CN119619487A

CN119619487A - A method for constructing colloidal gold for simultaneously labeling DNA and antibodies

Info

Publication number: CN119619487A
Application number: CN202411543011.4A
Authority: CN
Inventors: 董望; 吕慧芳; 井汇源; 李华玮; 彭志锋; 宋幸辉; 王金合; 王海花; 唐光武; 张艳
Original assignee: Henan University of Animal Husbandry and Economy
Current assignee: Henan University of Animal Husbandry and Economy
Priority date: 2024-10-31
Filing date: 2024-10-31
Publication date: 2025-03-14

Abstract

The invention relates to the technical field of medical biology, in particular to a method for constructing colloidal gold simultaneously marked DNA and antibody, which comprises the following steps: determining the optimal pH value of the colloidal gold-labeled antibody, preparing colloidal gold-labeled DNA, detecting the effect of the colloidal gold-labeled DNA, preparing colloidal gold-labeled DNA and antibody, detecting the effect of the colloidal gold-labeled DNA and antibody, and processing and testing experimental data. The invention connects colloidal gold with antibody by screening the optimal pH value, fixes DNA oligonucleotide on colloidal gold particles by freeze thawing method, finally connects DNA oligonucleotide with antibody and colloidal gold by freeze thawing method combined with electrostatic adsorption to construct nucleic acid-antibody-colloidal gold, the prepared colloidal gold-nucleic acid-antibody complex (Au-A-I-mAb) can combine with corresponding antigen, the DNA oligonucleotide can trigger hairpin to carry out HCR, and stronger fluorescence is generated, which indicates that the research method successfully prepares colloidal gold-nucleic acid-antibody complex and has good quality.

Description

Construction method for simultaneously labeling DNA and antibody by using colloidal gold

Technical Field

The invention relates to the technical field of medical biology, in particular to a method for constructing colloidal gold labeled DNA and antibody simultaneously.

Background

Gold nanoparticles (AuNPs), which refer to tiny particles of gold with a diameter of 1-100 nm, are generally present in the form of colloidal gold in aqueous solution, are considered to be the most stable metal nanoparticles, and are biocompatible (bioinert and low cytotoxicity). The nano gold has outstanding advantages due to its ultra-small size and high surface area to volume ratio, and can be functionalized by various molecules including polysaccharide, protein, peptide, fatty acid, plasmid or oligonucleotide, etc., so that the nano gold is widely applied to various biological sensing technologies and detection systems. The immune colloidal gold technology is a solid-phase labeling immune technology using colloidal gold as a marker and utilizing specific antigen-antibody reaction, and is widely applied to the research fields of biomedical immunohistochemistry, cytobiology and the like after a fluorescein, a radioisotope and an enzyme detection technology. The principle of colloidal gold labeling is essentially a coating process that biological macromolecules such as antibody proteins are adsorbed on the surfaces of colloidal gold particles, the adsorption mechanism is probably that negative charges carried on the surfaces of the colloidal gold particles and positive charges carried on protein molecules are mutually attracted by electrostatic force, firm combination is formed in Van der Waals attraction, and in addition, the roughness of the colloidal gold particles is an important condition favorable for forming adsorption, and the labeling process is mainly based on physical adsorption, so that the biological activity of the protein molecules is not obviously influenced. Currently, there are few methods for preparing colloidal gold-labeled antibodies and DNA at the same time, and it takes a long time.

Disclosure of Invention

Aiming at the defects and problems, the invention provides a method for constructing colloidal gold simultaneously marked DNA and antibody, wherein the colloidal gold is connected with the antibody by screening the optimal pH value, DNA oligonucleotides are fixed on colloidal gold particles by a freeze thawing method, finally the DNA oligonucleotides, the antibody and the colloidal gold are connected together by combining a freeze thawing method and electrostatic adsorption to construct nucleic acid-antibody-colloidal gold, and then the feasibility of the method is verified by combining an ELISA method.

