CN112986583A - Kit for detecting novel coronavirus neutralizing antibody and application thereof - Google Patents

Abstract

The invention relates to the technical field of biology, and particularly provides a kit for detecting a novel coronavirus neutralizing antibody and application thereof. The kit for detecting the novel coronavirus neutralizing antibody specifically comprises two schemes (a) or (b): (a) tracer-labelled RBD trimer antigen, ACE2 coated on a solid support, and a working solution containing 0.2-10mg/mL dodecyl dimethyl betaine; (b) tracer-labeled ACE2, RBD trimer antigen coated on a solid support, and, working solution containing 0.2-10mg/mL dodecyl dimethyl betaine; wherein the RBD tripolymer antigen is obtained by completely crosslinking RBD of the spike protein and S2 subunit by using a disulfide bond. The dodecyl dimethyl betaine can obviously improve the combination speed of the RBD tripolymer antigen and the new crown neutralizing antibody, improve the average luminous intensity of a positive sample and shorten the detection time.

Description

Kit for detecting novel coronavirus neutralizing antibody and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a kit for detecting a novel coronavirus neutralizing antibody and application thereof.

Background

The novel coronavirus SARS-CoV-2 (namely 2019-nCoV) is a virus newly discovered in 2019 and can cause human viral pneumonia or lung infection. The coronavirus genome is encoded sequentially as spike protein, envelope protein, membrane protein and nucleocapsid protein. Among them, the spike protein is the most important surface membrane protein of coronavirus, and contains two subunits, S1 subunit and S2 subunit. Wherein S1 mainly contains Receptor Binding Domain (RBD) responsible for recognizing cell receptors. The spike protein of SARS-CoV-2 interacts with the human Angiotensin-converting enzyme 2 (ACE 2) protein to infect human respiratory epithelial cells. The spike protein is responsible for the combination and membrane fusion of the new coronavirus and host cell membrane receptors, and is an important action site of a new corona neutralizing antibody and a key target spot of vaccine design.

The new corona vaccine is one of the effective methods for preventing the new coronavirus diseases at present. The key to the success of the preventive vaccine is whether the preventive vaccine can induce the human immune system to generate protective antibodies (namely neutralizing antibodies of the new coronavirus) capable of combining with the new coronavirus so as to achieve the aim of preventing infection, so that a stable and effective vaccine evaluation system is established, the protective effect of the immune response of the vaccine is detected, and the method has important significance to the development and quality control of the vaccine.

Common methods for detecting neutralizing antibodies against the novel coronavirus are: plaque reduction neutralization assay (PRNT), enzyme-linked immunosorbent assay (ELISA), colloidal gold assay, chemiluminescence assay, and the like. Among them, PRNT detection is a neutralizing antibody, but since the method is based on live toxicity, the method needs to be completed in a BSL-3 grade laboratory, and PRNT detection period is long, and the error of plaque counting is large, which brings difficulty to many researchers. Although the enzyme-linked immunosorbent assay (ELISA) and the colloidal gold assay are simple and convenient to operate, the sensitivity, stability and the like of detection need to be further improved. The full-automatic chemiluminescence immunoassay combines a high-sensitivity chemiluminescence determination technology and a magnetic particle separation technology on the basis of enzyme immunoassay, so that the detection sensitivity is greatly improved; and secondly, because a full-automatic instrument and a matched reagent are used, human factors are minimized, and the stability of the method and the repeatability of results are improved.

However, the detection time of the currently marketed new coronavirus neutralizing antibody luminescence detection kit is slow, generally more than 30min is needed, and the analysis sensitivity is poor.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention is to provide a kit for detecting novel coronavirus neutralizing antibodies.

The second purpose of the invention is to provide the application of the kit for detecting the novel coronavirus neutralizing antibody.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a kit for detecting novel coronavirus neutralizing antibodies, comprising (a) or (b):

(a) tracer-labelled RBD trimer antigen, ACE2 coated on a solid support, and a working solution containing 0.2-10mg/mL dodecyl dimethyl betaine;

(b) tracer-labeled ACE2, RBD trimer antigen coated on a solid support, and, working solution containing 0.2-10mg/mL dodecyl dimethyl betaine;

wherein the RBD trimer antigen is obtained by completely crosslinking RBD of the spike protein and S2 subunit by using disulfide bond.

Further, the working fluid includes: 0.01mol/L-0.2mol/L phosphate buffer solution, 0.1-50mg/mL bovine serum albumin, 5-50mg/mL sodium chloride, 10-50mg/mL sucrose, 0.2-10mg/mL dodecyl dimethyl betaine and 0.5-5mL/L proclin 300, and the pH value is 6.0-7.6.

Further, the working fluid includes: phosphate buffer solution with the concentration of 0.05mol/L, bovine serum albumin with the concentration of 10mg/mL, sodium chloride with the concentration of 8mg/mL, sucrose with the concentration of 10mg/mL, dodecyl dimethyl betaine with the concentration of 0.2-10mg/mL and proclin 300 with the concentration of 0.5mL/L, and the pH value is 7.4.

Further, the concentration of the dodecyl dimethyl betaine in the working solution is 5 mg/mL.

Further, the tracer comprises acridinium ester and derivatives thereof, horseradish peroxidase, alkaline phosphatase or ruthenium terpyridyl; the solid support comprises magnetic particles, a microtiter plate, a nitrocellulose membrane or polystyrene latex, and the magnetic particles comprise carboxyl magnetic beads, amino magnetic beads, NHS magnetic beads, hydroxyl magnetic beads, tosyl magnetic beads or epoxy magnetic beads.

Further, the ACE2 in (a) or the RBD trimer antigen in (b) are each independently coated on a solid support by a biotin-avidin system.

Further, the kit also comprises a calibrator and a quality control product of the novel coronavirus neutralizing antibody.

Further, the preservation solution of the calibrator and the quality control product independently comprise 0.01-0.2 mol/L phosphate buffer solution, 100-500mL/L calf serum, 5-100mL/L glycol, 0.2-10mL/L Tween20 and 0.5-5mL/L proclin 300, and the pH value is 6.0-7.6.

Further, the preservation solution of the calibrator and the quality control product independently comprise 0.05mol/L phosphate buffer solution, 100mL/L calf serum, 5mL/L ethylene glycol, 1mL/L Tween20 and 0.5mL/L proclin 300, and the pH is 7.4.

The kit is applied to the detection of a novel coronavirus neutralizing antibody or the evaluation of a novel coronavirus vaccine.

Compared with the prior art, the invention has the beneficial effects that:

the kit for detecting the novel coronavirus neutralizing antibody provided by the invention realizes qualitative or quantitative detection of the neutralizing antibody by detecting the binding amount of ACE2 and a constant amount of RBD by utilizing the principle that the neutralizing antibody in a sample to be detected and ACE2 competitively bind to the novel coronavirus RBD. The method specifically comprises two schemes (a) or (b): (a) tracer-labelled RBD trimer antigen, ACE2 coated on a solid support, and a working solution containing 0.2-10mg/mL dodecyl dimethyl betaine; (b) tracer-labeled ACE2, RBD trimer antigen coated on a solid support, and a working solution containing 0.2-10mg/mL dodecyl dimethyl betaine. The RBD tripolymer antigen is a prefusion state formed by completely crosslinking RBD of S protein and S2 subunit, has comprehensive epitope and high stability, and can greatly improve the reaction speed with a neutralizing antibody and improve the detection sensitivity. Meanwhile, the inventor discovers that the existence of dodecyl dimethyl betaine can obviously improve the protein combination speed of the RBD tripolymer antigen and the new crown neutralizing antibody in a reaction system for the first time in the development process of the kit, improves the average luminous intensity of a positive sample and shortens the detection time. This reaction is surprising in that the effect of the use of dodecyl dimethyl betaine was discovered by chance, since it was not predicted that dodecyl dimethyl betaine, which is known to be used as a surfactant, could have an effect on the detection of neutralizing antibodies. The test result shows that the dodecyl dimethyl betaine is favorable for opening the closed region of the RBD tripolymer antigen, the opened space structure is more favorable for being combined with a neutralizing antibody, and the detection time is shortened to 15 min.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.

The kit for detecting the novel coronavirus neutralizing antibody provided by the invention realizes qualitative or quantitative detection of the neutralizing antibody by detecting the binding amount of ACE2 and a constant amount of RBD by utilizing the principle that the neutralizing antibody in a sample to be detected and ACE2 competitively bind to the novel coronavirus RBD. The method specifically comprises two schemes (a) or (b): (a) tracer-labelled RBD trimer antigen, ACE2 coated on a solid support, and a working solution containing 0.2-10mg/mL dodecyl dimethyl betaine; (b) tracer-labeled ACE2, RBD trimer antigen coated on a solid support, and, working solution containing 0.2-10mg/mL dodecyl dimethyl betaine; wherein the RBD trimer antigen is obtained by completely crosslinking RBD of the spike protein and S2 subunit by using disulfide bond.

