CN115060913A - A protein chip for quantitatively detecting multiple cytokines and its preparation method and kit - Google Patents

Abstract

The present application relates to the field of biological detection technology, in particular to a protein chip for quantitative detection of various cytokines, a preparation method and a kit thereof. A protein chip for quantitatively detecting multiple cytokines includes a substrate and at least one antigen-specific antibody, and the antigen-specific antibody is immobilized on the substrate to form an antibody microarray. Kits for quantitative detection of multiple cytokines, including protein chips, adapters, cytokine standards, and fluorescent dye-labeled detection antibodies. The present application has the effects of high sensitivity, strong specificity, accuracy and efficiency, and is suitable for various scenarios.

Description

A protein chip for quantitatively detecting multiple cytokines and a preparation method thereof and kits

technical field

The present application relates to the field of biological detection technology, in particular to a protein chip for quantitative detection of various cytokines, a preparation method and a kit thereof.

Background technique

Cytokines are small-molecule polypeptides or glycoproteins mainly synthesized and secreted by immune cells, including interleukins, interferons, tumor necrosis factors, growth factors, and chemokines. Numerous cytokines are secreted in the body by paracrine, autocrine or endocrine, and have the characteristics of pleiotropic, overlapping, antagonistic, and synergistic, forming a very complex cytokine regulatory network, and are also involved in many important human body. The physiological functions of cytokines, such as mediating intercellular interactions, regulating cell growth, differentiation and maturation, function maintenance, regulating immune response, participating in inflammatory response, wound healing, and tumor growth and decline, etc., so the quantitative detection of cytokines is very necessary for disease diagnosis.

The traditional quantitative detection methods of cytokines mainly include: enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), western blot, liquid magnetic beads method (LUMINEX) and so on. Among them, enzyme-linked immunosorbent assay is the most common, with high sensitivity and specificity. However, the experimental steps are complex and the throughput is low. Due to the limitation of the bottom area of the ELISA plate, the amount of coated antibody or antigen is large, so the cost is high. In multiple cytokine detection, this method cannot be used for rapid detection; radioimmunoassay (RIA) has high sensitivity, but has radioactive contamination, which poses a potential threat to the health of the experimental operator; the immunoblotting method is cumbersome to operate, has a long cycle, a single detection index, and requires high experimental skills of the operator, and is also not suitable for multiplex Cytokine detection. In the liquid-phase magnetic bead method, antigen-specific antibodies are immobilized on magnetic beads with different fluorescent wavelengths. A different bead signal is read to represent the test substance, and another fluorescent signal represents the concentration on the bead. Because the magnetic beads immobilized with different antigen-specific antibodies are carried out in the same reaction system, the antigen-specific antibodies have cross-interference with each other, so that the reproducibility of the detection results is poor and the cost is high.

Therefore, it is necessary to develop a rapid, sensitive, simple and convenient multiplex cytokine detection kit.

SUMMARY OF THE INVENTION

In order to solve the problems of high cost and long detection period of the existing detection methods, the present application provides a protein chip kit for quantitative detection of various cytokines, which has high sensitivity, strong specificity, accuracy and efficiency, and is suitable for various scenarios.

In the first aspect, a protein chip kit for quantitatively detecting multiple cytokines provided by this application adopts the following technical scheme:

A protein chip for quantitatively detecting multiple cytokines comprises a substrate and at least one antigen-specific antibody, the antigen-specific antibody is immobilized on the substrate to form a plurality of mutually separated antibody microarrays.

By adopting the above technical solution, different antigen-specific antibodies are separated from each other and fixed on the surface of the substrate, so the interference of different antigen-specific antibodies with each other can be reduced, that is, the possibility of cross-contamination between samples is reduced, and high-throughput detection of cytokines is feasible. sex.

At the same time, the mutual interference of fluorescent signals of different microarray inspections is reduced, so multiple cytokines can be simultaneously detected at one time, and the sensitivity of detecting cytokines can reach the sensitivity of single-factor ELISA detection.

Optionally, the antigen-specific antibodies to each analyte are replicated at least three times in the microarray.

By adopting the above technical solution, the antigen to be detected is repeated at least three times, then at least three times the experimental result of ELISA can be obtained from a single sample.

Optionally, the substrate is PS, PC, PVC, PMMA or other materials of the same type.

Optionally, a plurality of the antigen-specific antibodies are specific for interferon gamma, interleukin-1, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-8, interleukin-10, interleukin-13 and tumor necrosis factor-alpha. One or a combination of antibodies.

