阅读说明：本技术 特异性抗体的检测标记物、其制备方法、其应用及试剂盒 (Specific antibody detection marker, preparation method, application and kit thereof ) 是由 王志新 李小冬 高省 于 2020-05-13 设计创作，主要内容包括：本发明适用于生物技术领域,提供了一种特异性抗体的检测标记物、其制备方法、其应用及试剂盒,该特异性抗体为2019-nCov特异性抗体,其检测标记物包括胶体金和2019-nCov抗原；所述2019-nCov抗原特异性地与所述2019-nCov特异性抗体结合。本发明将2019-nCov抗原通过物理吸附的方法固定在胶体金颗粒上,便可通过胶体金的颜色改变程度来反映与2019-nCov抗原进行特异性结合的2019-nCov特异性抗体的量,从而可以快速定性或定量检测新冠2019-nCov病毒感染后的抗体变化。(The invention is suitable for the technical field of biology, and provides a detection marker of a specific antibody, a preparation method, application and a kit thereof, wherein the specific antibody is a 2019-nCov specific antibody, and the detection marker comprises colloidal gold and a 2019-nCov antigen; the 2019-nCov antigen specifically binds to the 2019-nCov-specific antibody. According to the invention, the 2019-nCov antigen is fixed on the colloidal gold particles by a physical adsorption method, so that the amount of the 2019-nCov specific antibody specifically bound with the 2019-nCov antigen can be reflected by the color change degree of the colloidal gold, and thus, the antibody change after the new crown 2019-nCov virus infection can be rapidly detected qualitatively or quantitatively.)

1. A detection marker of a specific antibody, wherein the specific antibody is a 2019-nCov specific antibody, and the detection marker comprises colloidal gold and a 2019-nCov antigen; the 2019-nCov antigen specifically binds to the 2019-nCov-specific antibody.

2. The method for preparing a detection marker for a specific antibody according to claim 1, comprising the steps of:

respectively preparing a colloidal gold solution and a 2019-nCov antigen diluent for later use;

adjusting the pH value of the colloidal gold solution to 8-10, and mixing with the 2019-nCov antigen diluent to obtain a mixed solution;

adding a buffer solution containing glycine into the mixed solution for sealing treatment, and then separating to obtain a precipitate;

and dissolving the precipitate by using a dissolving solution to obtain the detection marker.

3. The method for preparing a detection marker for a specific antibody according to claim 2, wherein the method for preparing the colloidal gold solution comprises the steps of:

and (3) placing chloroauric acid and sodium citrate into boiling water for reaction to obtain the colloidal gold solution.

4. The method for preparing a detection marker for a specific antibody according to claim 3, wherein the step of reacting chloroauric acid with sodium citrate in boiling water to obtain the colloidal gold solution specifically comprises:

preparing a chloroauric acid solution with the mass concentration of 1-3% and a sodium citrate solution with the mass concentration of 1-3% for later use;

adding the chloroauric acid solution and the sodium citrate solution into boiling water, continuously boiling, cooling, and adding deionized water to supplement evaporated water to obtain the colloidal gold solution; the volume ratio of the boiling water to the chloroauric acid solution to the sodium citrate solution is (80-120): (0.5-1.5): 0.5-1.5).

5. The method for preparing a detection marker for a specific antibody according to claim 4, wherein the concentration of the 2019-nCov antigen in the 2019-nCov antigen dilution is 0.1-0.3 mg/mL; the volume ratio of the 2019-nCov antigen diluent to the colloidal gold solution is (0.05-0.07): 1.

6. The method for preparing a detection label for a specific antibody according to claim 2, wherein a final concentration of glycine is 10 to 30mmol/L in the step of adding a buffer solution containing glycine to the mixture solution, performing blocking treatment, and separating to obtain a precipitate.

7. The method for preparing a detection label for a specific antibody according to claim 2, wherein the lysis solution comprises tween-20, bovine serum albumin and glycine; the mass concentration of the Tween-20 in the dissolving solution is 0.1-0.3%, the mass concentration of the bovine serum albumin is 1-3%, and the concentration of the glycine is 10-30 mmol/L.

8. The detection marker prepared by the preparation method according to any one of claims 2 to 7, wherein the particle size of the colloidal gold is 70 to 90 nm.

9. Use of a detection marker according to claim 1 or 8 for the preparation of a reagent for the detection of 2019-nCov specific antibodies.

10. A kit for detecting 2019-nCov-specific antibodies, wherein the kit comprises a detection marker of claim 1 or 8.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a detection marker of a specific antibody, a preparation method, application and a kit thereof.

Background

2019-nCov is a novel coronavirus, and people infected with the coronavirus have common signs of respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like, and cause serious harm to human beings.