The invention solves the technical problems by adopting a scheme that the method for constructing the colloidal gold simultaneously marked DNA and antibody comprises the following steps:

(1) And (3) determining the optimal pH value of the colloidal gold labeled antibody:

(2) Preparing colloidal gold-labeled DNA, namely fixing sulfhydryl-modified oligonucleotide A-I (DNA) on the surface of gold particles by adopting a freeze thawing method to serve as a colloidal gold-nucleic acid complex (Au-A-I) for standby;

(3) Detecting the effect of the colloidal gold-labeled DNA;

(4) Preparing colloidal gold-labeled DNA and antibody, namely preparing a colloidal gold-nucleic acid-antibody complex by a freeze thawing method and an electrostatic adsorption method;

(5) Detecting the effect of the colloidal gold on the DNA and the antibody;

(6) Experimental data were processed and examined.

Further, in the step (1), the step of determining the optimal pH value of the colloidal gold-labeled antibody is as follows:

S11, taking 3 centrifuge tubes, respectively adding 1mL colloidal gold, and respectively adjusting the pH values to 7, 8 and 9 by using 0.2mol/L K ₂CO₃;

s12, sequentially adding colloidal gold with different pH values into a 96-well plate, adding 100 mu L of each well, adding 2 mu L of 1 mg/mL of anti-GAPDH monoclonal antibody solution into each well, uniformly mixing, and standing for 15: 15min at room temperature;

S13, adding 20 mu L of 10% NaCl solution into each hole, uniformly mixing, and standing at room temperature for 10 min;

s14, observing color change in the hole, and recording the lowest pH value for maintaining the red color of the solution.

Further, in the step (2), the specific operation steps are as follows:

s21, 1mL of colloidal gold is taken into a glass bottle, and the pH value of the glass bottle is adjusted to 7.5 by using 0.2 mol/L of K ₂CO₃;

S22, activating 8 mu L A-I by using 4 mu L of 2 mmol/L of TCEP, and reacting at room temperature for 30 min after uniformly mixing;

s23, dropwise adding the obtained A-I into 200 mu L of colloidal gold solution;

S24, placing the colloidal gold nucleic acid mixed solution in a refrigerator at the temperature of-20 ℃ for freezing for 1h, taking out and melting, and placing in a centrifuge for centrifugation at 13000 r/min for 5 min;

S25, after removing the supernatant, re-dissolving the supernatant by 200 mu L of 0.01 mol/L PBS, and centrifuging the supernatant by 13000r/min for 5 min;

s26, after removing the supernatant, the supernatant was dissolved in 400. Mu.L of 0.01 mol/L PBS to prepare a colloidal gold-nucleic acid complex.

Further, in the step (3), the detection step of the effect of the colloidal gold-labeled DNA is as follows:

S31, taking a black ELISA plate, adding 50 mu L of Au-A-I and 1 mu L A-H1 and A-H2 respectively into a1 st hole, and supplementing the mixture to 100 mu L by using PBS buffer solution;

s32, 1.5. Mu.L TCEP activated A-I and 1. Mu. L A-H1 and A-H2 each were added to well 2 and filled to 100. Mu.L with PBS buffer;

S33, 1 mu L A-I and 1 mu L A-H1 and A-H2 each were added to well 3 and filled to 100. Mu.L with PBS buffer;

s34, 1 mu L A-H1 and A-H2 each was added to well 4 and filled to 100. Mu.L with PBS buffer (5 nM Mg2+);

s35, reacting for 1h in a constant temperature incubator at 37 ℃, detecting the result by using a multifunctional enzyme-labeled instrument, and collecting result data.