In a preferred embodiment, the RBD trimer antigen of the invention is obtained by: the RBD of the spike protein and the subunit S2 are completely crosslinked by using a disulfide bond to form a closed stable configuration by adopting protein engineering, and the RBD tripolymer antigen can be stored for a long time at 4 ℃ and still maintains the original structure and antigenicity under the heating condition of 60 ℃.

It is understood that the technical scheme of detecting the RBD trimer antigen and the ACE2 binding amount can realize the detection of the novel coronavirus neutralizing antibody, so that the solid support for separating the RBD trimer antigen and the ACE2 conjugate can be connected with the RBD trimer antigen or with the ACE2, and correspondingly, the tracer for detection can be connected with the ACE2 or with the RBD trimer antigen.

In the invention, the RBD tripolymer antigen refers to a pre-fusion state formed by completely crosslinking the RBD of the S protein and the S2 subunit, compared with a single RBD antigen, the RBD tripolymer antigen has comprehensive epitope and high stability, can greatly improve the reaction speed with a neutralizing antibody and improve the detection sensitivity. Meanwhile, the inventor discovers for the first time in the development process of the kit that the existence of the dodecyl dimethyl betaine can obviously improve the protein binding speed of the RBD trimer antigen and the new crown neutralizing antibody in a reaction system, improve the average luminous intensity of a positive sample and shorten the detection time, and the reaction is surprising, and the use effect of the dodecyl dimethyl betaine is discovered accidentally because the dodecyl dimethyl betaine which is known to be used for a surfactant can not be predicted to influence the detection of the neutralizing antibody. The test result shows that the dodecyl dimethyl betaine is favorable for opening the closed zone of the RBD tripolymer antigen, and the opened space structure is more favorable for being combined with a neutralizing antibody.

It is noted that the concentration of dodecyl dimethyl betaine may be, but is not limited to, 0.2mg/mL, 0.5 mg/mL, 1mg/mL, 2mg/mL, 3 mg/mL, 4 mg/mL, 5mg/mL, 6 mg/mL, 7 mg/mL, 8mg/mL, 9 mg/mL, or 10 mg/mL. The working solution refers to a system existing when a reaction occurs, for example, RBD tripolymer antigen and ACE2 can be stored in the working solution to prepare a kit.

In a preferred embodiment, the working fluid comprises: 0.01mol/L-0.2mol/L phosphate buffer solution, 0.1-50mg/mL bovine serum albumin, 5-50mg/mL sodium chloride, 10-50mg/mL sucrose, 0.2-10mg/mL dodecyl dimethyl betaine and 0.5-5mL/Lproclin 300, and the pH value is 6.0-7.6.

The working solution can provide a stable environment for the reaction of the RBD tripolymer antigen and ACE2 or neutralizing antibody protein, and is favorable for opening the RBD tripolymer antigen closed zone so as to enable the RBD tripolymer antigen closed zone to be effectively combined with the neutralizing antibody. The concentration of the phosphate buffer solution can be but is not limited to 0.01mol/L, 0.05mol/L, 0.1mol/L, 0.15 mol/L or 0.2 mol/L; the concentration of bovine serum albumin can be, but is not limited to, 0.1mg/mL, 1mg/mL, 5mg/mL, 10mg/mL, 15mg/mL, 20mg/mL, 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL, 45mg/mL, or 50 mg/mL; the concentration of sodium chloride may be, but is not limited to, 5mg/mL, 10mg/mL, 15mg/mL, 20mg/mL, 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL, 45mg/mL, or 50 mg/mL; the concentration of sucrose may be, but is not limited to, 10mg/mL, 15mg/mL, 20mg/mL, 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL, 45mg/mL, or 50 mg/mL; the concentration of proclin 300 may be, but is not limited to, 0.5mL/L, 1mL/L, 2mL/L, 3mL/L, 4mL/L, or 5 mL/L; the pH may be, but is not limited to, 6.0, 6.5, 7.0, or 7.6.

In a preferred embodiment, the working fluid comprises: phosphate buffer solution with the concentration of 0.05mol/L, bovine serum albumin with the concentration of 10mg/mL, sodium chloride with the concentration of 8mg/mL, sucrose with the concentration of 10mg/mL, dodecyl dimethyl betaine with the concentration of 0.2-10mg/mL and proclin 300 with the concentration of 0.5mL/L, and the pH value is 7.4.

In a preferred embodiment, the concentration of dodecyl dimethyl betaine in the working solution is 5 mg/mL.

In a preferred embodiment, the tracer is a label used in a chemiluminescent immunoassay, such as acridinium esters and derivatives thereof in a direct chemiluminescent immunoassay; horseradish peroxidase or alkaline phosphatase in chemiluminescent enzyme immunoassays; adding oxidant and NaOH into terpyridyl ruthenium in electrochemical luminescence immunoassay; a substrate luminescent agent; and an electron donor for realizing a luminescence detection signal.

In a preferred embodiment, the solid support may be a material commonly used in the art to achieve separation and purification, such as magnetic microparticles, microtiter plates, nitrocellulose membranes, or polystyrene latex. Further preferred are magnetic particles, also called magnetic microspheres, magnetic beads or magnetic spheres, which may be magnetic particles commonly used in the art, such as nanoscale Fe₂O₃Or Fe₃O₄The magnetic particles and the organic polymer material are compounded to form micron-sized solid phase microspheres with superparamagnetism and extremely large protein adsorption capacity, and the micron-sized solid phase microspheres have the properties that the micron-sized solid phase microspheres can be quickly magnetized under the action of an external magnetic field and have zero residual magnetism after the magnetic field is removed. The kind of the organic polymer material is not particularly limited, and may be selected as needed. Specifically, the magnetic particles can be carboxyl magnetic beads, amino magnetic beads, NHS magnetic beads, hydroxyl magnetic beads, tosyl magnetic beads or epoxy magnetic beads, and the magnetic particles of different functional groups can be selected according to different connection modes of the magnetic particles and RBD trimer antigen or ACE 2. Preferably, the magnetic particles should have a diameter of 0.1 to 5 μm.

In a preferred embodiment, the ACE2 in (a) or the RBD trimer antigen in (b) are each independently coated on a solid support by a biotin-avidin system.

In a preferred embodiment, a calibrator and a quality control for the novel coronavirus neutralizing antibody are also included. The quantitative detection of the novel coronavirus neutralizing antibody is realized, and the quality control is carried out on the detection result. Preferably, the calibrator comprises a low-point calibrator solution with a concentration of neutralizing antibody of 3-9AU/mL and a high-point calibrator solution with a concentration of 200-500 AU/mL; the quality control product comprises low-point quality control product solution with neutralizing antibody concentration of 12-20AU/mL and high-point quality control product solution with concentration of 50-100 AU/mL.

In a preferred embodiment, the preservation solution of the calibrator and the quality control product independently comprise 0.01-0.2 mol/L phosphate buffer, 100-500mL/L calf serum, 5-100mL/L ethylene glycol, 0.2-10mL/LTween20 and 0.5-5mL/Lproclin 300, and the pH is 6.0-7.6. The preservation solution can realize stable preservation of the neutralizing antibody and prolong the effective period of the kit.

In a preferred embodiment, the preservation solutions of the calibrator and the quality control are each independently selected from 0.05mol/L phosphate buffer, 100mL/L calf serum, 5mL/L ethylene glycol, 1mL/LTween20 and 0.5mL/Lproclin 300, and the pH is 7.4.

In one embodiment, the kit of embodiment (a) of the present invention is exemplified.

The detection principle is as follows:

adding a sample to be detected, a calibrator or a quality control product and a constant amount of tracer labeled neo-crown RBD tripolymer antigen, wherein the tracer labeled neo-crown RBD tripolymer antigen is combined with a to-be-detected neo-crown neutralizing antibody, so that the reaction of the RBD tripolymer antigen and the biotin labeled ACE2 antigen can be prevented, when the concentration of the to-be-detected neo-crown neutralizing antibody is high, the content of the tracer labeled neo-crown RBD tripolymer antigen combined on the solid support ACE2 antigen is reduced, a luminescent catalyst or a luminescent substrate is added after washing with a washing solution, and a luminescent signal value detected by an instrument is low, which indicates that the concentration of the neutralizing antibody is high; on the contrary, when the concentration of the new crown neutralizing antibody to be detected is low, the detected luminous signal value is high, and the concentration of the neutralizing antibody is low. The concentration of the neutralizing antibody of the new coronavirus can be calculated by using a standard curve drawn by the concentration detection of the known neutralizing antibody (low-value and high-value calibrators) of the new coronavirus, so that the neutralizing capacity of the neutralizing antibody of the new coronavirus to be detected and whether a protective antibody and titer are generated after the neutralizing antibody is given to a vaccine can be calculated.

