Construction method for simultaneously labeling DNA (Deoxyribose Nucleic Acid) and antibody by colloidal gold

Abstract

The invention relates to the technical field of medical biology, in particular to a construction method for simultaneously labeling DNA and an antibody through colloidal gold. Comprising the following steps: determining the optimum pH value of a colloidal gold labeled antibody, preparing colloidal gold labeled DNA, detecting the effect of the colloidal gold labeled DNA, preparing the colloidal gold labeled DNA and the antibody, detecting the effect of the colloidal gold labeled DNA and the antibody, and processing and inspecting experimental data. Colloidal gold and an antibody are connected by screening an optimal pH value, DNA oligonucleotide is fixed on colloidal gold particles by a freeze-thawing method, and finally the DNA oligonucleotide, the antibody and the colloidal gold are connected together by the freeze-thawing method in combination with electrostatic adsorption to construct nucleic acid-antibody-colloidal gold; the antibody of the prepared colloidal gold-nucleic acid-antibody compound (Au-A-I-mAb) can be combined with a corresponding antigen, and DNA oligonucleotide of the colloidal gold-nucleic acid-antibody compound (Au-A-I-mAb) can trigger a hairpin to perform HCR and generate stronger fluorescence, so that the colloidal gold-nucleic acid-antibody compound is successfully prepared by the research method and has good quality.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical biotechnology, and in particular to a method for constructing colloidal gold for simultaneously labeling DNA and antibodies. Background Art

[0002] Gold nanoparticles (AuNPs) refer to tiny gold particles with a diameter of 1-100 nm, usually in the form of colloidal gold in aqueous solution. They are considered to be the most stable metal nanoparticles with biocompatibility (bioinertness and low cytotoxicity). Nanogold has outstanding advantages due to its ultra-small size and high surface area to volume ratio. Nanogold can be functionalized with various molecules, including polysaccharides, proteins, peptides, fatty acids, plasmids or oligonucleotides, making it widely used in various biosensor technologies and detection systems. Immunocolloidal gold technology is a solid-phase labeled immunotechnology that uses colloidal gold as a marker and specific antigen-antibody reactions. Following the fluorescein, radioisotope and enzyme detection technologies, immunocolloidal gold technology has been widely used in research fields such as biomedical immunohistochemistry and cell biology. The principle of colloidal gold labeling is essentially the coating process of biomacromolecules such as antibodies and proteins being adsorbed onto the surface of colloidal gold particles. The adsorption mechanism may be that the negative charge on the surface of colloidal gold and the positive charge on the protein molecules attract each other by electrostatic force, and a strong bond is formed within the van der Waals force. In addition, the roughness of colloidal gold particles is also an important condition conducive to adsorption. Since this labeling process mainly relies on physical adsorption, it has no obvious effect on the biological activity of protein molecules. At present, there are few preparation methods for simultaneously labeling antibodies and DNA with colloidal gold, and it takes a long time. Summary of the invention

[0003] In view of the above-mentioned defects and problems, the present invention provides a construction method for simultaneously labeling DNA and antibodies with colloidal gold, wherein the colloidal gold is connected to the antibody by screening the optimal pH value, the DNA oligonucleotide is fixed on the colloidal gold particles by the freeze-thaw method, and finally the DNA oligonucleotide is connected with the antibody and colloidal gold by the freeze-thaw method combined with electrostatic adsorption to construct nucleic acid-antibody-colloidal gold. Afterwards, the feasibility of the method is verified by combining with the ELISA method. This study provides a technical basis for constructing an identification and detection platform for multiple viral diseases based on HCR technology.

[0004] The solution adopted by the present invention to solve the technical problem is: a method for constructing colloidal gold to simultaneously label DNA and antibodies, comprising the following steps: (1) Determination of the optimal pH value of colloidal gold labeled antibodies: (2) Preparation of colloidal gold-labeled DNA: The thiol-modified oligonucleotide AI (DNA) was fixed to the surface of gold particles by freeze-thaw method to prepare colloidal gold-nucleic acid complex (Au-AI); (3) Detecting the effect of colloidal gold labeled DNA; (4) Preparation of colloidal gold labeled DNA and antibodies: Preparation of colloidal gold-nucleic acid-antibody complexes by freeze-thaw method and electrostatic adsorption method; (5) Detecting the effect of colloidal gold labeled DNA and antibodies; (6) Process and verify experimental data.