Optionally, the coating concentration of various antigen-specific antibodies can be 100-500 μg/ml.

In the second aspect, the preparation method of the above-mentioned protein chip provided by this application adopts the following technical scheme:

A preparation method of the above protein chip, comprising the following steps:

S1, take the substrate and modify it to obtain a modified substrate;

S2. Spot 100-1000pl of PBS buffer of specific antibody protein on the modified substrate, and incubate the spotted modified substrate at 20-37°C for 1-2 hours or in a wet box at 2-8°C overnight After sealing, store at 2-8°C for later use.

Optionally, the modification of the substrate can be one or a combination of random adsorption, EDC, EDC-NHS, active aldehyde group, active carboxyl group, active amino group, dopamine coating, and receptor-ligand method.

Optionally, each antigen-specific antibody is repeated at least three times in each antibody microarray, and the spotting manner may be to spot only one or a combination of several different antigen-specific antibodies on one chip.

Optionally, use 3%-10% nonfat dry milk, 5% BSA or ethanolamine to incubate for 1-2 hours at 16-37°C, then wash and block.

In the third aspect, a kit for quantitatively detecting multiple cytokines provided by the application adopts the following technical scheme:

A kit for quantitatively detecting multiple cytokines, comprising the above-mentioned protein chip, an adapter, a cytokine standard and a detection antibody labeled with a fluorescent dye.

The detection antibody can be mixed with one or more antigen-specific detection antibodies labeled with fluorescent dyes according to the experimental requirements. The fluorescent dyes can be: Cy3, Cy5, FITC and other fluorescent dyes with similar absorption wavelengths; the standard contains one or several Mixtures with graded concentrations of the cytokines.

Optionally, the adapter is an antibody microarray nested segmented adapter framework, each of which contains one antibody microarray.

To sum up, the present application includes at least one of the following beneficial technical effects:

1. Different antigen-specific antibodies are separated from each other and fixed on the surface of the substrate, so it can reduce the interference of different antigen-specific antibodies, which reduces the possibility of cross-contamination between samples, and has the feasibility of high-throughput detection of cytokines.

2. The kit is equipped with an adapter frame that can nest and divide antibody microarrays. Each small area contains an antibody microarray, and each small area contains an antibody microarray. In this way, multiple different samples can be tested simultaneously on the same slide. The dimensions of the multiple detachable frames are adapted to the size of the conventional orifice plate, and can be directly used for sample monitoring of the corresponding orifice plate. In contrast to conventional ELISA, the reaction system is a multiplex ELISA detection of multiple samples on the surface of a glass slide.

3. In order to ensure that the protein chip is easy to operate, does not cross-contaminate between samples, and is a high-throughput method, each specific antibody protein chip array is arranged on a plastic protein chip, and the arrangement of antibodies can be adjusted and improved according to different experimental needs. After the protein chip reaction is completed, the substrate can be directly scanned by a laser scanner, which can be scanned by the chip scanner produced by CapitalBioTechnology or the Juli Stage scanning splicing microscope produced by NanoEntek, and then scanned by digital processing software. The fluorescent signal was converted into a specific number, and then the concentration of cytokines was converted by software.

Description of drawings

1 is a schematic diagram of the overall structure of the adapter of the present application;

Fig. 2 is the exploded schematic diagram of the adapter of the present application;

Fig. 3 is the top view of the adapter of the present application;

Fig. 4 is the sectional view of A-A in Fig. 3;

inserts联用的示意图；Figure 5 is the application adapter with orifice plate or

Schematic diagram of the combination of inserts;

Fig. 6 is the standard curve that embodiment 2 quantitatively detects interleukin 6;

7 is a schematic diagram of the results of quantitative detection of interleukin-6 and comparison with ELISA in Example 2 under different usage scenarios;

FIG. 8 is the standard curve of quantitative detection of various cytokines in Example 3. FIG.

Description of reference numerals: 1, outer frame; 2, inner frame; 3, soft latex sheet; 4, mounting hole; 5, positioning ring; 6, step table; 7, positioning block; 8, protein chip; 9, sample label Area.