The preparation method comprises the following steps:

preparation of streptavidin-coated magnetic microspheres

1) Preparing an acetic acid buffer solution with pH of 3.6: weighing 2.55g of sodium acetate trihydrate, dissolving with 4500mL of purified water, adding 14mL of acetic acid, mixing uniformly, and diluting to 5000mL to obtain an acetic acid buffer solution with pH of 3.6.

2) Magnetic microsphere connection (magnetic microsphere connection CMC method): adding equal amount of the above pH3.6 acetic acid buffer solution into the magnetic microspheres for suspension, wherein the concentration of the magnetic microspheres is 20mg/mL, adding 10mg/mL of 1-cyclohexyl-2-morpholine ethyl carbodiimide p-toluenesulfonate (CMC), and adding 12 mu g of Streptavidin (SA) into 1mg of the magnetic microspheres to form a reaction system. And (3) putting the reaction system into a constant-temperature shaking water bath box to react for 24 hours at 37 ℃.

3) Cleaning the magnetic microspheres:

preparing a magnetic bead cleaning solution: BSA was dissolved in 0.05M PBS (pH7.4) to a concentration of 0.5g/mL, to obtain a magnetic bead washing solution.

Cleaning: pouring the reacted reaction system into a beaker, then placing the beaker on a magnet for precipitation, pouring out the supernatant, adding a magnetic bead cleaning solution with the volume 5 times that of the supernatant, stirring and cleaning the mixture, then placing the mixture on the magnet, and pouring out the supernatant after the supernatant is clear.

4) Suspension of magnetic microspheres:

preparation of magnetic bead suspension: BSA and Methylcellulose (MC) were dissolved in 0.05M PBS (pH7.4) so that the concentration of BSA was 0.5g/mL and the concentration of MC was 0.4g/mL, to obtain a magnetic bead suspension. And after washing, adding a coated volume of magnetic bead suspension with the suspension concentration of 20mg/mL to obtain a streptavidin-coated magnetic microsphere solution.

Preparation of ACE2 antigen labeled with biotin

1) Preparation of 0.1mol/L carbonate buffer (dialysate): adding Na into 5000ml beaker₂CO₃ 14.31g，NaHCO₃26.46 g, adding water to reach 4500mL to obtain 0.1mol/L carbonic acid buffer solution (dialysate). The prepared dialysate is placed on a magnetic stirrer for standby.

2) Selecting a dialysis bag with retention amount of 14000, measuring to a proper size, adjusting 1-2mg of ACE2 antigen to 1-2mL with dialysate, adding into the dialysate, and dialyzing for 2-3 hr under stirring at room temperature. Dissolving activated biotin in Dimethylformamide (DMF), mixing the two solutions according to the molar ratio of biotin to ACE2 antigen of 5:1-20:1, and reacting at 37 deg.C for 2 h; and dialyzing the reacted liquid with 0.1mol/L PBS at 4 ℃ for 24 hours to prepare the ACE2 antigen solution labeled with biotin.

3) The biotin-labeled ACE2 antigen obtained by the above reaction was purified by G-25 gel column.

Preparation of enzyme-labeled RBD trimer antigen

A, 1mL of 10-15mg/mL NaIO₄Mixing the water solution with 1mL of 5mg/mL ALP (alkaline phosphatase) or HRP (horse radish peroxidase) water solution at 2-8 deg.C, and reacting for 30-60 min to obtain activated ALP or HRP solution; then, adding 0.5mL of 90% ethylene glycol aqueous solution into the activated ALP or HRP solution, and reacting for 30-60 min at room temperature in a dark place to obtain an ALP or HRP mixed solution;

b, mixing RBD tripolymer antigen (preparation method is shown in literature, Xiaooli Xiong, and the like, A thermostabile, closed SARS-CoV-2 spike protein three polymer, Nature Structural & Molecular Biology volume 27, pages 934-941 (2020)) obtained by dialyzing with 0.05-0.1moL/L sodium carbonate-sodium bicarbonate buffer solution with pH of 8.5-9.5 with ALP or HRP mixed solution obtained in the step A according to the mass ratio of 1:1-1:2, dialyzing overnight at 2-8 ℃ to obtain dialyzed antigen;

c, adding 2-8mg/mL NaBH into the dialyzed antigen obtained in the step B₄Reacting the solution 80-120 μ L at 2-8 deg.C for 2-4h, placing into a dialysis bag, and dialyzing in 10-50mM PBS solution with pH of 7.4 at 2-8 deg.C overnight;

d, dropwise adding saturated ammonium sulfate with the same volume into the antibody solution obtained in the step C under stirring, and reacting at the temperature of 2-8 ℃ for 1-2 h; centrifuging at 3000-; then dissolving the precipitate in 1mL of 10-50mM PBS solution; dialyzing the solution in 10-50mM PBS solution, centrifuging at 10000-.

Preparation of kit

The novel coronavirus neutralizing antibody kit comprises: streptavidin coupled magnetic particle solution, biotin labeled ACE2 antigen solution, tracer labeled RBD trimer antigen solution, neutralizing antibody calibrator solution and quality control solution of the novel coronavirus.

The concentration of ACE2 antigen in the biotin-labeled ACE2 antigen solution is 25-5000ng/mL, and the working concentration of RBD trimer antigen in the tracer-labeled RBD trimer antigen solution is 50-5000 ng/mL. The solution of the two is the working solution provided by the invention, and the working solution specifically comprises: 0.01mol/L-0.2mol/L phosphate buffer solution, 0.1-50mg/mL bovine serum albumin, 5-50mg/mL sodium chloride, 10-50mg/mL sucrose, 0.2-10mg/mL dodecyl dimethyl betaine and 0.5-5mL/Lproclin 300, and the pH value is 6.0-7.6.

The neutralizing antibody calibrator solution for the novel coronavirus comprises a low-point calibrator solution with the neutralizing antibody concentration of 3-9AU/mL and a high-point calibrator solution with the concentration of 200-500 AU/mL; the quality control solution comprises a low-point quality control solution with a neutralizing antibody concentration of 12-20AU/mL and a high-point quality control solution with a concentration of 50-100 AU/mL. The preservation solution of the calibrator solution and the quality control solution comprises 0.01-0.2 mol/L phosphate buffer solution, 100-500mL/L calf serum, 5-100mL/L ethylene glycol, 0.2-10mL/LTween20 and 0.5-5mL/Lproclin 300, and the pH value is 6.0-7.6.

The method for detecting the novel coronavirus neutralizing antibody by using the kit comprises the following steps:

a) sample adding for one time: respectively adding 20 mu L of sample to be detected and the calibrator with high or low concentration into a reaction cup, then adding 50 mu L of RBD tripolymer antigen solution marked by the tracer, uniformly mixing, and carrying out warm bath at 37 ℃ for 5min to ensure that the neutralizing antibody of the new coronavirus in the sample to be detected and the RBD tripolymer antigen fully react to form a compound.

b) And (3) secondary sample adding: adding 50 mu L of biotin-labeled ACE2 antigen solution and 25 mu L of streptavidin-coated magnetic microsphere solution into the complex, uniformly mixing, and carrying out warm bath at 37 ℃ for 5min to enable the unreacted RBD trimer antigen in the complex to react with ACE2 antigen to form tracer-labeled RBD trimer antigen-biotin-labeled ACE2 antigen-streptavidin-coated magnetic microsphere complex.

c) And (3) detection: and (3) cleaning the compound precipitate by an external magnetic field, removing supernatant, cleaning by using a cleaning solution, adding a luminescent catalyst or a luminescent substrate, reacting for 15min in the whole reaction system, detecting the emitted relative light intensity, and calculating to obtain the content of the new coronavirus neutralizing antibody.

The invention also provides application of the kit in detection of the novel coronavirus neutralizing antibody or evaluation of a novel coronavirus vaccine.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

The novel coronavirus ACE2 antigen and RBD trimer antigen were purchased from Zhuhai standing grain medical diagnostic products, Inc. The ACE2 antigen is prepared by a eukaryotic expression system (HEK 293 cell) by utilizing a genetic engineering technology; RBD trimer antigen is prepared by protein engineering to design disulfide bond to crosslink Receptor Binding Domain (RBD) of spike protein with S2 protein to form stable trimer structure, specifically preparation method is described in Xiaoli Xiong et al, A thermostabile, closed SARS-CoV-2 spike protein tertiary, Nature Structural & Molecular Biology volume 27, pages 934-941 (2020).

