 D T4 T12 T20 T28 E T5 T13 T21 T29 F T6 T14 T22 T30 G T7 T15 T23 H T8 T16 T24

[0055] In the test results, the raw data obtained from the substrate plate test of the three solutions are shown in Table 2.

[0056] Table 2: Fluorescence intensity signal values ​​detected by substrate, in RFU (Relative Fluorescence Unit)

[0057]

[0058]

[0059] In the test of nonspecific adsorption, the background signal value of solution 2 was relatively high, with a background signal value of 63.6RFU in 10 tests and a coefficient of variation (CV) of 4.24%. Solution 1 was second, with a background signal value of 56RFU in 10 tests and a CV of 4.66%. Solution 3 performed the lowest, with a background signal value of 36.9RFU in 10 tests and a CV of 2.64%.

[0060] The background signal value mainly comes from the signal generated by nonspecific adsorption. In this experiment, a larger background signal value means that there are more nonspecific bindings, such as unnecessary interactions between the molecule to be tested and the solid phase carrier. The coefficient of variation is a statistic used to measure the degree of data dispersion. It is calculated by dividing the standard deviation by the mean and multiplying it by 100%. In this experiment, for the nonspecific adsorption test results of different bioactive substance dilutions, the repeatability and stability of the data are evaluated by calculating the CV.

[0061] Therefore, by comparing the experimental results of different bioactive sample diluents, it can be concluded that by optimizing the formula of the sample diluent, especially solution 3 containing 0.01% Tween 20, nonspecific adsorption can be effectively reduced and the detection accuracy can be improved. Therefore, the technical solution provided by the present invention can effectively evaluate the nonspecific adsorption during the experiment.

Claims

1. A digital method for evaluating nonspecific adsorption of bioactive substance dilutions, characterized in that: The following steps are involved: Determine the experimental detection process based on the biological detection method of digital fluorescent immunoadsorption; Replace the diluent of bioactive substances in the detection process; According to the detection results, the influence of different diluent formulas on nonspecific adsorption during the experiment was evaluated.

2. The method according to claim 1, characterized in that: The diluent has the following formula components: a PBS solution containing 0.1% BSA, or a PBS solution containing 0.1% BSA and 0.1% casein, or a PBS solution containing 0.1% BSA and 0.01% Tween 20.

3. The method according to claim 2, characterized in that: The experimental detection process includes coating of base antibodies, sample incubation, detection antibody incubation, and machine reading.

4. The method according to claim 3, characterized in that: The bioactive substance is interleukin-6, the antibody used is an interleukin-6 antibody pair, and the base plate of the solid phase carrier is a 96-well ELISA plate made of PS material.

5. The method according to claim 4, characterized in that: The coating of the substrate antibody comprises: S1-1: Add 100 μL of IL-6 antibody diluted with pH 9.6 carbonate buffer to the wells of the substrate plate, with an antibody concentration of 2 μg / mL, and place in a 37°C incubator for 2 h. After the reaction, wash the plate with PBST solution; S1-2: Add 150 μL of PBS solution containing 1% BSA and place at 37°C for 2 h for blocking. After blocking, wash the plate with PBST solution.

6. The method according to claim 5, characterized in that: The sample incubation includes: S2-1: Select IL-6 antigen standard and add it to different diluents to prepare different dilution samples of antigen standard; S2-2: Take the treated substrate plate, add 50 μL of each antigen standard diluted sample, place at 37°C for 1 hour, and wash the plate with PSBT solution after completion; S2-3: Add 150 μL of PBS solution containing 1% BSA and place at 37°C for 2 h for blocking. After blocking, wash the plate with PBST solution.

7. The method according to claim 6, characterized in that: The detection antibody incubation comprises: S3-1: Add 100 μL of fluorescent microspheres containing detection antibodies and place at 37°C for 1 hour. After completion, wash the plate with PBST solution.

8. The method according to claim 7, characterized in that: The on-machine readings include: S4-1: Place the processed substrate in a digital fluorescence immunoassay analyzer to read and analyze the fluorescence signal to obtain digital data of the experimental results; S4-2: Perform statistical analysis on the obtained data to determine the effects of different dilutions on nonspecific adsorption.

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