Digital method for evaluating non-specific adsorption of bioactive substance adsorption substrate plate

Abstract

The invention discloses a digital method for evaluating non-specific adsorption of a bioactive substance adsorption base plate, belongs to the technical field of immunodetection, and particularly relates to a digital method for evaluating non-specific adsorption of a bioactive substance adsorption base plate 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 the bioactive substance adsorption base plate, 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 present invention belongs to the technical field of immunoassay, and in particular relates to a bioactive substance adsorption substrate, and specifically is a solution for evaluating the non-specific adsorption problem of a bioactive substance adsorption substrate. Background Art

[0002] The bioactive substance adsorption substrate can effectively adsorb bioactive substances (Biomarker), which is of great significance in the fields of disease diagnosis, prognosis evaluation, and treatment effect monitoring.

[0003] Nonspecific adsorption refers to the nonspecific binding phenomenon that occurs between the molecule to be tested and the solid phase carrier during 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 hydrophobicity and charge distribution on the surface of the solid phase carrier, as well as the polarity of the molecule to be tested, will affect the degree of nonspecific adsorption. This adsorption phenomenon is a common problem in biomedical experiments, which may lead to 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 bioactive substances to a substrate, 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 non-specific adsorption of bioactive substances to a substrate comprises the following steps:

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

[0008] Replace the bioactive substance adsorption substrate in the detection process;

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

[0010] The substrate plates used include 96-well polystyrene (PS) ELISA plates, carboxylated 96-well ELISA plates, and 96-well polymethyl methacrylate (PMMA) ELISA plates.

[0011] In the present invention, the test detection process includes coating of base antibody, sample incubation, detection antibody incubation, and machine reading.

[0012] In the present invention, the bioactive substance is interleukin (IL)-6, and the antibody used is an interleukin-6 antibody pair.

[0013] In the present invention, the processing method of the base plate being a carboxylated 96-well ELISA plate comprises:

[0014] S1-1: Use a carboxylated 96-well ELISA plate, add 20 mg / mL EDC [1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride] and NHS (N-hydroxysuccinimide) solution 50 μL each, place in a 37°C incubator for activation for 1 hour, and then wash the plate with PBST (Phosphate Buffered Saline with Tween-20) solution;

[0015] S1-2: Add 100 μL of IL-6 antibody diluted with MES [2-(N-Morpholino)ethane sulfonicacid] solution to a concentration of 2 μg / mL, place in a 37°C incubator for 2 hours, and wash the plate with PBST solution after completion;

[0016] S1-3: Add 150 μL of PBS (Phosphate Buffered Saline) solution containing 1% BSA (Bovine Serum Albumin) and incubate at 37° C. for 2 h for blocking. After blocking, wash the plate with PBST solution.

[0017] The processing method of the 96-well plate made of PMMA substrate includes:

[0018] S2-1: Place a 96-well plate made of PMMA into a plasma treatment instrument for plasma surface activation. Add 50 μL of 20 mg / mL EDC and NHS solution to the activated substrate plate, place it in a 37°C incubator for activation for 1 hour, and then wash the plate with PBST solution.

[0019] S2-2: Add 100 μL of IL-6 antibody diluted with MES solution to a concentration of 2 μg / mL, place in a 37°C incubator for 2 h, and wash the plate with PBST solution after completion;

[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] The processing method of the substrate plate being a 96-well PS ELISA plate includes:

[0022] S3-1: Add 100 μL of IL-6 antibody diluted with pH 9.6 carbonate buffer to a concentration of 2 μg / mL, place in a 37°C incubator for 2 h, and wash the plate with PBST solution after completion;

[0023] S3-2: 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.

[0024] In the present invention, the method for evaluating the effects of different substrates on nonspecific adsorption includes:

[0025] S4-1: Take the above-mentioned different treated substrate plates respectively, add 50 μL of IL-6 antigen standard, the concentrations of the antigen standard are: 1600, 800, 400, 200, 100, 50, 25, 0 pg / mL, place at 37°C for 1 hour, and wash the plate with PSBT solution after completion;

[0026] S4-2: 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.

[0027] S4-3: Place the test plate on the digital fluorescence immunoassay analyzer for reading and analysis.