[0005] Furthermore, in step (1), the step of determining the optimal pH value of the colloidal gold labeled antibody is: S11. Take 3 centrifuge tubes, add 1 mL of colloidal gold to each tube, and adjust the pH to 7, 8, and 9 respectively using 0.2 mol / L K2CO3; S12, add colloidal gold of different pH values ​​to a 96-well plate, 100 μL per well, and add 2 μL of 1 mg / mL anti-GAPDH monoclonal antibody solution to each well, mix well, and let stand at room temperature for 15 min; S13, add 20 μL of 10% NaCl solution to each well, mix well and let stand at room temperature for 10 min; S14. Observe the color change in the well and record the lowest pH value that maintains the red color of the solution.

[0006] Furthermore, in step (2), the specific operation steps are as follows: S21, take 1 mL of colloidal gold into a glass bottle, and adjust its pH to 7.5 using 0.2 mol / L K2CO3; S22, activate 8 μL AI with 4 μL 2 mmol / L TCEP, mix well and react at room temperature for 30 min; S23, adding the activated AI to 200 μL of colloidal gold solution; S24, place the colloidal gold-nucleic acid mixture in a -20°C refrigerator for 1 hour, take it out to melt, and centrifuge it at 13,000 r / min for 5 minutes; S25, discard the supernatant and re-dissolve with 200 μL 0.01 mol / L PBS, and then centrifuge at 13000 r / min for 5 min; S26. Discard the supernatant and dissolve the solution in 400 μL 0.01 mol / L PBS for use as colloidal gold-nucleic acid complex.

[0007] Furthermore, in step (3), the detection steps of the effect of colloidal gold labeling DNA are as follows: S31. Take a black ELISA plate, add 50 μL Au-AI and 1 μL A-H1 and A-H2 to the first well, and fill up to 100 μL with PBS buffer; S32, add 1.5 μL TCEP-activated AI and 1 μL each of A-H1 and A-H2 to the second well, and fill up to 100 μL with PBS buffer; S33, add 1 μL AI and 1 μL each of A-H1 and A-H2 to well 3, and fill to 100 μL with PBS buffer; S34, add 1 μL each of A-H1 and A-H2 to well 4 and fill to 100 μL with PBS buffer (5 nM Mg2+); S35. React in a 37°C constant temperature incubator for 1 hour, detect the results with a multifunctional microplate reader, and collect the result data.

[0008] Furthermore, the specific operation steps of step (4) are as follows: S41, take 1 mL of colloidal gold into a glass bottle and adjust its pH to 7.5 using 0.2 mol / L K2CO3; S42, activate 8 μL AI with 4 μL 2 mmol / L TCEP, mix well and react at room temperature for 30 min; S43, adding the activated AI dropwise to 200 μL of colloidal gold solution, freezing the colloidal gold-nucleic acid mixture in a -20°C refrigerator for 1 hour, and then taking it out to melt; S44, add 4 μL of anti-GAPDH mAb (1 mg / mL) to the colloidal gold-nucleic acid mixture and incubate at room temperature with shaking for 4 h; S45. After the incubation, 22 uL of 10% BSA solution was thoroughly mixed with the colloidal gold-nucleic acid-antibody mixture and mixed at room temperature for 30 min to block excess sites; S46. After the sealing and mixing is completed, the reaction product is placed in a centrifuge and centrifuged at 13000 r / min for 5 min. After the supernatant is discarded, it is re-dissolved with 200 μL 0.01 mol / L PBS, and then centrifuged at 13000 r / min for 5 min. After the supernatant is discarded, it is re-dissolved with 200 μL 0.01 mol / L PBS and used as colloidal gold-nucleic acid-antibody complex for later use.

