CN114578046A - Novel coronavirus (SARS-CoV-2) antibody detection reagent - Google Patents

Abstract

The invention discloses a new application of poly-ApoA I in a novel coronavirus antibody immunodetection reagent. By adding poly-ApoA I, the background signal value of the detection reagent can be significantly reduced, thereby effectively improving the sensitivity of the novel coronavirus antibody detection reagent.

Description

Novel coronavirus (SARS-CoV-2) antibody detection reagent

technical field

The present invention relates to the field of biotechnology, in particular, to a novel coronavirus (SARS-CoV-2) IgM/IgG antibody detection reagent.

Background technique

Apolipoprotein ApoA I (apolipoproteinAI) is composed of 243 amino acid residues and has a molecular mass of 28.3KD. It is mainly synthesized in the liver and is an important component of high-density lipoprotein (HDL). It is also an important component of HDL function. main performer. ApoA I plays an important role in cholesterol transport and metabolism, anti-atherosclerosis, anti-endotoxin, anti-inflammatory, anti-viral and inhibition of tissue damage caused by acute phase inflammatory response.

The novel coronavirus (SARS-CoV-2) is a large family of viruses known to cause colds as well as more serious diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). The new coronavirus is a new strain of coronavirus that has never been found in the human body before. Its transmission routes include direct transmission, aerosol transmission, and contact transmission.

The detection of the virus mainly adopts the method of fluorescent quantitative PCR for nucleic acid detection. In practice, due to differences in specimen types and collection methods, the positive rate of nucleic acid detection is likely to decrease; at the same time, the nucleic acid detection time is long, and the laboratory biosafety level is required. High, inconvenient for the general development of grass-roots hospitals. After the virus infects the human body, its IgM antibody is produced in about 5 to 7 days, and the IgG antibody can be produced in 10 to 15 days. Therefore, the detection of IgG and IgM content of SARS-CoV-2 in human serum is of great significance for the census and diagnosis of new pneumonia.

At present, there are many new crown IgG/IgM rapid detection kits on the market, and most of them are detected by indirect methods. However, due to the defects of the method itself and the non-specific adsorption of antigens, there are generally high background values and poor sensitivity. Defects. Therefore, it is of great application value to explore a technology that reduces the background signal of IgG/IgM detection and improves the sensitivity of the reagent.

In view of this, the present invention is proposed.

SUMMARY OF THE INVENTION

The purpose of the present invention is to solve the problems of low detection sensitivity and inaccurate detection results due to high background in the existing new coronavirus detection antibody test reagents. The present invention finds that adding poly ApoAI to the detection reagent can effectively reduce the background signal of the new crown detection reagent and improve the sensitivity of the detection reagent.

ApoAI is the protein part of plasma lipoprotein, which can bind and transport blood lipids to various tissues of the body for metabolism and utilization. ApoAI is composed of 243 amino acid residues and is a single polypeptide chain with a molecular weight of 28.3KD.

Poly-ApoAI refers to the polymerized form of ApoAI, and the molecular weight of poly-ApoAI is greater than 76.6KD.

Polymeric ApoAI can be polymerized from ApoAI monomers to form multimers by physical or chemical methods. For example, ApoAI multimer can be obtained by heat treatment, ApoAI multimer can be obtained by irradiating ApoAI, and protein denaturing agent can be added to obtain ApoAI multimer.

Unexpectedly, it has been found that adding poly ApoAI to the antibody reagent for the detection of new crown can significantly reduce the detection background signal and improve the detection sensitivity.

In order to realize the above-mentioned purpose of the present invention, the following technical solutions are specially adopted:

The invention provides the use of poly-ApoAI in the preparation of a novel coronavirus (SARS-CoV-2) antibody detection reagent.

The invention provides the use of adding poly ApoAI in the preparation of a novel coronavirus (SARS-CoV-2) antibody detection reagent, which can reduce background interference in detection, reduce noise and improve detection sensitivity.

In some embodiments of the present invention, the poly-ApoAI can be added to the sample diluent in the novel coronavirus (SARS-CoV-2) antibody detection reagent, and can also be added to the labeling solution.