Detailed ways

Example 1: Preparation of protein chip using interleukin-6 as an example

The preparation method of a protein chip using interleukin-6 as an example is as follows:

(1) The substrate of the protein chip 8 can be PS, PC, PVC, PMMA or other materials of the same type. The substrate material in Example 1 of the present application is PMMA. In addition, the modification of the substrate can be one or a combination of random adsorption, EDC, EDC-NHS, active aldehyde group, active carboxyl group, active amino group, dopamine coating, and receptor-ligand method. Example 1 of the present application adopts random adsorption.

(2) Spotting of anti-interleukin-6 antibody (DA012, Nearshore Protein Technology Co., Ltd.) was performed on the modified PMMA substrate. Among them, the coating concentration of various antigen-specific antibodies was 500 μg/ml, and 1000pl of PBS buffer (containing 10% glycerol) containing specific antibody proteins was spotted on the substrate, and each antigen-specific antibody was placed on each antibody. There are three replicates in the microarray, and the spotting method can be to spot only one or a combination of several different antigen-specific antibodies on a chip. A total of 16 chips that can fit into a 96-well plate were spotted, and the chips that had been spotted were incubated at 37°C for 2 hours, and stored at 2°C for later use or blocked.

It should be noted that the coating concentration and content of multiple antigen-specific antibodies can be different. In some embodiments, the coating concentration of multiple antigen-specific antibodies is 100-500 μg/ml, which will contain 100-1000pl specificity Antibody protein in PBS buffer (containing 10%-50% glycerol) was spotted on the substrate.

The incubation temperature can also be 20-37°C, the incubation time is 1-2 hours, or the incubation temperature can be 2-8°C overnight in a humid box; the storage temperature can also be 2-8°C.

Specifically, the blocking process was as follows: incubate with 5% nonfat dry milk at 37° C. for 2 hours, wash with PBS buffer (containing 10% glycerol) for 3 times for 5 minutes each time, in which, stand for 2 minutes and shake for 3 minutes.

Of course, in another embodiment, it can be 3%-10% skimmed milk powder, or 5% BSA or ethanolamine, incubated at 16-37°C for 1-2 hours and then treated with PBS buffer (containing 10%-50% glycerol) cleaning.

(3) After standing for 15 minutes, put the chip into the adapter frame structure that fits with the chip, put the mucosa on the chip, and vacuum seal the entire protein chip 8 and place it at 4° C. for later use.

A kit for quantitatively detecting a variety of cytokines, comprising the above-mentioned protein chip 8 taking interleukin 6 as an example, an adapter, a cytokine standard and a detection antibody labeled with a fluorescent dye.

1-4 , the adapter includes a soft latex sheet 3 , an inner frame 2 and an outer frame 1 . The outer frame 1 is provided with a plurality of mounting holes 4 for inserting the inner frame 2 , a positioning ring 5 is protruded on the inner periphery of the lower end of the outer frame 1 , and a stepped platform 6 is protruded from the lower end of the inner frame 2 . When the inner frame 2 is inserted into the installation hole 4 , the step platform 6 is clamped with the positioning ring 5 .

The soft latex sheet 3 is inserted into the inner frame 2 and abuts on the upper end surface of the step platform 6 . The outer periphery of the lower end of the soft latex sheet 3 is provided with a plurality of slope-shaped positioning blocks 7 , and the protein chip 8 is clamped between the positioning blocks 7 and the stepped platform 6 .

When installing, first insert the inner frame 2 into the outer frame 1, and then place the protein chip 8 on the outer frame 1 through the sloped soft latex sheet 3 on both sides to fasten the protein chip 8 on the outer frame 1, so that the holes do not cross-contaminate each other. matrix antibody chip.

Referring to Fig. 3, in addition, there are alignment lines and sample labeling areas 9 on the combined plastic hard frame and the protein chip 8; the size of each mounting hole 4 of the outer frame 1 is adapted to different orifice plates, or can be customized with microfluidics Adapters adapted to the size of the control chip.

inserts联用即可。此外，蛋白芯片8也可直接用于细胞实验，放入其中。Referring to Figure 5, the adapter can be used for cell experiments. It is only necessary to place the antibody-coated surface of the protein chip 8 on the bottom of the inner frame 2 during installation, and then place the entire structure upside down in the well plate or with

inserts can be used together. In addition, the protein chip 8 can also be directly used for cell experiments and put into it.