In addition, the self-protection mechanism is initiated when the normal body is attacked by foreign substances, the generation of specific antibodies by the immune system of the body is one of the self-protection modes of the body, and the generation of specific antibodies capable of recognizing the foreign attacking substances is a typical expression of the immune mechanism. The body will produce specific immunoglobulin IgM in about 3-5 days, specific immunoglobulin IgA in 10-30 days and specific immunoglobulin IgG in 3-5 months, and the produced specific IgM, IgA or IgG is combined with foreign matter and eliminated. If the attack force of the foreign substances is increased continuously, and the generated specific IgM, IgA or IgG cannot remove the attacking substances in time, the immune system of the organism is broken down, and the organism is seriously damaged until the life is threatened.

Therefore, the clinical significance of timely and rapidly detecting the 2019-nCov virus specific antibody is very important.

Disclosure of Invention

The purpose of the embodiments of the present invention is to provide a detection marker for a specific antibody, which is intended to solve the problems mentioned in the background art.

The embodiment of the invention is realized by a detection marker of a specific antibody, wherein the specific antibody is a 2019-nCov specific antibody, and the detection marker comprises colloidal gold and a 2019-nCov antigen; the 2019-nCov antigen specifically binds to the 2019-nCov-specific antibody.

Another object of the embodiments of the present invention is to provide a method for preparing a detection marker for the specific antibody, which comprises the following steps:

respectively preparing a colloidal gold solution and a 2019-nCov antigen diluent for later use;

adjusting the pH value of the colloidal gold solution to 8-10, and mixing with the 2019-nCov antigen diluent to obtain a mixed solution;

adding a buffer solution containing glycine into the mixed solution for sealing treatment, and then separating to obtain a precipitate;

and dissolving the precipitate by using a dissolving solution to obtain the detection marker.

As a preferable scheme of the embodiment of the present invention, the preparation method of the colloidal gold solution includes the following steps:

and (3) placing chloroauric acid and sodium citrate into boiling water for reaction to obtain the colloidal gold solution.

As another preferable scheme of the embodiment of the present invention, the step of placing chloroauric acid and sodium citrate in boiling water for reaction to obtain the colloidal gold solution specifically includes:

preparing a chloroauric acid solution with the mass concentration of 1-3% and a sodium citrate solution with the mass concentration of 1-3% for later use;

adding the chloroauric acid solution and the sodium citrate solution into boiling water, continuously boiling, cooling, and adding deionized water to supplement evaporated water to obtain the colloidal gold solution; the volume ratio of the boiling water to the chloroauric acid solution to the sodium citrate solution is (80-120): (0.5-1.5): 0.5-1.5).

As another preferable scheme of the embodiment of the invention, the concentration of the 2019-nCov antigen in the diluent of the 2019-nCov antigen is 0.1-0.3 mg/mL; the volume ratio of the 2019-nCov antigen diluent to the colloidal gold solution is (0.05-0.07): 1.

In another preferable embodiment of the invention, in the step of adding a buffer solution containing glycine to the mixed solution to perform blocking treatment and then separating to obtain a precipitate, the final concentration of glycine is 10 to 30 mmol/L.

As another preferable scheme of the embodiment of the present invention, the dissolving solution includes tween-20, bovine serum albumin and glycine; the mass concentration of the Tween-20 in the dissolving solution is 0.1-0.3%, the mass concentration of the bovine serum albumin is 1-3%, and the concentration of the glycine is 10-30 mmol/L.

The embodiment of the invention also aims to provide the detection marker prepared by the preparation method, wherein the particle size of the colloidal gold is 70-90 nm.

Another object of the embodiments of the present invention is to provide an application of the above detection marker in preparation of a reagent for detecting 2019-nCov specific antibody.

It is another object of embodiments of the present invention to provide a kit for detecting 2019-nCov-specific antibodies, which comprises the above-mentioned detection marker.

According to the detection marker of the specific antibody, the 2019-nCov antigen expressed by genetic engineering is fixed on the colloidal gold particles by a physical adsorption method, and the original color of the colloidal gold (changed from brownish red with the wavelength of 550nm to bluish black with the wavelength of 680 nm) is changed after the antigen fixed on the colloidal gold is specifically combined with the 2019-nCov specific antibody generated by an organism, so that the amount of the 2019-nCov specific antibody specifically combined with the 2019-nCov antigen can be reflected by the color change degree of the colloidal gold, and the change of the antibody after the new crown 2019-nCov virus infection can be quickly, qualitatively or quantitatively detected.

Drawings

FIG. 1 is an electron micrograph of gold in a colloidal solution obtained in example 3 of the present invention.

FIG. 2 is a diagram showing the light absorption spectrum of gold in a colloidal solution obtained in example 3 of the present invention.

FIG. 3 is a light absorption spectrum of a colloidal gold-antigen-antibody conjugate obtained in example 4 of the present invention.

FIG. 4 is a standard graph for detecting 2019-nCov-specific antibodies obtained in example 4 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.