In some embodiments of the present invention, the working concentration of poly-ApoAI is 0.01mg/ml-2mg/ml, and in some embodiments, the working concentration of poly-ApoAI is 0.01mg/ml, 0.02mg/ml, 0.04mg/ml , 0.08mg/ml, 1.6mg/ml, 1.7mg/ml, 1.8mg/ml, 1.9mg/ml, 2.0mg/ml; in other embodiments, the working concentration of poly-ApoAI is 0.1mg/ml- 1.5 mg/ml, in some embodiments the working concentration of polyApoAI is 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 1.5 mg/ml, 1.4 mg/ml, 1.3 mg/ml; in other In some embodiments, the working concentration of polyApoAI is 0.4mg/ml-1.2mg/ml, and in some experiments the working concentration of polyApoAI is 0.4mg/ml, 0.5mg/ml, 0.6mg/ml, 0.7mg /mll, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml, 1.1 mg/ml, 1.2 mg/ml.

The present invention also provides a novel coronavirus (SARS-CoV-2) antibody detection reagent, comprising a sample diluent and a labeling solution, wherein the sample diluting solution and/or the labeling solution contain poly ApoAI.

In the novel coronavirus (SARS-CoV-2) antibody detection reagent provided by the present invention, the working concentration of poly ApoAI is 0.01mg/ml-2mg/ml, and in some embodiments, the working concentration of poly ApoAI is 0.01mg/ml ml, 0.02mg/ml, 0.04mg/ml, 0.08mg/ml, 1.6mg/ml, 1.7mg/ml, 1.8mg/ml, 1.9mg/ml, 2.0mg/ml; in other embodiments, more The working concentration of poly-ApoAI is 0.1mg/ml-1.5mg/ml, in some embodiments the working concentration of poly-ApoAI is, 0.1mg/ml, 0.2mg/ml, 0.3mg/ml, 1.5mg/ml, 1.4 mg/ml, 1.3 mg/ml; in other embodiments, the working concentration of poly-ApoAI is 0.4 mg/ml-1.2 mg/ml, and in some experiments the working concentration of poly-ApoAI is 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml, 1.1 mg/ml, 1.2 mg/ml.

The term "working concentration" as used herein refers to the concentration used in the actual assay.

The present invention also provides a detection kit for detecting SARS-CoV-2 antibody by lateral immunochromatography.

The present invention also provides an immune detection kit for detecting novel coronavirus (SARS-CoV-2) antibodies, wherein the sample diluent in the kit contains poly-ApoAI.

The present invention also provides an immunoassay kit for detecting novel coronavirus (SARS-CoV-2) antibodies, wherein the marker liquid in the kit contains poly ApoAI.

Detailed ways

Below in conjunction with embodiment, the present invention is further elaborated:

Unless otherwise specified, the chemical reagents used in the examples of the present invention are all analytically pure and purchased from Sigma-aldrich Company. The chemiluminescence detector used was Hamamatsu BHP9507 chemiluminescence detector from Beijing Hamamatsu Photonics Technology Co., Ltd., China.

Example 1 Preparation of poly-ApoAI:

1. Collect 50L of serum, add 250ml of 10% polyanion and 2500ml of 2mol/L calcium chloride, mix well, set for 30 minutes at 4000 rpm, centrifuge for 20 minutes, and collect the supernatant.

2. Add guanidine hydrochloride to the upper layer to 6 mol/L, incubate at 37°C for 3 hours, separate the apolipoprotein by centrifugation, remove guanidine hydrochloride by dialysis with normal saline, adjust the density to 1.21g/cm3 with KBr, centrifuge again, and collect the centrifuge tube The lower third of the solution was dialyzed with physiological saline to remove KBr, and then dialyzed with 6 mol/L urea.

3. Use DEAE-SepharoseCL6B column gradient chromatography to collect the main peak which is pure ApoAI, remove urea by dialysis, measure the protein content with Shimadzu-UV2550 spectrophotometer at 280nm wavelength, the total ApoAI content is about 25g, adjust the ApoAI concentration to 10g/ ml.

4. Incubate 10g/ml of ApoAI in a 37°C water bath for 10h, use Sepharose G-25 column gradient chromatography, and collect the main peak as multimeric ApoAI.