Example 1

Preparing a biotin-labeled ACE2 antigen solution and an alkaline phosphatase-labeled RBD tripolymer antigen solution: phosphate buffer solution with pH of 7.4 and concentration of 0.05mol/L is prepared, and the buffer solution contains 10mg/mL of bovine serum albumin, 8mg/mL of sodium chloride, 10mg/mL of sucrose, 5mg/mL of dodecyl dimethyl betaine and 0.5mL/L of proclin 300. Then biotin-labeled ACE2 antigen and alkaline phosphatase-labeled RBD trimer antigen were diluted to 0.5mg/L with the working solution, respectively.

Preparing a neutralizing antibody calibrator and a quality control solution of the novel coronavirus: phosphate buffer with the pH of preservation solution of 7.4 and the concentration of 0.05mol/L is prepared, and the buffer contains 100mL/L calf serum, 5mL/L ethylene glycol, 1mL/LTween20 and 0.5mL/Lproclin 300. Then, the preservative solution is used for preparing a calibrator and a quality control product of the novel coronavirus neutralizing antibody. Calibration solution: a low point calibrator solution with a neutralizing antibody concentration of 10AU/mL and a high point calibrator solution with a concentration of 200 AU/mL; quality control solution: a low-point quality control solution with a neutralizing antibody concentration of 20AU/mL and a high-point quality control solution with a concentration of 100 AU/mL.

Reagent subpackaging and assembling

Subpackaging 5.0 mL/bottle of streptavidin-coupled magnetic particle working solution, 9.5 mL/bottle of biotin-labeled ACE2 antigen working solution, 9.5 mL/bottle of alkaline phosphatase-labeled RBD trimer antigen working solution and 1.0 mL/branch of calibrator/quality control working solution, assembling together, and storing at 2-8 ℃.

Comparative example 1

The RBD trimer antigen in example 1 was replaced with the RBD antigen alone, and the rest of the conditions were the same as in example 1.

Comparative example 2

The biotin-labeled ACE2 antigen solution and the alkaline phosphatase-labeled RBD trimer antigen solution in example 1 were removed of dodecyl dimethyl betaine, and the other conditions were the same as in example 1.

Comparative example 3

The dodecyl dimethyl betaine in the biotin-labeled ACE2 antigen solution and the alkaline phosphatase-labeled RBD trimer antigen solution in example 1 was replaced with octadecyl trimethyl ammonium chloride (cationic surfactant) at the same concentration, and the remaining conditions were the same as in example 1.

Comparative example 4

The same conditions as in example 1 were used except that dodecyl dimethyl betaine in the biotin-labeled ACE2 antigen solution and the alkaline phosphatase-labeled RBD trimer antigen solution in example 1 was replaced with sodium dodecylbenzenesulfonate (anionic surfactant) at the same concentration.

Comparative example 5

The dodecyl dimethyl betaine in the biotin-labeled ACE2 antigen solution and the alkaline phosphatase-labeled RBD trimer antigen solution in example 1 was replaced with tween 80 (nonionic surfactant) at the same concentration, and the remaining conditions were the same as in example 1.

Comparative example 6

The same conditions as in example 1 were used except that dodecyl dimethyl betaine in the biotin-labeled ACE2 antigen solution and the alkaline phosphatase-labeled RBD trimer antigen solution in example 1 was replaced with dodecyl aminopropionic acid (amino acid type zwitterionic surfactant) at the same concentration.

Comparative example 7

The dodecyl dimethyl betaine in the biotin-labeled ACE2 antigen solution and the alkaline phosphatase-labeled RBD trimer antigen solution in example 1 was replaced with octadecyl dihydroxyethyl amine oxide (amine oxide type zwitterionic surfactant) at the same concentration, and the remaining conditions were the same as in example 1.

Comparative examples 8, 9 and examples 2,3

The concentrations of dodecyl dimethyl betaine in the biotin-labeled ACE2 antigen solution and alkaline phosphatase-labeled RBD trimer antigen solution in example 1 were changed to 0.15mg/mL, 0.2mg/mL, 10mg/mL and 12mg/mL, respectively, corresponding to comparative example 8, example 2, example 3 and comparative example 9, and the other conditions were the same as in example 1.

50 samples of the novel coronavirus vaccine collected at a hospital in Guangdong were tested as positive samples (numbered P1-P50) and 30 samples of healthy persons not injected with the novel coronavirus vaccine were tested as negative samples (numbered N1-N30) using the kits of example 1 and comparative examples 1,2,3,4, and 5, and the intensity of the reaction light of the six kits with the samples was compared. And (3) taking a full-automatic chemiluminescence immunoassay analyzer as a detection instrument, and loading the kit on the instrument for testing. The results are shown in Table 1.