[0009] Furthermore, the detection process of step (5) is as follows: S51, take a black ELISA plate, add 50 μL Au-AI-mAb and 1 μL A-H1 and A-H2 to the first well, and fill up to 100 μL with PBS buffer; S52, add 50 μL Au-AI and 1 μL each of A-H1 and A-H2 to the second well, and fill up to 100 μL with PBS buffer; The steps of S53 to S55 are the same as the steps of S33 to S35; S56, coated 50 uL of PK-15 cell lysate containing GAPDH antigen in the wells of a black ELISA plate, incubated overnight at 4°C, washed the plate three times with PBST and patted dry; S57, incubate with fast blocking solution I (200 uL / well) at room temperature for 30 min, wash the plate three times with PBST and pat dry; S58, add 50 uL Au-AI-mAb, Au-AI or PBS to the wells respectively, incubate at room temperature for 1 h, wash the plate 5 times with PBST and pat dry; S59, add 1 μL each of A-H1 and A-H2, and make up to 100 μL with PBS buffer; S510, react in a 37°C constant temperature incubator for 1 h, detect the results with a multifunctional microplate reader, and collect the result data.

[0010] Furthermore, in step (6), the experiment was repeated multiple times, and the results were the mean ± standard deviation (SD) of multiple repetitions. The data were analyzed by one-way ANOVA and Bonferroni post hoc test using SPSS software.

[0011] The beneficial effects of the present invention are as follows: the present invention connects colloidal gold with antibodies by screening the optimal pH value, fixes DNA oligonucleotides on colloidal gold particles by freeze-thaw method, and finally connects DNA oligonucleotides with antibodies and colloidal gold by freeze-thaw method combined with electrostatic adsorption to construct nucleic acid-antibody-colloidal gold; the colloidal gold-nucleic acid-antibody complex (Au-AI-mAb) prepared by the present invention has antibodies that can bind to corresponding antigens, and its DNA oligonucleotides can trigger hairpins to perform HCR and generate strong fluorescence, indicating that the research method successfully prepares the colloidal gold-nucleic acid-antibody complex with good quality, confirms the feasibility of its application in ELISA method, and provides a technical basis for constructing an identification and detection platform for multiple viral diseases based on HCR technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the fluorescence detection result of the effect of colloidal gold labeling DNA; Figure 2 This is the fluorescence detection result of the effect of colloidal gold simultaneously labeling DNA and antibodies; Figure 3 This is the application result of colloidal gold labeling DNA and antibodies simultaneously; Figure 4 This is the result of determining the optimal pH value for colloidal gold labeled antibodies. DETAILED DESCRIPTION

[0013] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0014] See also Figure 1-4 The present invention provides a technical solution for constructing a method for simultaneously labeling DNA and antibodies with colloidal gold: Embodiment 1: The construction method is completed by the following steps: (1) Determine the optimal pH value of colloidal gold labeled antibody. The specific determination steps are: S11. Take 3 centrifuge tubes, add 1 mL of colloidal gold to each tube, and adjust the pH to 7, 8, and 9 respectively using 0.2 mol / L K2CO3; S12, add colloidal gold of different pH values ​​to a 96-well plate, 100 μL per well, and add 2 μL of 1 mg / mL anti-GAPDH monoclonal antibody solution to each well, mix well, and let stand at room temperature for 15 min; S13, add 20 μL of 10% NaCl solution to each well, mix well and let stand at room temperature for 10 min; S14. Observe the color change in the well and record the lowest pH value that maintains the red color of the solution.

[0015] like Figure 4 As shown, the stability of colloidal gold of 3 tubes of colloidal gold-antibody complexes at pH values ​​of 7, 8, and 9 was tested. The results showed that there was no obvious precipitation or color change in the color of the 3 tubes of colloidal gold solution, indicating that the colloidal gold-antibody complex had good stability at pH values ​​of 7, 8, and 9, and had strong tolerance to pH values. Combined with previous references, a higher pH value is conducive to improving the stability of colloidal gold, and a lower pH value is conducive to increasing the amount of adsorbed antibodies. Therefore, in order to increase the amount of antibodies bound to colloidal gold, this study selected the optimal pH value of 7.5.