[0028] The present invention provides a digital method to evaluate nonspecific adsorption during the experiment. 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

[0029] Figure 1 The figure is an outline diagram of the base plate used in the present invention;

[0030] Figure 2 is a line graph of the detection standard of the carboxylated substrate of the present invention;

[0031] Figure 3It is a line graph of the detection standard of the PMMA material substrate of the present invention;

[0032] Figure 4 It is a line graph of the detection standard of the PS material ELISA plate of the present invention. DETAILED DESCRIPTION

[0033] 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.

[0034] Example 1

[0035] A digital method for evaluating the nonspecific adsorption of bioactive substances on substrates, according to the biological detection method of digital fluorescent immunoadsorption, determine the experimental detection process; replace the bioactive substance adsorption substrate in the detection process; according to the detection effect, evaluate the influence of different substrates 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 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:

[0036] (1) Preparation of substrate

[0037] According to the experimental requirements and expected results, select the appropriate substrate plate format, such as carboxylated 96-well ELISA plate, PMMA 96-well plate, PS ELISA plate, and other substrate plates with different surface properties, and perform pretreatment.

[0038] (2) Base antibody coating

[0039] ① Antibody dilution: Use an appropriate buffer, such as MES solution or pH 9.6 carbonate buffer, to dilute the substrate antibody interleukin-6 antibody to the desired concentration, such as 2 μg / mL;

[0040] 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;

[0041] ③ Washing the plate: After the reaction is completed, wash the plate with PBST solution to remove unbound antibodies.

[0042] (3) Blocking redundant sites

[0043] ① Preparation of blocking solution: Use PBS solution containing 1% BSA as blocking solution;

[0044] 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;

[0045] ③ Washing the plate: After blocking, wash the plate again with PBST solution to remove excess blocking solution.

[0046] (4) Sample incubation

[0047] ① Sample preparation: dilute the sample containing interleukin-6 to the appropriate concentration range: 1600, 800, 400, 200, 100, 50, 25, 0 pg / mL;

[0048] ② Incubation: Add 50 μL of the diluted sample 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;

[0049] ③ Washing the plate: After incubation, wash the plate with PBST solution to remove unbound sample components.

[0050] (5) Detection antibody incubation

[0051] ① Preparation of detection antibodies: dilute the fluorescently labeled interleukin-6 detection antibody to an appropriate concentration;

[0052] 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;

[0053] ③ Washing: After incubation, wash the plate with PBST solution to remove unbound detection antibodies.

[0054] (6) Reading on the computer

[0055] ① Instrument preparation: Ensure that the digital fluorescence immunoassay analyzer is in normal working condition and set the instrument parameters according to experimental requirements;

[0056] 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.

[0057] In this embodiment, the substrate plate formats selected include commercial 96-well PS plates, carboxylated 96-well ELISA plates, and PMMA 96-well ELISA plates. Figure 1 Different substrates require different treatments:

[0058] For carboxylated substrate plates, add 50 μL of 20 mg / mL EDC and NHS solutions, place them in a 37°C incubator for activation for 1 hour, and then wash the plates with PBST solution; for PMMA substrate plates, place them in a plasma instrument for plasma surface activation, add 50 μL of 20 mg / mL EDC and NHS solutions to the activated substrate plates, place them in a 37°C incubator for activation for 1 hour, and then wash the plates with PBST solution; for PS ELISA plates, no special pretreatment is required.

[0059] Based on the above substrate and test process, the experimental data was recorded and the sample loading layout was shown in Table 1 below, where S1-S8 are standards and T1-T10 are blank sample dilution controls:

[0060] Table 1: Sample layout of substrate board

[0061]

[0062]

[0063] In the test results, the raw data obtained from the three types of substrate tests are as follows:

[0064] Table 2 below shows the carboxylated substrate:

[0065] Table 2: Fluorescence intensity signal values ​​of carboxylated substrates, in RFU (Relative Fluorescence Unit)

[0066] 1 2 3 4 A 6145 6345 15 24 B 3744 3564 14 26 C 1645 1385 18 D 628 668 28 E 345 325 34 F 146 104 49 G 52 62 19 H 25 15 18

[0067] Table 3 below shows the base plate made of PMMA material:

[0068] Table 3: Fluorescence intensity signal values ​​of PMMA substrate, in RFU

[0069] 1 2 3 4 A 4258 4250 4 4 B 1894 2074 5 5 C 765 743 8 D 394 384 4 E 154 154 2 F 62 76 1 G 24 22 5 H 3 7 8

[0070] The following table shows 4 ELISA plates made of PS material:

[0071] Table 4: Fluorescence intensity signal value of PS ELISA plate, unit is RFU

[0072]

[0073]

[0074] The linearity of the three bioactive adsorption substrate standards is as follows Figure 2-4As shown, they all have a good linear relationship; in the test of nonspecific adsorption, the PS material ELISA plate is relatively high, with a background signal value of 59.8RFU in 10 tests and a coefficient of variation (CV) of 14.38%, followed by the carboxylated bioactive substance fineness substrate plate, with a background signal value of 24.5RFU in 10 tests and a CV of 10.64%, while the PMMA material bioactive substance adsorption substrate plate has the lowest performance, with a background signal value of 4.6RFU in 10 tests and a CV of 2.22%.

[0075] 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 adsorption substrates, the repeatability and stability of the data are evaluated by calculating CV.

[0076] Therefore, by comparing the experimental results of different bioactive substance adsorption substrates, it can be concluded that the bioactive substance adsorption substrate made of PMMA material performs best in reducing nonspecific adsorption, with the lowest mean background signal value and the smallest CV, indicating that its repeatability and stability are good. The ELISA plate made of PS material performs relatively poorly in the nonspecific adsorption test, with a higher mean background signal value and a larger CV. Therefore, the technical solution provided by the present invention can effectively evaluate nonspecific adsorption during the experiment.

Claims

1. A digital method for evaluating non-specific adsorption of bioactive substances to substrates, characterized in that: The following steps are involved: Determine the experimental detection process according to the biological detection method of digital fluorescent immunosorbent; replace the bioactive substance adsorption substrate in the detection process; According to the detection results, the influence of different substrates on nonspecific adsorption during the experiment was evaluated.

2. The method according to claim 1, characterized in that: The base plate includes a 96-well polystyrene ELISA plate, a carboxylated 96-well ELISA plate, and a 96-well polymethyl methacrylate plate.

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 biologically active substance is interleukin-6, and the antibody used is an interleukin-6 antibody pair.

5. The method according to claim 4, characterized in that: The processing method of the base plate being a carboxylated 96-well ELISA plate includes: S1-1: Use a carboxylated 96-well ELISA plate, add 50 μL of 20 mg / mL EDC and NHS solution respectively, place in a 37°C incubator for activation for 1 hour, and then wash the plate with PBST solution; S1-2: Add 100 μL of IL-6 antibody diluted with MES solution to a concentration of 2 μg / mL, place in a 37°C incubator for 2 h, and wash the plate with PBST solution after completion; S1-3: Add 150 μL of PBS solution containing 1% BSA and incubate at 37°C for 2 h for blocking. Afterwards, wash the plate with PBST solution.

6. The method according to claim 4, characterized in that: The processing method of the 96-well plate made of polymethyl methacrylate as the base plate includes: S2-1: Place a 96-well plate made of polymethyl methacrylate into a plasma treatment instrument for plasma surface activation. Add 50 μL of 20 mg / mL EDC and NHS solution to the activated substrate plate, place it in a 37°C incubator for activation for 1 hour, and wash the plate with PBST solution after activation. S2-2: Add 100 μL of IL-6 antibody diluted with MES solution to a concentration of 2 μg / mL, place in a 37°C incubator for 2 h, and wash the plate with PBST 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 4, characterized in that: The processing method of the substrate plate being a 96-well polystyrene ELISA plate includes: S3-1: Add 100 μL of IL-6 antibody diluted with pH 9.6 carbonate buffer to a concentration of 2 μg / mL, place in a 37°C incubator for 2 h, and wash the plate with PBST solution after completion; S3-2: Add 150 μL of PBS solution containing 1% BSA and incubate at 37°C for 2 h for blocking. Afterwards, wash the plate with PBST solution.

8. The method according to claim 5, 6 or 7, characterized in that: The evaluation method of the effects of different substrates on nonspecific adsorption includes: S4-1: Take the above-mentioned different treated substrate plates respectively, add 50 μL of IL-6 antigen standard, the concentrations of the antigen standard are: 1600, 800, 400, 200, 100, 50, 25, 0 pg / mL, place at 37°C for 1 hour, and wash the plate with PSBT solution after completion; S4-2: 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; S4-3: Place the detection plate on a digital fluorescent immunoassay analyzer for reading and analysis.

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