TABLE 1

Sample numbering	Example 1 (light) Strength)	Comparative example 1 (light) Strength)	Signal value Deviation of	Comparative example 2 (light) Strength)	Signal value Deviation of	Comparative example 3 (light) Strength)	Signal value Deviation of	Comparative example 4 (light) Strength)	Signal value Deviation of	Comparative example 5 (light) Strength)	Signal value Deviation of
												N1	2746	2641	-3.82%	2029	-26.11%	2563	-6.66%	2403	-12.49%	2586	-5.83%
N2	2032	2491	22.59%	2514	23.72%	2430	19.59%	2419	19.05%	1850	-8.96%
												N3	1670	1531	-8.32%	2202	31.86%	1612	-3.47%	2609	56.23%	2268	35.81%
N4	1457	1578	8.30%	1711	17.43%	2338	60.47%	1878	28.89%	1431	-1.78%
												N5	2414	2808	16.32%	2700	11.85%	2209	-8.49%	1904	-21.13%	2222	-7.95%
N6	2697	1943	-27.96%	1555	-42.34%	1705	-36.78%	1810	-32.89%	2287	-15.20%
												N7	2319	2530	9.10%	2347	1.21%	2472	6.60%	2406	3.75%	1947	-16.04%
N8	1689	2087	23.56%	2340	38.54%	1909	13.03%	2520	49.20%	2116	25.28%
												N9	1533	1690	10.24%	1802	17.55%	2039	33.01%	2220	44.81%	1928	25.77%
N10	2409	2656	10.25%	1912	-20.63%	2251	-6.56%	1881	-21.92%	1984	-17.64%
												N11	2506	2605	3.95%	1795	-28.37%	2068	-17.48%	1900	-24.18%	2253	-10.10%
N12	2516	2322	-7.71%	1623	-35.49%	2319	-7.83%	2162	-14.07%	1997	-20.63%
												N13	2254	2504	11.09%	2342	3.90%	2428	7.72%	2496	10.74%	2027	-10.07%
N14	1611	1754	8.88%	1803	11.92%	1947	20.86%	2431	50.90%	2059	27.81%
												N15	1713	1758	2.63%	1896	10.68%	2157	25.92%	2149	25.45%	1958	14.30%
N16	2482	2646	6.61%	1848	-25.54%	2080	-16.20%	1892	-23.77%	2071	-16.56%
												N17	2512	2504	-0.32%	1713	-31.81%	2206	-12.18%	1945	-22.57%	2174	-13.46%
N18	2379	2473	3.95%	2127	-10.59%	2422	1.81%	2465	3.61%	2013	-15.38%
												N19	2082	2365	13.59%	2028	-2.59%	2091	0.43%	2459	18.11%	2046	-1.73%
N20	1645	1705	3.65%	1888	14.77%	2105	27.96%	2406	46.26%	2000	21.58%
												N21	2226	2644	18.78%	1856	-16.62%	2154	-3.23%	1956	-12.13%	2021	-9.21%
N22	2493	2596	4.13%	1740	-30.20%	2103	-15.64%	1909	-23.43%	2144	-14.00%
												N23	2412	2484	2.99%	1934	-19.82%	2291	-5.02%	2346	-2.74%	2031	-15.80%
N24	2227	2404	7.95%	2037	-8.53%	2130	-4.36%	2459	10.42%	2041	-8.35%
												N25	1654	1754	6.05%	1999	20.86%	2105	27.27%	2436	47.28%	2001	20.98%
N26	1950	2548	30.67%	1860	-4.62%	2136	9.54%	2197	12.67%	2014	3.28%
												N27	2361	2603	10.25%	1752	-25.79%	2151	-8.89%	1941	-17.79%	2089	-11.52%
N28	2415	2512	4.02%	1881	-22.11%	2173	-10.02%	2196	-9.07%	2049	-15.16%
												N29	2402	2466	2.66%	1955	-18.61%	2290	-4.66%	2385	-0.71%	2036	-15.24%
N30	1804	2453	35.98%	2024	12.20%	2122	17.63%	2459	36.31%	2014	11.64%
												Negative sample bias Mean value	/	/	7.67%	/	-5.11%	/	3.48%	/	7.49%	/	-2.14%
P1	177547	95343	-46.30%	134048	-24.50%	132450	-25.40%	149672	-15.70%	155531	-12.40%
												P2	15659	6921	-55.80%	13263	-15.30%	11102	-29.10%	13138	-16.10%	13404	-14.40%
P3	82733	39877	-51.80%	66931	-19.10%	59402	-28.20%	67758	-18.10%	71068	-14.10%
												P4	179677	95408	-46.90%	150569	-16.20%	128828	-28.30%	144640	-19.50%	160991	-10.40%
P5	145008	78449	-45.90%	115861	-20.10%	102376	-29.40%	121082	-16.50%	127027	-12.40%
												P6	91226	48988	-46.30%	72981	-20.00%	65500	-28.20%	74805	-18.00%	78454	-14.00%
P7	120713	61201	-49.30%	93673	-22.40%	89690	-25.70%	99468	-17.60%	107917	-10.60%
												P8	62842	28153	-55.20%	52976	-15.70%	45938	-26.90%	53227	-15.30%	54547	-13.20%
P9	67524	27955	-58.60%	52669	-22.00%	48347	-28.40%	55370	-18.00%	57936	-14.20%
												P10	63458	39661	-37.50%	50576	-20.30%	46261	-27.10%	53559	-15.60%	56858	-10.40%
P11	180318	9366	-94.81%	135599	-24.80%	131452	-27.10%	151647	-15.90%	162286	-10.00%
												P12	90410	57049	-36.90%	72961	-19.30%	67265	-25.60%	74950	-17.10%	77753	-14.00%
P13	21146	7200	-65.95%	16684	-21.10%	15838	-25.10%	17805	-15.80%	18080	-14.50%
												P14	97837	54202	-44.60%	78367	-19.90%	73280	-25.10%	78857	-19.40%	83553	-14.60%
P15	110922	71767	-35.30%	93396	-15.80%	78755	-29.00%	90069	-18.80%	97279	-12.30%
												P16	121182	75496	-37.70%	96219	-20.60%	88705	-26.80%	98036	-19.10%	105550	-12.90%
P17	84423	46939	-44.40%	65681	-22.20%	59856	-29.10%	69480	-17.70%	74968	-11.20%
												P18	177162	80432	-54.60%	137478	-22.40%	129683	-26.80%	143678	-18.90%	156434	-11.70%
P19	33179	7157	-78.43%	28036	-15.50%	23557	-29.00%	27472	-17.20%	29529	-11.00%
												P20	145376	78067	-46.30%	123424	-15.10%	106997	-26.40%	120371	-17.20%	127495	-12.30%
P21	114728	55184	-51.90%	93733	-18.30%	82834	-27.80%	92241	-19.60%	99010	-13.70%
												P22	138757	71182	-48.70%	110867	-20.10%	98517	-29.00%	111283	-19.80%	121690	-12.30%
P23	165619	100199	-39.50%	130508	-21.20%	119246	-28.00%	138457	-16.40%	147235	-11.10%
												P24	165077	73129	-55.70%	137014	-17.00%	121167	-26.60%	132392	-19.80%	141141	-14.50%
P25	92490	55309	-40.20%	74824	-19.10%	66963	-27.60%	75379	-18.50%	79079	-14.50%
												P26	104994	70976	-32.40%	89035	-15.20%	74126	-29.40%	84520	-19.50%	91450	-12.90%
P27	39830	8857	-77.76%	33656	-15.50%	29594	-25.70%	31944	-19.80%	35608	-10.60%
												P28	181669	121537	-33.10%	139158	-23.40%	136070	-25.10%	152057	-16.30%	159142	-12.40%
P29	29491	7488	-74.61%	24301	-17.60%	21794	-26.10%	24330	-17.50%	25244	-14.40%
												P30	52972	30141	-43.10%	40630	-23.30%	37822	-28.60%	44338	-16.30%	45980	-13.20%
P31	132867	59126	-55.50%	104301	-21.50%	97790	-26.40%	111077	-16.40%	118916	-10.50%
												P32	112262	50855	-54.70%	87789	-21.80%	82737	-26.30%	92841	-17.30%	96545	-14.00%
P33	126121	52088	-58.70%	95852	-24.00%	90429	-28.30%	103419	-18.00%	111869	-11.30%
												P34	124839	65540	-47.50%	98123	-21.40%	91882	-26.40%	103866	-16.80%	109733	-12.10%
P35	33017	8425	-74.48%	25192	-23.70%	23805	-27.90%	27932	-15.40%	29484	-10.70%
												P36	134549	69965	-48.00%	101046	-24.90%	94992	-29.40%	112752	-16.20%	118807	-11.70%
P37	130021	73462	-43.50%	107657	-17.20%	96346	-25.90%	109088	-16.10%	110778	-14.80%
												P38	163369	9454	-94.21%	135760	-16.90%	120240	-26.40%	136086	-16.70%	141641	-13.30%
P39	168910	92394	-45.30%	131074	-22.40%	121108	-28.30%	140026	-17.10%	147121	-12.90%
												P40	45064	22667	-49.70%	37313	-17.20%	32942	-26.90%	38259	-15.10%	38349	-14.90%
P41	135402	68920	-49.10%	107915	-20.30%	95594	-29.40%	112519	-16.90%	120914	-10.70%
												P42	18800	8704	-53.70%	14796	-21.30%	13423	-28.60%	15303	-18.60%	16807	-10.60%
P43	163139	80428	-50.70%	138179	-15.30%	114850	-29.60%	135079	-17.20%	142910	-12.40%
												P44	114516	68824	-39.90%	96766	-15.50%	80390	-29.80%	91613	-20.00%	98026	-14.40%
P45	186302	107124	-42.50%	145688	-21.80%	135628	-27.20%	152022	-18.40%	160220	-14.00%
												P46	69181	29402	-57.50%	57559	-16.80%	49257	-28.80%	57005	-17.60%	61986	-10.40%
P47	130060	58657	-54.90%	101967	-21.60%	95204	-26.80%	105219	-19.10%	117054	-10.00%
												P48	86733	47443	-45.30%	65483	-24.50%	61060	-29.60%	73116	-15.70%	76845	-11.40%
P49	14172	7384	-47.90%	11862	-16.30%	10416	-26.50%	11607	-18.10%	12556	-11.40%
												P50	153755	78569	-48.90%	123465	-19.70%	115162	-25.10%	127002	-17.40%	131461	-14.50%
Deviation of positive sample Mean value	/	/	-51.83%	/	-19.74%	/	-27.48%	/	-17.50%	/	-12.52%

Under the condition that the average luminous intensity of the negative samples is close to the average luminous intensity of the positive samples, the higher the average luminous intensity of the negative samples is, the higher the analysis sensitivity is, the shorter the time required for reaching the same signal is, and the shorter the detection time is. As can be seen from the above results, the test results of the kits of example 1 and comparative example 1 of the present invention are: the light intensity difference of 30 negative samples is small, the light intensity difference of 50 positive samples is obvious, wherein the detection light intensity of the kit in the example 1 is 51.83% higher than the average light intensity of the kit in the comparative example 1. The kit of the embodiment 1 of the invention has shorter time for reaching the same signal value compared with the kit of the comparative example 1, the reaction time is shortened to 15min compared with 30min of similar products sold in the market, and simultaneously, the RBD tripolymer antigen is proved to be capable of greatly improving the reaction speed with a neutralizing antibody of the new coronavirus and ACE2 antigen compared with the single RBD antigen.