[0016] (2) Preparation of colloidal gold-labeled DNA. The thiol-modified oligonucleotide AI (DNA) was fixed to the surface of gold particles by freeze-thaw method, and used as colloidal gold-nucleic acid complex (Au-AI) for later use. The specific operation steps are as follows: S21, take 1mL of colloidal gold (20 nm) into a glass bottle and adjust its pH to 7.5 using 0.2 mol / L K2CO3; S22, activate 8 μL AI with 4 μL 2 mmol / L TCEP, mix well and react at room temperature for 30 min; S23, adding the activated AI to 200 μL of colloidal gold solution (pH=7.5); S24, place the colloidal gold-nucleic acid mixture in a -20°C refrigerator and freeze for 1 hour, then take it out to melt, and centrifuge it at 13,000 r / min for 5 minutes; S25, discard the supernatant and re-dissolve with 200 μL 0.01 mol / L PBS, and then centrifuge at 13000 r / min for 5 min; S26. Discard the supernatant and dissolve the solution in 400 μL 0.01 mol / L PBS for use as colloidal gold-nucleic acid complex (Au-AI).

[0017] (3) Detect the effect of colloidal gold labeling DNA. The detection steps are as follows: S31. Take a black ELISA plate, add 50 μL Au-AI and 1 μL A-H1 and A-H2 to the first well, and wash with PBS buffer (5 nM Mg 2+ ) Fill to 100 μL; S32, add 1.5 μL TCEP to activate AI and 1 μL A-H1 and A-H2 respectively to the second well, and wash with PBS buffer (5 nM mg 2+ ) Fill to 100 μL; S33, add 1 μL AI and 1 μL A-H1 and A-H2 to well 3, and wash with PBS buffer (5 nM Mg 2+ ) Fill to 100 μL; S34, add 1 μL of A-H1 and A-H2 to well 4 and wash with PBS buffer (5 nM Mg 2+ ) to make up to 100 μL.

[0018] S35. React in a 37°C constant temperature incubator for 1 hour, detect the results with a multifunctional microplate reader, set the fluorescence parameters as follows: excitation wavelength at 494 nm, emission wavelength at 522 nm, and collect the result data.

[0019] The colloidal gold-nucleic acid complex prepared by freeze-thaw method was tested by HCR experiment. Figure 1 As shown, the negative control has no initiating chain, so hairpin A (A-H1 and A-H2) cannot be initiated, so the fluorescence intensity is extremely low; the positive control TCEP activates AI or AI, which can trigger HCR of hairpin A and produce strong fluorescence; the colloidal gold-nucleic acid complex (Au-AI) in the experimental group can also trigger HCR of hairpin A and produce strong fluorescence, but its fluorescence value is weaker than that of the positive control. The results show that the colloidal gold-nucleic acid complex prepared by the freeze-thaw method can successfully connect DNA to colloidal gold without affecting its original properties (such as the ability to trigger hairpins), but the initiation ability of its DNA oligonucleotide is reduced.

[0020] (4) Preparation of colloidal gold labeled DNA and antibodies. Based on the exploration of the above experimental steps, colloidal gold-nucleic acid-antibody complexes were prepared by freeze-thaw method and electrostatic adsorption method. The specific operation steps are as follows: S41, take 1mL (20 nm) of colloidal gold into a glass bottle and adjust its pH to 7.5 using 0.2 mol / L K2CO3; S42, activate 8 μL AI with 4 μL 2 mmol / L TCEP, mix well and react at room temperature for 30 min; S43, add the activated AI dropwise to 200 μL colloidal gold solution (pH = 7.5), freeze the colloidal gold-nucleic acid mixture in a -20°C refrigerator for 1 hour, then take it out and melt it; S44, add 4 μL of anti-GAPDH mAb (1 mg / mL) to the colloidal gold-nucleic acid mixture and incubate at room temperature with shaking for 4 h; S45. After the incubation, 22uL of 10% BSA solution was fully mixed with the colloidal gold-nucleic acid-antibody mixture and mixed at room temperature for 30min to block the excess sites; S46. After the sealing and mixing is completed, the reaction product is placed in a centrifuge and centrifuged at 13000 r / min for 5 min. After the supernatant is discarded, it is re-dissolved with 200 μL 0.01 mol / L PBS, and then centrifuged at 13000 r / min for 5 min. After the supernatant is discarded, it is re-dissolved with 200 μL 0.01 mol / L PBS and used as colloidal gold-nucleic acid-antibody complex for later use.