Example 2: Sensitivity of quantitative detection of interleukin-6 and its use in actual samples compared with ELISA test results

The method for quantitative detection of interleukin-6 using this kit is as follows:

(1) The IL-6 standard is from Nearshore Protein Technology Co., Ltd., product number C009. The standard is diluted with PBS buffer (containing 10% glycerol) to 30, 60, 90, 120, 150, 180 pg/ml, and set negative at the same time The control was PBS buffer (containing 10% glycerol). In fact, in other embodiments, the recombinant protein concentration used for the standard curve can be selected from different ranges, and is not limited to this concentration.

(2) Add the diluted 8 IL-6 standard products to the upper eight chips in the 2×8 protein chip 8 in Example 1, and write down the numbers.

inserts上层和下层细胞培养液样品2和3，已知浓度的IL-6稀释液样品4-8的浓度分别为(4，20，80，150，200pg/ml)，从样品1-8中各取100μl用于ELISA试剂盒检测。ELISA试剂盒为达科为生物技术有限公司的IL-6检测试剂盒，货号为1110602，具体操作步骤根据说明书进行操作。(3) Prepare test samples 1-8 and perform ELISA detection: cell supernatant sample 1 in 96-well plate, in 96-well plate

Inserts upper and lower cell culture fluid samples 2 and 3, with known concentrations of IL-6 dilution samples 4-8 at concentrations (4, 20, 80, 150, 200 pg/ml), respectively, from samples 1-8 Take 100 μl for ELISA kit detection. The ELISA kit is the IL-6 detection kit from Daktronics Biotechnology Co., Ltd., the product number is 1110602, and the specific operation steps are carried out according to the instructions.

(4) The protein chip 8 and the protein chip 8 with the adapter were placed in samples 1-3 and incubated respectively. See Figure 5 for different sample placement methods; samples 4-8 were respectively added to the different chips shown in Figure 3 and incubated.

(5) The chip was washed three times with PBS buffer (containing 10% glycerol + 0.05% Tween 20) after the chip was taken out from the sample.

(6) Add CY3-labeled detection antibody and incubate for 1 h.

(7) After washing 3 times with PBS buffer (containing 10% glycerol + 0.05% Tween 20), scan with Juli Stage, and analyze and calculate with professional software.

Results: Using the standard curve and detection results of the kit of this application and ELISA detection, refer to Figure 6 and Figure 7, wherein,

(1) The standard curve of the protein chip has good linearity and R ² >99%.

inserts上层和下层细胞样品2和3)的蛋白芯片检测结果和ELISA结果基本一致，证明该检测方法的准确度高，实际可行。(2) Among the 8 samples tested, the actual samples 1-3 (96-well plate cell sample 1, 96-well plate

The protein chip detection results of the upper and lower cell samples 2 and 3) of the inserts are basically consistent with the ELISA results, which proves that the detection method has high accuracy and is practical.

(3) The known concentration of IL-6 dilution samples 4-8, the concentrations are (4, 20, 80, 150, 200pg/ml), and the accuracy and repeatability of the protein chip detection of samples 5-8 are excellent In addition, sample 4 was not detected by ELISA, but the protein chip was accurately detected, which proved that the protein chip has higher sensitivity.

Example 3: Comparison of Sensitivity for Quantitative Detection of Various Cytokines in Actual Samples and ELISA Test Results Using this kit to quantitatively detect interleukin 6, interleukin 8, tumor necrosis factor alpha and interleukin 1 beta, the preparation method of protein chip is the same as that in Example 1 :

(1) Add the diluted standard mixtures of 8 different concentrations of IL-6, IL-8, TNF-α and IL-1β to the upper eight chips in the 2×8 chips in Example 1, respectively, and write the number.

inserts上层和下层细胞样品2和3，和已知浓度的细胞因子稀释液混合样品(IL-6，IL-8,TNF-α和IL-1β均已同样的浓度混合)4-8的浓度分别为(10，50，100，150，200pg/ml)，从样品1-8中各取100μl用于ELISA试剂盒检测。ELISA试剂盒为达科为生物技术有限公司的检测试剂盒(1110802,1110602,1110122,1117202)，具体操作步骤根据说明书进行操作。(2) Prepare test samples 1-8: 96-well plate cell sample 1, 96-well plate

Inserts upper and lower cell samples 2 and 3, and mixed samples of known concentrations of cytokine dilution (IL-6, IL-8, TNF-α and IL-1β have all been mixed at the same concentration) 4-8 concentrations respectively For (10, 50, 100, 150, 200 pg/ml), take 100 μl from each of samples 1-8 for ELISA kit detection. The ELISA kit is the detection kit (1110802, 1110602, 1110122, 1117202) of Daktronics Biotechnology Co., Ltd. The specific operation steps are operated according to the instructions.