The detection results of five kits of comparative example 1 and comparative examples 2,3,4 and 5 are as follows: the light intensity difference of 30 negative samples is small, and the light intensity difference of 50 positive samples is obvious, wherein the detection light intensity of the kit in the embodiment 1 is 19.74% higher than the average light intensity of the comparison example 2, 27.48% higher than the comparison example 3, 17.50% higher than the comparison example 4, and 12.52% higher than the comparison example 5, which indicates that the reaction speed of the antigen antibody in the kit can be greatly improved compared with the dodecyl dimethyl betaine which does not contain a surfactant, contains a cation, an anion or a nonionic surfactant. Because the RBD trimer antigen has a closed state and an open state, the RBD trimer antigen is more favorable for being specifically combined with the new corona neutralizing antibody in the open state. The use of the surfactant can affect the surface charge of the RBD tripolymer antigen, so that the spatial structure of the RBD tripolymer antigen is changed, the closed zone of the RBD tripolymer antigen is promoted to be opened, and the combination speed of the RBD tripolymer antigen and a new crown neutralizing antibody is accelerated. It is presumed that the use of octadecyl trimethyl ammonium chloride (cationic surfactant), sodium dodecylbenzenesulfonate (anionic surfactant), tween 80 (nonionic surfactant) or no surfactant may result in insufficient opening of the closed zone of the RBD trimer antigen, affecting its steric structure, and that the binding with the neocorona neutralizing antibody is unfavorable compared to the use of dodecyl dimethyl betaine, resulting in a lower average light intensity being detected.

The reagent kits of example 1 and comparative examples 6 and 7 were used to detect 50 positive samples and 30 negative samples, and the intensity of the reaction light of the three reagent kits and the samples was compared. And (3) taking a full-automatic chemiluminescence immunoassay analyzer as a detection instrument, and loading the kit on the instrument for testing. The results are shown in Table 2.

TABLE 2

Sample numbering	Example 1 (light intensity)	COMPARATIVE EXAMPLE 6 (light intensity)	Deviation of signal value	COMPARATIVE EXAMPLE 7 (light intensity)	Deviation of signal value
						N1	2746	2589	-5.70%	2688	-2.10%
N2	2032	2113	4.00%	1878	-7.60%
						N3	1670	1723	3.20%	1546	-7.40%
N4	1457	1425	-2.20%	1390	-4.60%
						N5	2414	2334	-3.30%	2477	2.60%
N6	2697	2532	-6.10%	2651	-1.70%
						N7	2319	2196	-5.30%	2370	2.20%
N8	1689	1706	1.00%	1660	-1.70%
						N9	1533	1512	-1.40%	1564	2.00%
N10	2409	2315	-3.90%	2301	-4.50%
						N11	2506	2599	3.70%	2546	1.60%
N12	2516	2383	-5.30%	2365	-6.00%
						N13	2254	2358	4.60%	2249	-0.20%
N14	1611	1522	-5.50%	1667	3.50%
						N15	1713	1694	-1.10%	1609	-6.10%
N16	2482	2497	0.60%	2455	-1.10%
						N17	2512	2623	4.40%	2354	-6.30%
N18	2379	2243	-5.70%	2474	4.00%
						N19	2082	2130	2.30%	2072	-0.50%
N20	1645	1706	3.70%	1535	-6.70%
						N21	2226	2099	-5.70%	2215	-0.50%
N22	2493	2578	3.40%	2548	2.20%
						N23	2412	2448	1.50%	2234	-7.40%
N24	2227	2118	-4.90%	2091	-6.10%
						N25	1654	1626	-1.70%	1685	1.90%
N26	1950	1843	-5.50%	2005	2.80%
						N27	2361	2186	-7.40%	2274	-3.70%
N28	2415	2231	-7.60%	2253	-6.70%
						N29	2402	2212	-7.90%	2448	1.90%
N30	1804	1815	0.60%	1692	-6.20%
						Negative sample deviation mean	/	/	-1.77%	/	-2.08%
P1	177547	150915	-15.00%	145589	-18.00%
						P2	15659	12950	-17.30%	12809	-18.20%
P3	82733	68255	-17.50%	67924	-17.90%
						P4	179677	149312	-16.90%	145718	-18.90%
P5	145008	122242	-15.70%	120067	-17.20%
						P6	91226	76356	-16.30%	74714	-18.10%
P7	120713	100554	-16.70%	98864	-18.10%
						P8	62842	51719	-17.70%	50902	-19.00%
P9	67524	56923	-15.70%	54019	-20.00%
						P10	63458	52226	-17.70%	51211	-19.30%
P11	180318	149664	-17.00%	148041	-17.90%
						P12	90410	75040	-17.00%	72780	-19.50%
P13	21146	17572	-16.90%	17382	-17.80%
						P14	97837	80716	-17.50%	80520	-17.70%
P15	110922	93064	-16.10%	90845	-18.10%
						P16	121182	99490	-17.90%	100581	-17.00%
P17	84423	71422	-15.40%	69902	-17.20%
						P18	177162	149170	-15.80%	145981	-17.60%
P19	33179	27605	-16.80%	26875	-19.00%
						P20	145376	120953	-16.80%	117900	-18.90%
P21	114728	97289	-15.20%	92471	-19.40%
						P22	138757	114752	-17.30%	114058	-17.80%
P23	165619	136304	-17.70%	135476	-18.20%
						P24	165077	135693	-17.80%	133052	-19.40%
P25	92490	78154	-15.50%	74917	-19.00%
						P26	104994	88195	-16.00%	86830	-17.30%
P27	39830	32780	-17.70%	31904	-19.90%
						P28	181669	154237	-15.10%	149150	-17.90%
P29	29491	24920	-15.50%	23799	-19.30%
						P30	52972	44338	-16.30%	43013	-18.80%
P31	132867	112405	-15.40%	109748	-17.40%
						P32	112262	92616	-17.50%	89810	-20.00%
P33	126121	105816	-16.10%	104554	-17.10%
						P34	124839	104865	-16.00%	102618	-17.80%
P35	33017	28064	-15.00%	26810	-18.80%
						P36	134549	113156	-15.90%	110061	-18.20%
P37	130021	109608	-15.70%	105447	-18.90%
						P38	163369	134616	-17.60%	135270	-17.20%
P39	168910	142560	-15.60%	137155	-18.80%
						P40	45064	37674	-16.40%	36998	-17.90%
P41	135402	113738	-16.00%	108728	-19.70%
						P42	18800	15585	-17.10%	15472	-17.70%
P43	163139	135405	-17.00%	132958	-18.50%
						P44	114516	96079	-16.10%	94819	-17.20%
P45	186302	156866	-15.80%	151091	-18.90%
						P46	69181	57628	-16.70%	57213	-17.30%
P47	130060	109250	-16.00%	104178	-19.90%
						P48	86733	73116	-15.70%	71381	-17.70%
P49	14172	11834	-16.50%	11522	-18.70%
						P50	153755	130384	-15.20%	126233	-17.90%
Mean of deviation of positive samples	/	/	-16.42%	/	-18.36%

As can be seen from the above results, the test results of the three kits of comparative example 1 and comparative examples 6 and 7 are: the light intensity difference of 30 negative samples is small, and the light intensity difference of 50 positive samples is obvious, wherein the detection light intensity of the kit in the embodiment 1 is 16.42% higher than the average light intensity of the comparative example 6, and 18.36% higher than the average light intensity of the comparative example 7, which shows that the reaction speed of the RBD trimer antigen and the neocorona neutralizing antibody in the kit can be greatly improved by the dodecyl dimethyl betaine zwitterionic surfactant used in the invention compared with the amino acid zwitterionic surfactant and the amine oxide zwitterionic surfactant, and the average luminous intensity of the dodecyl amino propionic acid (amino acid zwitterionic surfactant) and the octadecyl dihydroxyethyl amine oxide (amine oxide zwitterionic surfactant) is lower than that of the Tween 80 (nonionic surfactant).

It is speculated that dodecyl dimethyl betaine can promote the opening of the closed zone of the RBD trimer antigen and accelerate the binding speed with the new crown neutralizing antibody. Other classes of amphoteric surfactants may leave the seal zone of the RBD trimer antigen insufficiently open, affecting its steric structure, and less favorable for binding to neocorona neutralizing antibodies than with dodecyl dimethyl betaine, resulting in lower average light intensity detected.

50 positive samples diluted 10 times were simultaneously detected using the kits of example 1 and comparative example 1, and the detection rates of the two kits were compared to compare the analytical sensitivities thereof. The results are shown in Table 3.