Example 2 Test of poly ApoAI in chemiluminescence novel coronavirus (SARS-CoV-2) IgG antibody detection:

1. Using conventional preparation methods known to those skilled in the art, prepare a group of chemiluminescence kits containing poly-ApoAI in the sample dilution solution and a group of conventional chemiluminescence kits without poly-ApoAI.

component Ingredients/Uses R1 Sample diluent (experimental group contains 0.5mg/ml poly-ApoAI) R2 Magnetic bead-coated SARS-CoV-2 N or S antigen R3 Alkaline phosphatase-labeled anti-human IgG antibody Chemiluminescent substrate AMPPD cleaning concentrate Used to configure cleaning solution (PBST) quality control Goat polyclonal antibody cross-linked with human antibody

R1 can use conventional formula, containing phosphate buffer, inorganic salts, surfactants, preservatives.

2. Detect by the following steps:

(1) Add 10ul serum sample or 10ul quality control substance, 50ul R1 and 50ul R2 to the reaction cup;

(2) Incubate at 37°C for 5min, and add 100ul of R3 after washing four times;

(3) Incubate at 37°C for 5 min, wash four times and add chemiluminescence substrate for reading.

3. Test results:

From the above results, it can be seen that the background signal value of the negative serum in the poly-APOAI group was reduced by 40%-50%, and the signal-to-noise ratio (P/N) was increased by more than 90%, which significantly improved the sensitivity of the reagent.

Example 3 Test of poly ApoAI in chemiluminescence novel coronavirus (SARS-CoV-2) IgM antibody detection:

1. Using conventional preparation methods known to those skilled in the art, prepare a group of chemiluminescence kits containing poly-ApoAI in the sample dilution solution and a group of conventional chemiluminescence kits without poly-ApoAI.

R1 can use conventional formulations, containing Tris buffer pair, inorganic salts, surfactants, and preservatives.

2. Detect by the following steps:

(1) Add 10ul serum sample or 10ul quality control substance, 50ul R1 and 50ul R2 to the reaction cup;

(2) Incubate at 37°C for 5min, and add 100ul of R3 after washing four times;

(3) Incubate at 37°C for 5 min, wash four times and add chemiluminescence substrate for reading.

3. Test results:

From the above results, it can be seen that the background signal and quality control signal of the negative serum in the poly-APOAI group were significantly reduced, the negative serum samples decreased by 77%-78%, the signal-to-noise ratio increased by 43%-55%, and the reagents were significantly improved. sensitivity.

Example 4 Test of different concentrations of poly-APOAI in chemiluminescence novel coronavirus (SARS-CoV-2) IgG or IgM antibody detection:

According to the method of Example 3 or Example 4 above, diluents containing different concentrations of poly-APOAI were prepared and tested in the detection of chemiluminescence novel coronavirus (SARS-CoV-2) IgG or IgM antibodies. The results are as follows:

IgM detection result signal-to-noise ratio (P/N):

In the IgM detection, the addition of 0.01mg/ml-2.0mg/ml of poly-APOAI to the sample dilution can reduce the luminescence value of negative serum and quality control products, and the signal-to-noise ratio is higher than that of no poly-APOAI added. It has been improved, and the sensitivity of the detection reagent has been enhanced.

Signal-to-noise ratio (P/N) of IgG detection results:

In IgG detection, adding 0.01mg/ml-2.0mg/ml of poly-APOAI to the sample diluent can reduce the luminescence value of negative serum and improve the luminescence value of quality control products. The noise ratio has been improved, and the sensitivity of the detection reagent has been enhanced.

Example 5 Application of poly-APOAI in lateral immunochromatography detection reagent for novel coronavirus (SARS-CoV-2) antibody

Purchase an existing novel coronavirus (2019-nCoV) IgM/IgG antibody detection kit (colloidal gold method) kit (manufacturer A) on the market, take the sample diluent in the kit and divide it into 2 parts, and add poly APOAI to 1mg/ml, one serving without any treatment. The sera of 300 people with negative nucleic acid tests were tested, and the results were as follows:

negative result positive result Accuracy control 285 15 95% poly-APOAI 297 3 99%

From the above results, it can be seen that adding polymeric APOAI to the sample diluent can improve the accuracy of colloidal gold in detecting IgM/IgG of the new coronavirus (2019-nCoV) and effectively reduce false positive results.

Embodiment 6 Add poly-APOAI to the labeling solution to detect the test in the detection of IgG antibody of novel coronavirus (SARS-CoV-2).

1. Using conventional preparation methods known to those skilled in the art, prepare a set of chemiluminescence kits containing poly-ApoAI in a labeling solution (R3) A set of conventional chemiluminescence kits without poly-ApoAI.