[0021] (5) Detect the effect of colloidal gold labeling DNA and antibodies. The specific detection process is as follows: S51. Take a black ELISA plate, add 50 μL Au-AI-mAb and 1 μL A-H1 and A-H2 to the first well, and wash with PBS buffer (containing 5 nM Mg 2+ ) Fill to 100 μL; S52, add 50 μL Au-AI and 1 μL A-H1 and A-H2 to the second well, and fill to 100 μL with PBS buffer. S53, add 1 μL AI and 1 μL A-H1 and A-H2 to well 3, and wash with PBS buffer (containing 5 nM Mg 2+ ) Fill to 100 μL; S54, add 1 μL of A-H1 and A-H2 to well 4 and wash with PBS buffer (containing 5 nM Mg 2+ ) Fill to 100 μL; S55, react in a 37℃ constant temperature incubator for 1 h, and detect the results with a multifunctional microplate reader. The fluorescence parameters are set as follows: the excitation wavelength is 494 nm, the emission wavelength is 522 nm, and the result data are collected; S56, coated 50 uL PK-15 cell lysate (containing GAPDH antigen) in the wells of a black ELISA plate, incubated overnight at 4°C, washed the plate three times with PBST and patted dry; S57, incubate with fast blocking solution I (200 uL / well) at room temperature for 30 min, wash the plate three times with PBST and pat dry; S58, add 50 uL Au-AI-mAb, Au-AI or PBS to the wells respectively, incubate at room temperature for 1 h, wash the plate 5 times with PBST and pat dry; S59, add 1 μL of A-H1 and A-H2, and wash with PBS buffer (containing 5 nM Mg 2+ ) Fill to 100 μL; S510, react in a 37°C constant temperature incubator for 1 h, detect the results with a multifunctional microplate reader, set the fluorescence parameters as follows: excitation wavelength at 494 nm, emission wavelength at 522 nm, and collect the result data.

[0022] The colloidal gold-nucleic acid-antibody complex was prepared by freeze-thaw method and electrostatic adsorption method, and the preparation effect was tested by HCR experiment. Figure 2 As shown in the figure, the negative control has no initiator chain, and hairpin A (A-H1 and A-H2) cannot be initiated, so the fluorescence intensity is extremely low; the positive control AI can trigger HCR of hairpin A and produce strong fluorescence; the colloidal gold-nucleic acid-antibody complex (Au-AI-mAb) in the experimental group can also trigger HCR of hairpin A and produce strong fluorescence, but its fluorescence value is weaker than that of the positive control, and its fluorescence value is further weakened compared with the colloidal gold-nucleic acid complex (Au-AI) in the experimental group. The results show that the colloidal gold-nucleic acid complex prepared by the freeze-thaw method can successfully connect DNA and antibodies to colloidal gold without affecting its original properties (such as the ability to trigger hairpins), but the initiation ability of its DNA oligonucleotide is significantly reduced.

[0023] (6) Processing and testing of experimental data. The experiment was repeated three times, and the results are the mean ± standard deviation (SD) of the three replicates. SPSS software (version 18.0) was used for one-way analysis of variance and Bonferroni post hoc test. P < 0.05 was considered statistically significant.

[0024] The main reagents and materials for the above tests are: Gold nanoparticles (20 nm) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; anti-GAPDH monoclonal antibody was purchased from Proteintech; PK-15 cell lysate was from our laboratory; A set of oligonucleotide hybridization sequences (including AI, A-H1, A-H2) were prepared by Shanghai Sangon Biotechnology Co., Ltd., China, and the purification method was HPLC. The sequences are shown in Table 2; fast blocking solution I, 20×ELISA coating buffer, 10×PBST WB rinse solution, and black ELISA plate were purchased from Shanghai Sangon Biotechnology Co., Ltd., China. All reagents and solvents were of analytical grade. All solutions and reagents used in the experiment were prepared with ultrapure water.