TABLE 3

Sample numbering	Example 1 concentration	Comparative example 1 concentration
			Range of normal value	<20AU/mL	<20AU/mL
P-1	180.25	172.32
			P-2	15.9	7.03
P-3	83.99	88.11
			P-4	182.41	175.66
P-5	147.22	79.64
			P-6	92.62	97.06
P-7	122.55	117.28
			P-8	63.8	28.58
P-9	68.55	71.77
			P-10	64.42	40.27
P-11	183.06	17.39
			P-12	91.79	92.61
P-13	21.47	12.34
			P-14	99.33	103.8
P-15	112.61	114.08
			P-16	123.03	128.81
P-17	85.71	87.08
			P-18	179.86	184.72
P-19	33.68	13.78
			P-20	147.59	145.08
P-21	116.48	56.02
			P-22	140.87	140.17
P-23	168.14	101.73
			P-24	167.59	168.76
P-25	93.9	91.18
			P-26	106.59	107.45
P-27	40.44	19.25
			P-28	184.44	189.78
P-29	29.94	17.75
			P-30	53.78	30.6
P-31	134.89	131.79
			P-32	113.97	118.07
P-33	128.04	128.17
			P-34	126.74	127.63
P-35	33.52	16.46
			P-36	136.6	134.14
P-37	132	137.02
			P-38	165.86	7.01
P-39	171.48	171.65
			P-40	45.75	43.65
P-41	137.46	142
			P-42	19.09	8.84
P-43	165.62	171.25
			P-44	116.26	122.07
P-45	189.14	181.95
			P-46	70.23	68.83
P-47	132.04	126.23
			P-48	88.05	84.44
P-49	14.39	7.5
			P-50	156.1	162.65
Detection rate	94.00%	80.00%

As can be seen from the above results, the detection results of the kits of example 1 and comparative example 1 of the present invention are: after 50 vaccine samples are diluted by 10 times, 49 positive samples can still be detected by using the kit in the embodiment 1, and the detection rate is 94%, while the kit in the comparative example 1 can only detect 40 positive samples, and the detection rate is 80%, and the omission rate of the kit in the embodiment 1 is reduced by 70% compared with that of the kit in the comparative example 1. The RBD trimer antigen used in the invention has stronger binding capacity with the neutralizing antibody of the new coronavirus than the RBD antigen alone, and the kit of the example 1 has better analysis sensitivity.

50 positive samples and 30 negative samples were tested using the kits of examples 1 to 3 and comparative examples 2, 8 and 9, and the intensity of reaction light was compared between the six kits and the samples. And (3) taking a full-automatic chemiluminescence immunoassay analyzer as a detection instrument, and loading the kit on the instrument for testing. The results are shown in Table 4.

TABLE 4

Sample numbering	Example 1 (light) Strength)	Comparative example 2 (light) Strength)	Signal value Deviation of	Comparative example 8 (light) Strength)	Signal value Deviation of	Example 2 (light) Strength)	Signal value Deviation of	Example 3 (light) Strength)	Signal value Deviation of	Comparative example 9 (light) Strength)	Signal value Deviation of
												N1	2746	2029	-26.11%	2563	-6.66%	2403	-12.49%	2586	-5.83%	1418	-48.36%
N2	2032	2514	23.72%	2430	19.59%	2419	19.05%	1850	-8.96%	2001	-1.53%
												N3	1670	2202	31.86%	1612	-3.47%	2609	56.23%	2268	35.81%	2364	41.56%
N4	1457	1711	17.43%	2338	60.47%	1878	28.89%	1431	-1.78%	2760	89.43%
												N5	2414	2700	11.85%	2209	-8.49%	1904	-21.13%	2222	-7.95%	2340	-3.07%
N6	2697	1555	-42.34%	1705	-36.78%	1810	-32.89%	2287	-15.20%	1408	-47.79%
												N7	2319	2347	1.21%	2472	6.60%	2406	3.75%	1947	-16.04%	1438	-37.99%
N8	1689	2340	38.54%	1909	13.03%	2520	49.20%	2116	25.28%	2121	25.58%
												N9	1533	1802	17.55%	2039	33.01%	2220	44.81%	1928	25.77%	2710	76.78%
N10	2409	1912	-20.63%	2251	-6.56%	1881	-21.92%	1984	-17.64%	2643	9.71%
												N11	2506	1795	-28.37%	2068	-17.48%	1900	-24.18%	2253	-10.10%	1957	-21.91%
N12	2516	1623	-35.49%	2319	-7.83%	2162	-14.07%	1997	-20.63%	1420	-43.56%
												N13	2254	2342	3.90%	2428	7.72%	2496	10.74%	2027	-10.07%	1490	-33.90%
N14	1611	1803	11.92%	1947	20.86%	2431	50.90%	2059	27.81%	2551	58.35%
												N15	1713	1896	10.68%	2157	25.92%	2149	25.45%	1958	14.30%	2649	54.64%
N16	2482	1848	-25.54%	2080	-16.20%	1892	-23.77%	2071	-16.56%	2364	-4.75%
												N17	2512	1713	-31.81%	2206	-12.18%	1945	-22.57%	2174	-13.46%	1526	-39.25%
N18	2379	2127	-10.59%	2422	1.81%	2465	3.61%	2013	-15.38%	1442	-39.39%
												N19	2082	2028	-2.59%	2091	0.43%	2459	18.11%	2046	-1.73%	1980	-4.90%
N20	1645	1888	14.77%	2105	27.96%	2406	46.26%	2000	21.58%	2576	56.60%
												N21	2226	1856	-16.62%	2154	-3.23%	1956	-12.13%	2021	-9.21%	2536	13.93%
N22	2493	1740	-30.20%	2103	-15.64%	1909	-23.43%	2144	-14.00%	2058	-17.45%
												N23	2412	1934	-19.82%	2291	-5.02%	2346	-2.74%	2031	-15.80%	1455	-39.68%
N24	2227	2037	-8.53%	2130	-4.36%	2459	10.42%	2041	-8.35%	1892	-15.04%
												N25	1654	1999	20.86%	2105	27.27%	2436	47.28%	2001	20.98%	2282	37.97%
N26	1950	1860	-4.62%	2136	9.54%	2197	12.67%	2014	3.28%	2561	31.33%
												N27	2361	1752	-25.79%	2151	-8.89%	1941	-17.79%	2089	-11.52%	2266	-4.02%
N28	2415	1881	-22.11%	2173	-10.02%	2196	-9.07%	2049	-15.16%	1745	-27.74%
												N29	2402	1955	-18.61%	2290	-4.66%	2385	-0.71%	2036	-15.24%	1624	-32.39%
N30	1804	2024	12.20%	2122	17.63%	2459	36.31%	2014	11.64%	1966	8.98%
												Negative sample bias Mean value	/	/	-5.11%	/	3.48%	/	7.49%	/	-2.14%	/	1.40%
P1	177547	134048	-24.50%	142215	-19.90%	162456	-8.50%	166184	-6.40%	151092	-14.90%
												P2	15659	13263	-15.30%	12699	-18.90%	14030	-10.40%	15174	-3.10%	13248	-15.40%
P3	82733	66931	-19.10%	66269	-19.90%	75370	-8.90%	78265	-5.40%	70323	-15.00%
												P4	179677	150569	-16.20%	146257	-18.60%	161889	-9.90%	167818	-6.60%	152366	-15.20%
P5	145008	115861	-20.10%	116441	-19.70%	132247	-8.80%	139933	-3.50%	123547	-14.80%
												P6	91226	72981	-20.00%	72342	-20.70%	83837	-8.10%	86938	-4.70%	78181	-14.30%
P7	120713	93673	-22.40%	95725	-20.70%	109849	-9.00%	114315	-5.30%	103210	-14.50%
												P8	62842	52976	-15.70%	49834	-20.70%	55992	-10.90%	58883	-6.30%	52913	-15.80%
P9	67524	52669	-22.00%	54087	-19.90%	61920	-8.30%	64485	-4.50%	57260	-15.20%
												P10	63458	50576	-20.30%	50576	-20.30%	58318	-8.10%	59651	-6.00%	54257	-14.50%
P11	180318	135599	-24.80%	147139	-18.40%	163909	-9.10%	169860	-5.80%	152910	-15.20%
												P12	90410	72961	-19.30%	74046	-18.10%	82816	-8.40%	86613	-4.20%	76668	-15.20%
P13	21146	16684	-21.10%	17192	-18.70%	19264	-8.90%	19920	-5.80%	18059	-14.60%
												P14	97837	78367	-19.90%	78661	-19.60%	88640	-9.40%	94217	-3.70%	83944	-14.20%
P15	110922	93396	-15.80%	89625	-19.20%	99830	-10.00%	103601	-6.60%	94838	-14.50%
												P16	121182	96219	-20.60%	96461	-20.40%	109670	-9.50%	114032	-5.90%	103853	-14.30%
P17	84423	65681	-22.20%	68636	-18.70%	75643	-10.40%	79442	-5.90%	71169	-15.70%
												P18	177162	137478	-22.40%	144387	-18.50%	158560	-10.50%	168835	-4.70%	150942	-14.80%
P19	33179	28036	-15.50%	26211	-21.00%	29795	-10.20%	31221	-5.90%	28368	-14.50%
												P20	145376	123424	-15.10%	116592	-19.80%	129675	-10.80%	138253	-4.90%	123133	-15.30%
P21	114728	93733	-18.30%	91668	-20.10%	105091	-8.40%	109336	-4.70%	96486	-15.90%
												P22	138757	110867	-20.10%	113642	-18.10%	124188	-10.50%	130709	-5.80%	117805	-15.10%
P23	165619	130508	-21.20%	132164	-20.20%	148726	-10.20%	155682	-6.00%	141935	-14.30%
												P24	165077	137014	-17.00%	133217	-19.30%	147414	-10.70%	159299	-3.50%	138830	-15.90%
P25	92490	74824	-19.10%	75749	-18.10%	83611	-9.60%	86756	-6.20%	79264	-14.30%
												P26	104994	89035	-15.20%	85675	-18.40%	94285	-10.20%	97959	-6.70%	88930	-15.30%
P27	39830	33656	-15.50%	31665	-20.50%	36006	-9.60%	37161	-6.70%	33537	-15.80%
												P28	181669	139158	-23.40%	148787	-18.10%	164047	-9.70%	171496	-5.60%	153874	-15.30%
P29	29491	24301	-17.60%	23298	-21.00%	27132	-8.00%	27663	-6.20%	25156	-14.70%
												P30	52972	40630	-23.30%	42748	-19.30%	48681	-8.10%	51065	-3.60%	45026	-15.00%
P31	132867	104301	-21.50%	105629	-20.50%	118384	-10.90%	127685	-3.90%	112273	-15.50%
												P32	112262	87789	-21.80%	90371	-19.50%	102720	-8.50%	105302	-6.20%	96433	-14.10%
P33	126121	95852	-24.00%	102662	-18.60%	115527	-8.40%	119058	-5.60%	108212	-14.20%
												P34	124839	98123	-21.40%	100495	-19.50%	112605	-9.80%	116475	-6.70%	106488	-14.70%
P35	33017	25192	-23.70%	26381	-20.10%	30277	-8.30%	31069	-5.90%	27932	-15.40%
												P36	134549	101046	-24.90%	107370	-20.20%	120152	-10.70%	125803	-6.50%	115039	-14.50%
P37	130021	107657	-17.20%	104407	-19.70%	117279	-9.80%	121960	-6.20%	111298	-14.40%
												P38	163369	135760	-16.90%	130859	-19.90%	148339	-9.20%	157651	-3.50%	140497	-14.00%
P39	168910	131074	-22.40%	136986	-18.90%	153877	-8.90%	160802	-4.80%	141884	-16.00%
												P40	45064	37313	-17.20%	36367	-19.30%	40513	-10.10%	42721	-5.20%	38575	-14.40%
P41	135402	107915	-20.30%	110217	-18.60%	120779	-10.80%	126872	-6.30%	115092	-15.00%
												P42	18800	14796	-21.30%	15360	-18.30%	16864	-10.30%	18180	-3.30%	16036	-14.70%
P43	163139	138179	-15.30%	130348	-20.10%	146825	-10.00%	155308	-4.80%	139810	-14.30%
												P44	114516	96766	-15.50%	91269	-20.30%	104668	-8.60%	108561	-5.20%	96193	-16.00%
P45	186302	145688	-21.80%	151277	-18.80%	167299	-10.20%	177360	-4.80%	157425	-15.50%
												P46	69181	57559	-16.80%	55898	-19.20%	62609	-9.50%	65999	-4.60%	58527	-15.40%
P47	130060	101967	-21.60%	103138	-20.70%	117574	-9.60%	123297	-5.20%	110031	-15.40%
												P48	86733	65483	-24.50%	70080	-19.20%	78580	-9.40%	82049	-5.40%	73810	-14.90%
P49	14172	11862	-16.30%	11338	-20.00%	12967	-8.50%	13322	-6.00%	11961	-15.60%
												P50	153755	123465	-19.70%	123158	-19.90%	140840	-8.40%	144837	-5.80%	130999	-14.80%
Deviation of positive sample Mean value	/	/	-19.74%	/	-19.52%	/	-9.46%	/	-5.32%	/	-14.97%