R1 can use conventional formula, containing carbonate buffer pair, inorganic salts, surfactants, preservatives.

2. Detect by the following steps:

(1) Add 10ul serum sample or 10ul quality control substance, 50ul R1 and 50ul R2 to the reaction cup;

(2) Incubate at 37°C for 5min, and add 100ul of R3 after washing four times;

(3) Incubate at 37°C for 5 min, wash four times and add chemiluminescence substrate for reading.

3. Test results:

From the above results, it can be seen that the background signal value of the negative serum in the poly-APOAI group was reduced by 40%-50%, and the signal-to-noise ratio (P/N) was increased by more than 95%, which significantly improved the sensitivity of the reagent.

Embodiment 7 Add poly-APOAI to the labeling solution to detect the test in the detection of IgM antibody of novel coronavirus (SARS-CoV-2)

1. Using conventional preparation methods known to those skilled in the art, prepare a set of chemiluminescence kits containing poly-ApoAI in a labeling solution (R3) A set of conventional chemiluminescence kits without poly-ApoAI.

R1 can use conventional formula, containing phosphate buffer, inorganic salts, surfactants, preservatives.

2. Detect by the following steps:

(1) Add 10ul serum sample or 10ul quality control substance, 50ul R1 and 50ul R2 to the reaction cup;

(2) Incubate at 37°C for 5min, and add 100ul of R3 after washing four times;

(3) Incubate at 37°C for 5 min, wash four times and add chemiluminescence substrate for reading.

3. Test results:

From the above results, it can be seen that the background signal and quality control signal of negative serum in the poly-APOAI group were significantly reduced. sensitivity.

It can be seen from the detection results of the above different methodologies that the addition of poly-APOAI in the sample diluent or in the labeling solution can improve the detection sensitivity or accuracy of the antibody without being affected by the detection methodology.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. The application of poly-ApoAI in preparing novel coronavirus (SARS-CoV-2) antibody detection reagent is disclosed.

2. The use according to claim 1, wherein the working concentration of poly-apoai is between 0.01mg/ml and 2 mg/ml;

preferably the working concentration of the ApoA i is 0.1mg/ml to 1.5 mg/ml;

preferably the working concentration of the ApoA i is between 0.4mg/ml and 1.2 mg/ml.

3. The use of claim 1, wherein the poly-apoai is added to a sample diluent or to a labeling solution in a reagent for detecting antibodies to the type coronavirus (SARS-CoV-2).

4. The use according to claim 1, wherein the addition of said multimeric ApoAI reduces the background interference of said novel coronavirus (SARS-CoV-2) antibody detection reagent.

5. Use according to claim 1, characterized in that the novel coronavirus (SARS-CoV-2) antibody assay is the detection of IgG or IgM or IgA antibodies.

6. A novel coronavirus (SARS-CoV-2) antibody detection sample diluent, wherein the sample diluent contains poly-ApoAI;

preferably the working concentration of the ApoA i is 0.01mg/ml to 2 mg/ml;

preferably the working concentration of the ApoA i is 0.1mg/ml to 1.5 mg/ml;

preferably, the working concentration of the ApoA i is between 0.4mg/ml and 1.2 mg/ml.

7. A novel coronavirus (SARS-CoV-2) antibody detection marker fluid is characterized in that the marker fluid contains poly-ApoAI;

preferably the working concentration of the ApoA i is 0.01mg/ml to 2 mg/ml;

preferably the working concentration of the ApoA i is 0.1mg/ml to 1.5 mg/ml;

preferably the working concentration of the ApoA i is between 0.4mg/ml and 1.2 mg/ml.

8. The detection kit for detecting the novel coronavirus (SARS-CoV-2) antibody by lateral immunochromatography is characterized by comprising a lateral immunochromatography card and a sample diluent, wherein the sample diluent contains poly-ApoAI.

9. An immunoassay kit for detecting a novel coronavirus (SARS-CoV-2) antibody, the kit comprises a sample diluent and a marking solution, and is characterized in that the sample diluent and/or the marking solution contain poly-ApoA I.

10. The kit of claim 9, wherein the working concentration of poly-apoai is 0.01mg/ml to 2 mg/ml;

preferably the working concentration of the ApoA i is 0.1mg/ml to 1.5 mg/ml;

preferably the working concentration of the ApoA i is between 0.4mg/ml and 1.2 mg/ml.