Further, the specific operation steps of the step (4) are as follows:

S41, 1mL of colloidal gold is put into a glass bottle, and the pH value is adjusted to 7.5 by using 0.2 mol/L of K ₂CO₃;

S42, activating 8 mu L A-I by using 4 mu L of 2 mmol/L TCEP, uniformly mixing and reacting at room temperature for 30 min;

S43, dripping the activated A-I into 200 mu L of colloidal gold solution, putting the colloidal gold nucleic acid mixed solution into a refrigerator at the temperature of minus 20 ℃ for freezing for 1 hour, and taking out and melting;

S44, dropwise adding 4 mu L of anti-GAPDH mAb (1 mg/mL) into the colloidal gold nucleic acid mixed solution, and carrying out shaking incubation for 4 hours at room temperature;

S45, after incubation, fully mixing 22 uL of 10% BSA solution with the colloidal gold-nucleic acid-antibody mixed solution, and mixing for 30min at room temperature to block redundant sites;

After S46, sealing and mixing are finished, the reaction product is placed in a centrifugal machine to be centrifuged at 13000 r/min for 5min, supernatant is removed and then is re-dissolved by 200 mu L of 0.01 mol/L PBS, then the reaction product is centrifuged at 13000 r/min for 5min, and 200 mu L of 0.01 mol/L PBS is removed and the supernatant is removed and is dissolved to be used as a colloidal gold-nucleic acid-antibody complex for standby.

Further, the detection flow of the step (5) is as follows:

S51, taking a black ELISA plate, adding 50 mu L of Au-A-I-mAb and 1 mu L A-H1 and A-H2 respectively into a1 st hole, and supplementing the mixture to 100 mu L by using PBS buffer solution;

S52, 50. Mu.L of Au-A-I and 1. Mu. L A-H1 and A-H2 each were added to well 2 and filled to 100. Mu.L with PBS buffer;

the steps S53-S55 are the same as the steps S33-S35;

S56, coating 50 uL PK-15 cell lysate containing GAPDH antigen in a black enzyme-labeled plate hole, incubating overnight at 4 ℃, washing the PBST plate for 3 times, and drying;

S57, incubating for 30min at room temperature by using a rapid sealing liquid I (200 uL/hole), washing the plate 3 times by using PBST, and beating to dry;

S58, respectively adding 50 uL Au-A-I-mAb, au-A-I or PBS into the holes, incubating for 1h at room temperature, washing the PBST plate for 5 times, and drying;

s59, adding 1 mu L A-H1 and A-H2 respectively, and supplementing to 100 mu L by using PBS buffer solution;

S510, reacting 1h in a 37 ℃ constant temperature incubator, detecting the result by using a multifunctional enzyme-labeled instrument, and collecting result data.

Further, in the step (6), the experiment was repeated several times, and the result is the mean ± Standard Deviation (SD) of the repeated several times, and the data were subjected to single factor analysis of variance and Bonferroni post-hoc test using SPSS software.

The invention has the beneficial effects that the colloidal gold is connected with the antibody by screening the optimal pH value, the DNA oligonucleotide is fixed on the colloidal gold particles by a freeze thawing method, and finally the DNA oligonucleotide, the antibody and the colloidal gold are connected together by combining the freeze thawing method and electrostatic adsorption to construct nucleic acid-antibody-colloidal gold, the prepared colloidal gold-nucleic acid-antibody complex (Au-A-I-mAb) can combine with the corresponding antigen, and the DNA oligonucleotide can trigger hairpin to carry out HCR to generate stronger fluorescence, so that the research method successfully prepares the colloidal gold-nucleic acid-antibody complex, has good quality, proves the feasibility of being applied to an ELISA method, and provides a technical basis for constructing a discrimination detection platform for various virus diseases based on the HCR technology.

Drawings

FIG. 1 shows the fluorescence detection result of the effect of labeling DNA with colloidal gold;

FIG. 2 shows the fluorescence detection results of the effect of simultaneously labeling DNA and antibody with colloidal gold;

FIG. 3 shows the results of the application of colloidal gold to simultaneously label DNA and antibodies;

FIG. 4 shows the measurement results of the optimal pH of the colloidal gold-labeled antibody.

Detailed Description

The invention will be further described with reference to the drawings and examples.