(4) Place chips in samples 1-8 respectively and incubate for 1 h or place the chips in the culture equipment for a specified time.

(5) The chip was washed three times with PBS buffer (containing 10% glycerol + 0.05% Tween 20) after the chip was taken out from the sample.

(6) Add CY3-labeled mixed detection antibody and incubate for 1 h.

(7) After washing 3 times with PBS buffer (containing 10% glycerol + 0.05% Tween 20), scan with Juli Stage, and analyze and calculate with professional software.

Result: Refer to Figure 8 and Table 1 for the standard curve and detection results and ELISA detection results of the kit of the present invention, wherein,

(1) The standard curve R ² of the protein chip was >99%.

inserts上层和下层细胞样品2和3)的蛋白芯片检测结果和ELISA结果基本一致。(2) Among the 8 samples tested, the actual samples 1-3 (96-well plate cell sample 1, 96-well plate

The protein chip detection results of the upper and lower cell samples 2 and 3) of the inserts were basically consistent with the ELISA results.

(3) Cytokine dilution samples 4-8 with known concentrations, the concentrations are (10, 50, 100, 150, 200 pg/ml), respectively. The average value of the three results is shown in Table 1.

Table 1 Detection results table of various cytokines

It can be seen from Table 1 that the concentration accuracy of protein chip detection is relatively higher than that of ELISA; in addition, sample 4 is difficult to detect by ELISA due to the low concentration, but the protein chip can be detected, which proves that the protein chip has high sensitivity; according to the results, it can be seen that four detection methods There is no cross-reaction between the systems.

The above are all preferred embodiments of the present application, and are not intended to limit the protection scope of the present application. Therefore: all equivalent changes made according to the structure, shape and principle of the present application should be covered within the scope of the present application. Inside.

Claims (10)

1. A protein chip for quantitatively detecting multiple cytokines, characterized in that it comprises a substrate and at least one antigen-specific antibody, and the antigen-specific antibody is immobilized on the substrate to form an antibody microarray. 2 . The protein chip for quantitatively detecting multiple cytokines according to claim 1 , wherein the antigen-specific antibodies for each substance to be detected are repeated at least three times in the microarray. 3 . 3. The protein chip for quantitatively detecting multiple cytokines according to claim 1 or 2, wherein the multiple antigen-specific antibodies are interferon gamma, interleukin-1, interleukin-2, interleukin-4 , interleukin 5, interleukin 6, interleukin 8, interleukin 10, interleukin 13 and tumor necrosis factor alpha specific antibodies or a combination of several. 4 . The protein chip for quantitatively detecting multiple cytokines according to claim 3 , wherein the coating concentration of multiple antigen-specific antibodies is 100-500 μg/ml. 5 . 5. a preparation method of the described protein chip of any one of claim 1-4, is characterized in that, comprises the following steps: S1, take the substrate and modify it to obtain a modified substrate; S2. Spot 100-1000pl of PBS buffer of specific antibody protein on the modified substrate, and incubate the spotted modified substrate at 20-37°C for 1-2 hours or in a wet box at 2-8°C overnight After sealing, store at 2-8°C for later use. 6. The method for preparing a protein chip according to claim 5, wherein the modification of the substrate can be random adsorption, EDC, EDC-NHS, active aldehyde group, active carboxyl group, active amino group, dopamine coating, receptor - One or a combination of ligand methods. 7. The method for preparing a protein chip according to claim 5, wherein each antigen-specific antibody is repeated three times in each antibody microarray, and the spotting method is to spot only one or more on a protein chip. A combination of several different antigen-specific antibodies. 8. according to the preparation method of the described protein chip of claim 5, it is characterized in that: described closed process is: adopt 3%-10% skimmed milk powder, 5% BSA or ethanolamine 16-37 ℃ of incubation 1-2 hours Afterwards, clean the seal for later use. 9. A kit for quantitatively detecting multiple cytokines, characterized in that it comprises the detection of the protein chip (8) according to any one of claims 1-4, an adapter, a cytokine standard and a fluorescent dye label Antibody. 10 . The kit for quantitatively detecting multiple cytokines according to claim 9 , wherein the adaptor is an adaptor frame of antibody microarray nesting and partitioning, and each small area of the adaptor contains one antibody microarray. 11 .

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