[0025] The main instruments used in the experiment are shown in Table 1.

[0026] Example

[0027] On the basis of Example 1, in order to further explore the effect and application of colloidal gold labeling DNA and antibodies at the same time, the present invention combines the ELISA method to determine the quality of the colloidal gold-nucleic acid-antibody complex. Figure 3 PK-15 cell lysate (containing GAPDH antigen) was coated in the black ELISA plate wells. After adding PBS or Au-AI, A hairpins (A-H1 and A-H2) were added after washing, and the fluorescence intensity was extremely low; while after adding Au-AI-mAb, A hairpins (A-H1 and A-H2) were added after washing, and a higher fluorescence intensity was detected. The results show that the colloidal gold-nucleic acid-antibody complex (Au-AI-mAb) can bind to the antigen at the bottom of the well, thereby being fixed at the bottom of the well and cannot be washed off after washing. After adding A hairpins (A-H1 and A-H2), its initiator chain (AI) can trigger the hairpins to perform HCR and produce strong fluorescence, indicating that the method of this study successfully prepared the colloidal gold-nucleic acid-antibody complex with good quality.

[0028] The above description is only a preferred embodiment of the present invention and does not limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for constructing colloidal gold for simultaneously labeling DNA and antibodies, characterized in that: The following steps are involved: (1) Determination of the optimal pH value of colloidal gold labeled antibodies: (2) Preparation of colloidal gold-labeled DNA: The thiol-modified oligonucleotide AI (DNA) was fixed to the surface of gold particles by freeze-thaw method to prepare colloidal gold-nucleic acid complex (Au-AI); (3) Detecting the effect of colloidal gold labeled DNA; (4) Preparation of colloidal gold labeled DNA and antibodies: Preparation of colloidal gold-nucleic acid-antibody complexes by freeze-thaw method and electrostatic adsorption method; (5) Detecting the effect of colloidal gold labeled DNA and antibodies; (6) Process and verify experimental data.

2. The method for constructing a colloidal gold-labeled DNA and antibody simultaneously according to claim 1, characterized in that: In the step (1), the step of determining the optimal pH value of the colloidal gold labeled antibody is: S11. Take 3 centrifuge tubes, add 1 mL of colloidal gold to each tube, and adjust the pH to 7, 8, and 9 respectively using 0.2 mol / L K2CO3; S12, add colloidal gold of different pH values ​​to a 96-well plate, 100 μL per well, and add 2 μL of 1 mg / mL anti-GAPDH monoclonal antibody solution to each well, mix well, and let stand at room temperature for 15 min; S13, add 20 μL of 10% NaCl solution to each well, mix well and let stand at room temperature for 10 min; S14. Observe the color change in the well and record the lowest pH value that maintains the red color of the solution.

3. The method for constructing a colloidal gold-labeled DNA and antibody simultaneously according to claim 1, characterized in that: In step (2), the specific operation steps are as follows: S21, take 1 mL of colloidal gold into a glass bottle, and adjust its pH to 7.5 using 0.2 mol / L K2CO3; S22, activate 8 μL AI with 4 μL 2 mmol / L TCEP, mix well and react at room temperature for 30 min; S23, adding the activated AI to 200 μL of colloidal gold solution; S24, place the colloidal gold-nucleic acid mixture in a -20°C refrigerator and freeze for 1 hour, then take it out to melt, and centrifuge it at 13,000 r / min for 5 minutes; S25, discard the supernatant and re-dissolve with 200 μL 0.01 mol / L PBS, and then centrifuge at 13000 r / min for 5 min; S26. Discard the supernatant and dissolve the solution in 400 μL 0.01 mol / L PBS for use as colloidal gold-nucleic acid complex.