Referring to fig. 1-4, the invention provides a method for constructing colloidal gold labeled DNA and antibody simultaneously, which comprises the following steps:

the first embodiment is that the construction method is completed by the following steps:

(1) And (5) determining the optimal pH value of the colloidal gold labeled antibody. The specific measurement steps are as follows:

S12, sequentially adding colloidal gold with different pH values into a 96-well plate, adding 100 mu L of each well, adding 2 mu L of 1 mg/mL of anti-GAPDH monoclonal antibody solution into each well, uniformly mixing, and standing for 15: 15 min at room temperature;

As shown in fig. 4, the colloidal gold-antibody complex of 3 tubes was tested for stability at pH values of 7, 8, 9. The result shows that the color in the 3-tube colloidal gold solution has no obvious precipitate or color change, and the colloidal gold-antibody complex has good stability at pH values of 7, 8 and 9 and has strong tolerance to the pH value. In combination with the earlier references, a higher pH is advantageous for increasing the stability of the colloidal gold and a lower pH is advantageous for increasing the amount of adsorbed antibody. Therefore, to make the amount of antibody bound to colloidal gold higher, the optimum pH value was chosen to be 7.5 in this study.

(2) Colloidal gold-labeled DNA was prepared. The thiol-modified oligonucleotide A-I (DNA) is immobilized on the surface of gold particles by freeze thawing method and is used as colloidal gold-nucleic acid complex (Au-A-I) for standby. The specific operation steps are as follows:

S21, 1mL of colloidal gold (20 nm) is taken into a glass bottle, and the pH value is adjusted to 7.5 by using K ₂CO₃ of 0.2 mol/L;

S23, dropwise adding the obtained a-I to 200 μl of colloidal gold solution (ph=7.5);

S24, placing the colloidal gold nucleic acid mixed solution in a refrigerator at the temperature of-20 ℃ for freezing for 1h, taking out for melting, and placing in a centrifuge for centrifugation at 13000 r/min for 5min;

S25, removing the supernatant, re-dissolving with 200 mu L of 0.01 mol/L PBS, and centrifuging for 5min at 13000 r/min;

S26, after the supernatant was discarded, it was dissolved in 400. Mu.L of 0.01 mol/L PBS to prepare a colloidal gold-nucleic acid complex (Au-A-I).

(3) The effect of colloidal gold-labeled DNA was examined. The detection steps are as follows:

S31, taking a black ELISA plate, adding 50 mu L of Au-A-I and 1 mu L A-H1 and A-H2 respectively into a1 st hole, and supplementing to 100 mu L by using PBS buffer solution (5 nM Mg ²⁺);

S32, 1.5. Mu.L TCEP activated A-I and 1. Mu. L A-H1 and A-H2 each were added to well 2 and made up to 100. Mu.L with PBS buffer (5 nM Mg ²⁺);

s33, 1. Mu. L A-I and 1. Mu. L A-H1 and A-H2 each were added to well 3 and filled to 100. Mu.L with PBS buffer (5 nM Mg ²⁺);

S34, 1. Mu. L A-H1 and A-H2 each was added to well 4 and the wells were filled to 100. Mu.L with PBS buffer (5 nM Mg ²⁺).

And S35, reacting for 1h in a constant temperature incubator at 37 ℃, detecting the result by using a multifunctional enzyme-labeled instrument, setting fluorescence parameters as follows, wherein excitation wavelength is 494nm, emission wavelength is 522nm, and collecting result data.

The colloidal gold-nucleic acid complex prepared by adopting a freeze thawing method is tested for preparation effect by carrying out HCR experiments. As shown in FIG. 1, the negative control has very low fluorescence intensity because the A hairpin (A-H1 and A-H2) cannot be initiated due to the absence of the initiating chain, the positive control TCEP activates A-I or A-I to initiate HCR of the A hairpin to generate strong fluorescence, and the test group colloidal gold-nucleic acid complex (Au-A-I) also can initiate HCR of the A hairpin to generate stronger fluorescence, but has weaker fluorescence value compared with the positive control. The results indicate that the colloidal gold-nucleic acid complex prepared by the freeze-thawing method can successfully bind DNA to colloidal gold without affecting its original properties (e.g., the ability to trigger hairpin), but its DNA oligonucleotide triggering ability is reduced.