From the above results, it can be seen that when the concentration of dodecyl dimethyl betaine used in the present invention is in the range of 0.2 to 10mg/mL, the light intensity deviation of the kits of examples 2 and 3 is within 10% compared to the kit of example 1. When the concentration is too low or too high, the detection results of the kits of comparative examples 8 and 9 are closer to those of the kit without adding the dodecyl dimethyl betaine surfactant, wherein the optimal concentration of the dodecyl dimethyl betaine is 5 mg/mL. Presumably, when the concentration of the dodecyl dimethyl betaine in the working solution is 5mg/mL, the closed zone of the RBD tripolymer antigen is opened completely, and the formed space structure is more favorable for being combined with the new crown neutralizing antibody.

The invention discloses a chemiluminescence kit for detecting a novel coronavirus neutralizing antibody. By utilizing a signal amplification system of streptavidin-biotin, RBD tripolymer antigen is adopted to replace single RBD antigen, 5mg/mL dodecyl dimethyl betaine is added, the average light intensity, the analysis sensitivity and the detection speed of the kit are greatly improved, and meanwhile, the quantitative detection of a novel coronavirus neutralizing antibody can be realized; particularly, the kit is used for a full-automatic chemiluminescence system, the steps of sample adding, incubation, cleaning, detection and the like can be automatically performed in an experiment, the result deviation caused by manual operation is avoided, the working efficiency is improved, and only a test sample needs to be arranged in the test software, so that the novel coronavirus neutralizing antibody in the sample can be quantitatively detected, and the detection is quicker, more reliable and more stable.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims (10)

1. A kit for detecting a novel coronavirus neutralizing antibody, comprising (a) or (b):

(a) tracer-labelled RBD trimer antigen, ACE2 coated on a solid support, and a working solution containing 0.2-10mg/mL dodecyl dimethyl betaine;

(b) tracer-labeled ACE2, RBD trimer antigen coated on a solid support, and, working solution containing 0.2-10mg/mL dodecyl dimethyl betaine;

wherein the RBD trimer antigen is obtained by completely crosslinking RBD of the spike protein and S2 subunit by using disulfide bond.

2. The kit of claim 1, wherein the working fluid comprises: 0.01mol/L-0.2mol/L phosphate buffer solution, 0.1-50mg/mL bovine serum albumin, 5-50mg/mL sodium chloride, 10-50mg/mL sucrose, 0.2-10mg/mL dodecyl dimethyl betaine and 0.5-5mL/Lproclin 300, and the pH value is 6.0-7.6.

3. The kit of claim 2, wherein the working fluid comprises: phosphate buffer solution with the concentration of 0.05mol/L, bovine serum albumin with the concentration of 10mg/mL, sodium chloride with the concentration of 8mg/mL, sucrose with the concentration of 10mg/mL, dodecyl dimethyl betaine with the concentration of 0.2-10mg/mL and proclin 300 with the concentration of 0.5mL/L, and the pH value is 7.4.

4. The kit of claim 3, wherein the concentration of dodecyl dimethyl betaine in the working solution is 5 mg/mL.

5. The kit of claim 1, wherein the tracer comprises acridinium esters and derivatives thereof, horseradish peroxidase, alkaline phosphatase, or ruthenium terpyridyl; the solid support comprises magnetic particles, a microtiter plate, a nitrocellulose membrane or polystyrene latex, and the magnetic particles comprise carboxyl magnetic beads, amino magnetic beads, NHS magnetic beads, hydroxyl magnetic beads, tosyl magnetic beads or epoxy magnetic beads.

6. The kit of claim 1, wherein each of ACE2 in (a) or RBD trimer antigen in (b) is independently coated on a solid support via a biotin-avidin system.

7. The kit of any one of claims 1 to 6, further comprising a calibrator and a quality control for the novel coronavirus neutralizing antibodies.

8. The kit as claimed in claim 7, wherein the preservation solutions of the calibrator and the quality control are each independently selected from the group consisting of 0.01mol/L to 0.2mol/L phosphate buffer, 100mL/L calf serum, 5mL/L to 100mL/L ethylene glycol, 0.2 mL/10 mL/L LTween20 and 0.5mL/L proclin 300, and the pH is 6.0 to 7.6.

9. The kit of claim 8, wherein the preservation solutions of the calibrator and the quality control are each independently selected from the group consisting of 0.05mol/L phosphate buffer, 100mL/L calf serum, 5mL/L ethylene glycol, 1mL/L LTween20, and 0.5mL/L proclin 300, and has a pH of 7.4.

10. Use of a kit according to any one of claims 1 to 9 for the detection of neutralizing antibodies against novel coronaviruses or for the evaluation of novel coronavirus vaccines.