4. The method for constructing a colloidal gold-labeled DNA and antibody simultaneously according to claim 1, characterized in that: In step (3), the detection steps of the effect of colloidal gold labeling DNA are as follows: S31. Take a black ELISA plate, add 50 μL Au-AI and 1 μL A-H1 and A-H2 to the first well, and wash with PBS buffer (5 nM Mg 2+ ) Fill to 100 μL; S32, add 1.5 μL TCEP-activated AI and 1 μL each of A-H1 and A-H2 to the second well, and fill up to 100 μL with PBS buffer; S33, add 1 μL AI and 1 μL each of A-H1 and A-H2 to well 3, and fill to 100 μL with PBS buffer; S34, add 1 μL each of A-H1 and A-H2 to well 4 and fill to 100 μL with PBS buffer; S35. React in a 37°C constant temperature incubator for 1 hour, detect the results with a multifunctional microplate reader, and collect the result data.

5. The method for constructing a colloidal gold-labeled DNA and antibody simultaneously according to claim 1, characterized in that: The specific operation steps of step (4) are as follows: S41, take 1 mL of colloidal gold into a glass bottle and adjust its pH to 7.5 using 0.2 mol / L K2CO3; S42, activate 8 μL AI with 4 μL 2 mmol / L TCEP, mix well and react at room temperature for 30 min; S43, adding the activated AI dropwise to 200 μL of colloidal gold solution, freezing the colloidal gold-nucleic acid mixture in a -20°C refrigerator for 1 hour, and then taking it out to melt; S44, add 4 μL of anti-GAPDH mAb (1 mg / mL) to the colloidal gold-nucleic acid mixture and incubate at room temperature with shaking for 4 h; S45. After the incubation, 22uL of 10% BSA solution was fully mixed with the colloidal gold-nucleic acid-antibody mixture and mixed at room temperature for 30min to block the excess sites; S46. After the sealing and mixing is completed, the reaction product is placed in a centrifuge and centrifuged at 13000 r / min for 5 min. After the supernatant is discarded, it is re-dissolved with 200 μL 0.01 mol / L PBS, and then centrifuged at 13000 r / min for 5 min. After the supernatant is discarded, it is re-dissolved with 200 μL 0.01 mol / L PBS and used as colloidal gold-nucleic acid-antibody complex for later use.

6. The method for constructing a colloidal gold-labeled DNA and antibody simultaneously according to claim 4, characterized in that: The detection process of step (5) is as follows: S51, take a black ELISA plate, add 50 μL Au-AI-mAb and 1 μL A-H1 and A-H2 to the first well, and fill up to 100 μL with PBS buffer; S52, add 50 μL Au-AI and 1 μL each of A-H1 and A-H2 to the second well, and fill up to 100 μL with PBS buffer; S53, add 1 μL AI and 1 μL each of A-H1 and A-H2 to well 3, and fill to 100 μL with PBS buffer; S54, add 1 μL each of A-H1 and A-H2 to well 4 and fill to 100 μL with PBS buffer; S55, react in a 37°C constant temperature incubator for 1 hour, detect the results with a multifunctional microplate reader, and collect the result data; S56, coated 50 uL of PK-15 cell lysate containing GAPDH antigen in the wells of a black ELISA plate, incubated overnight at 4°C, washed the plate three times with PBST and patted dry; S57, incubate with fast blocking solution I (200 uL / well) at room temperature for 30 min, wash the plate three times with PBST and pat dry; S58, add 50 uL Au-AI-mAb, Au-AI or PBS to the wells respectively, incubate at room temperature for 1 h, wash the plate 5 times with PBST and pat dry; S59, add 1 μL each of A-H1 and A-H2, and make up to 100 μL with PBS buffer; S510, react in a 37°C constant temperature incubator for 1 h, detect the results with a multifunctional microplate reader, and collect the result data.

7. The method for constructing a colloidal gold-labeled DNA and antibody simultaneously according to claim 1, characterized in that: In step (6), the experiment was repeated multiple times, and the results were the mean ± standard deviation (SD) of multiple repetitions. The data were analyzed by one-way analysis of variance and Bonferroni post hoc test using SPSS software.

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