(4) Colloidal gold-labeled DNA and antibodies were prepared. According to the exploration of the test steps, the colloidal gold-nucleic acid-antibody complex is prepared by a freeze thawing method and an electrostatic adsorption method, and the specific operation steps are as follows:

S41, 1mL (20 nm) of colloidal gold is taken into a glass bottle, and the pH value is adjusted to 7.5 by using 0.2 mol/L of K ₂CO₃;

S43, dripping the activated A-I into 200 mu L of colloidal gold solution (pH=7.5), putting the colloidal gold nucleic acid mixed solution into a refrigerator at-20 ℃ for freezing for 1h, and taking out for melting;

S45, after incubation, 22uL of 10% BSA solution and the colloidal gold-nucleic acid-antibody mixed solution are fully mixed, and mixed for 30min at room temperature to block redundant sites;

(5) The effect of colloidal gold-labeled DNA and antibody was examined. The specific detection flow is as follows:

S51, taking a black ELISA plate, adding 50 mu L of Au-A-I-mAb and 1 mu L A-H1 and A-H2 respectively into a1 st hole, and supplementing the mixture to 100 mu L by using PBS buffer solution (containing 5 nM Mg ²⁺);

S52, 50. Mu.L of Au-A-I and 1. Mu. L A-H1 and A-H2 each were added to well 2, and filled to 100. Mu.L with PBS buffer

S53, 1. Mu. L A-I and 1. Mu. L A-H1 and A-H2 each were added to well 3 and filled to 100. Mu.L with PBS buffer (5 nM Mg ²⁺);

S54, adding 1 mu L A-H1 and A-H2 into the 4 th hole, and supplementing to 100 mu L by using PBS buffer solution (containing 5 nM Mg ²⁺);

S55, detecting a result by using a multifunctional enzyme-labeled instrument after reaction 1h in a 37 ℃ constant-temperature incubator, setting fluorescence parameters as follows, wherein excitation wavelength is 494 nm, emission wavelength is 522 nm, and collecting result data;

S56, coating 50 uL PK-15 cell lysate (containing GAPDH antigen) in a black enzyme-labeled plate hole, incubating overnight at 4 ℃, washing the PBST plate for 3 times and drying;

s59, adding 1 mu L A-H1 and A-H2 respectively, and supplementing to 100 mu L by using PBS buffer solution (containing 5 nM Mg ²⁺);

And S510, carrying out reaction 1h in a 37 ℃ constant temperature incubator, detecting the result by using a multifunctional enzyme-labeled instrument, setting fluorescence parameters as follows, wherein excitation wavelength is 494 nm, emission wavelength is 522 nm, and collecting result data.

The colloidal gold-nucleic acid-antibody complex is prepared by adopting a freeze thawing method and an electrostatic adsorption method, and the preparation effect is detected by carrying out an HCR experiment. As shown in FIG. 2, the negative control has very low fluorescence intensity because the A hairpin (A-H1 and A-H2) cannot be initiated without initiating chain, the positive control A-I can initiate the A hairpin to generate HCR and generate strong fluorescence, and the test group colloidal gold-nucleic acid-antibody complex (Au-A-I-mAb) can also initiate the A hairpin to generate HCR and generate stronger fluorescence, but the fluorescence value is weaker compared with the positive control and further weakened compared with the test group colloidal gold-nucleic acid complex (Au-A-I). The results show that the colloidal gold-nucleic acid complex prepared by the freeze thawing method can successfully link DNA and antibodies to colloidal gold without affecting the original characteristics (such as hairpin initiation ability) of the complex, but the initiation ability of DNA oligonucleotides is obviously reduced.

(6) Experimental data were processed and examined. Experiments were repeated three times and the result is the mean ± Standard Deviation (SD) of the three replicates. Data were analyzed by single factor analysis of variance and Bonferroni post hoc test using SPSS software (version 18.0). P <0.05 is statistically significant for the differences.

The main reagents and materials of the above test:

Gold nanoparticles (20 nm) were purchased from Shanghai Ala Biotechnology Co., ltd., anti-GAPDH monoclonal antibody was purchased from proteintech Co., PK-15 cell lysate was obtained from this laboratory, A set of oligonucleotide hybridization sequences (comprising A-I, A-H1, A-H2) were prepared from Shanghai Biotechnology Co., ltd. In China, the purification method was HPLC, the sequences were shown in Table 2, and quick blocking solution I, 20 XELISA coating buffer, 10 XPBST WB rinse solution, and black ELISA plate were purchased from Shanghai Biotechnology Co., ltd. In China. All reagents and solvents were of analytically pure grade. All solutions and reagents used in the experiments were prepared using ultrapure water.

The main instruments used for the experiment are shown in table 1.

Examples

Based on the first embodiment, in order to further explore the effect and application of the colloidal gold for simultaneously labeling DNA and antibody, the invention combines ELISA method to determine the quality of the colloidal gold-nucleic acid-antibody complex. As shown in FIG. 3, PK-15 cell lysate (containing GAPDH antigen) was coated in a black microplate well, and after PBS or Au-A-I was added, A hairpins (A-H1 and A-H2) were added after washing the plate, fluorescence intensity was extremely low, while Au-A-I-mAb was added, and after washing the plate, A hairpins (A-H1 and A-H2) were added, higher fluorescence intensity was detected. The results show that the colloidal gold-nucleic acid-antibody complex (Au-A-I-mAb) can be combined with the antigen at the bottom of the hole, so that the complex can not be eluted after washing the plate, and the priming chain (A-I) can trigger the hairpin to carry out HCR after adding the A hairpin (A-H1 and A-H2) to generate stronger fluorescence, thus indicating that the research method successfully prepares the colloidal gold-nucleic acid-antibody complex and has good quality.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

Claims

1. A method for constructing colloidal gold for simultaneously labeling DNA and antibodies, characterized in that it comprises 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 A-I (DNA) was fixed to the surface of gold particles by freeze-thaw method to prepare colloidal gold-nucleic acid complex (Au-A-I);

(3) Detecting the effect of colloidal gold labeling of 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 according to claim 1, characterized in that in step (1), the step of determining the optimal pH value of the colloidal gold-labeled antibody is:

S11. Take three 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 K ₂ CO ₃ ;

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 K ₂ CO ₃ ;

S22, activate 8 μL A-I with 4 μL 2 mmol/L TCEP, mix well and react at room temperature for 30 min;

S23, adding the activated A-I dropwise into 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. After discarding the supernatant, dissolve it with 400 μL 0.01 mol/L PBS and use it as colloidal gold-nucleic acid complex.

4. The method for constructing a colloidal gold-labeled DNA and antibody according to claim 1, characterized in that in step (3), the steps for detecting the effect of colloidal gold-labeled DNA are as follows:

S31. Take a black ELISA plate, add 50 μL Au-AI and 1 μL each of A-H1 and A-H2 to the first well, and fill up to 100 μL with PBS buffer (5 nM Mg ²⁺ );

S32, add 1.5 μL TCEP to activate A-I 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 A-I and 1 μL each of A-H1 and A-H2 to well 3, and fill up 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 enzyme marker, 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 K ₂ CO ₃ ;

S42, activate 8 μL A-I with 4 μL 2 mmol/L TCEP, mix well and react at room temperature for 30 min;

S43, adding the activated A-I 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 discarding the supernatant, it is re-dissolved with 200 μL 0.01 mol/L PBS, and then centrifuged at 13000 r/min for 5 min. After discarding the supernatant, 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-A-I-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-A-I 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 A-I 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-A-I-mAb, Au-A-I 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 according to claim 1, characterized in that in the step (6), the experiment is repeated multiple times, and the results are the mean ± standard deviation (SD) of multiple repetitions, and the data are subjected to one-way analysis of variance and Bonferroni post hoc test using SPSS software.