CN113721032A - Novel quantitative detection kit for coronavirus neutralizing antibody and application thereof - Google Patents
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Abstract
The invention discloses a novel coronavirus SARS-CoV-2 neutralizing antibody quantitative detection kit, which consists of a microporous plate 1 coated with a new coronavirus S1 protein, a microporous plate 2 coated with an AXL protein and an ACE2 protein, a neutralizing antibody standard solution, a positive control, a negative control, a sample diluent, a recombinant RBD + NTD protease conjugate, a 20 multiplied concentrated washing solution, a display solution A, a display solution B, a stop solution and an instruction book, wherein the microporous plate 1 is arranged in a box body. The invention also discloses application of the kit in detecting biological samples containing the novel coronavirus neutralizing antibody. The neutralizing antibody titer in a trace cell neutralizing test can be effectively estimated according to the concentration of the neutralizing antibody determined by the kit, and the method is used for evaluating the generation of the neutralizing antibody after the novel coronavirus vaccine is inoculated. The kit has good sensitivity and specificity and has important value for accurately evaluating the vaccine effect clinically.
Description
Technical Field
The invention relates to detection of a coronavirus neutralizing antibody, in particular to a novel quantitative detection kit of a coronavirus (SARS-CoV-2) neutralizing antibody and application thereof, belonging to the technical field of clinical examination.
Background
Since the new coronavirus (SARS-CoV-2, hereinafter referred to as new crown) was reported in 2019, the new crown epidemic spread continuously all over the world and the number of infection cases has been increasing. To effectively cope with epidemic situations, the development of new corona vaccines is being accelerated in all countries of the world, and up to now, several vaccines have been approved successively in each country and region for population vaccination. By 8 months in 2021, china has completed 19 hundred million doses of new crown vaccination.
In the evaluation of the clinical effectiveness of vaccines, the protective efficacy of the vaccines (i.e., the percentage of the incidence of disease prevented after vaccination) is the most critical evaluation index. Meanwhile, the detection of the neutralizing antibody also plays an important role in evaluating the immune response level of a human body after vaccination and the like.
The laboratory gold standard for detection of neutralizing antibodies is an infection inhibition assay, which mainly includes a Plaque Reduction Neutralization Test (PRNT) using live virus and a microcytotic neutralization test (CPE) that is analyzed by detecting the amount of cytopathic effect. In both methods, a certain amount of live virus is mixed with equivalent serum of different dilutions, and then a monolayer of cells prepared in advance is inoculated, and the degree of pathological changes of the cells is evaluated through different indexes to evaluate the titer of neutralizing antibodies. Neutralization assays with live viruses cultured in vitro reflect the titer of total neutralizing antibodies in human blood samples that block viral infection. However, the test method needs a professional cell culture process, has high requirements on the experimental grade, long period and complex operation, and is not suitable for high-throughput screening of common vaccination population.
At present, many scientific research institutions and enterprises at home and abroad take the neutralizing antibody detection reagent as a research and development direction and conduct much exploration. Generally, the detection of neutralizing antibodies is carried out by adopting an enzyme-linked immunosorbent assay based on the competitive inhibition principle based on single epitope or multiple epitopes. The proposed intended use of the product is related to the evaluation of the effectiveness after vaccination. However, the above products or technologies are all neutralizing antibody in vitro diagnostic and qualitative products, and the correlation between the concentration value of the neutralizing antibody and the protective efficacy of the vaccine cannot be effectively evaluated. Through retrieval, the detection kit which can accurately and quantitatively determine the novel coronavirus neutralizing antibody in the body of a new corona vaccine injection population, determine the affinity of the neutralizing antibody and facilitate the evaluation of the vaccine protection efficacy, and the preparation method and the application thereof are not reported.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a novel coronavirus (SARS-CoV-2) neutralizing antibody quantitative detection kit and application thereof.
The invention relates to a novel coronavirus SARS-CoV-2 neutralizing antibody quantitative detection kit, which consists of a microporous plate 1 which is arranged in a box body and is coated with a new coronavirus S1 protein, a microporous plate 2 which is coated with an AXL protein and an ACE2 protein, a neutralizing antibody standard solution, a positive control, a negative control, a sample diluent, a recombinant RBD + NTD protease conjugate, a 20 multiplied concentrated washing solution, a display solution A, a display solution B, a stop solution and an instruction book;
the method is characterized in that:
the microporous plate 1 is a polystyrene microporous plate, and each hole of the microporous plate is coated with a new coronavirus S1 protein with the concentration of 1-5 mug/ml;
the microporous plate 2 is a polystyrene microporous plate, and each hole of the microporous plate is coated with an AXL protein (tyrosine protein kinase receptor UFO) with the concentration of 1-5 mug/ml and an ACE2 protein (angiotensin converting enzyme 2) with the concentration of 2-5 mug/ml, wherein the coating proportion of the AXL protein and the ACE2 protein in each micropore is 1: 2-1: 4;
the neutralizing antibody standard solution is a mixed solution containing an RBD neutralizing antibody and an NTD neutralizing antibody, wherein the molar ratio of the RBD neutralizing antibody to the NTD neutralizing antibody is 2: 1; the standard solution is 7 standard solutions with concentration gradients of 0AU/mL, 0.1563AU/mL, 0.3125AU/mL, 0.625AU/mL, 1.25AU/mL, 2.5AU/mL and 5AU/mL respectively, wherein 0AU/mL is standard buffer solution: phosphate buffer, pH 7.4;
the positive control is a mixed solution of 3ug/ml RBD neutralizing antibody and 1ug/ml NTD neutralizing antibody;
the negative control is a negative serum of a normal human new coronavirus SARS-CoV-2 antibody;
the formula of the sample diluent is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、CTAB 1g、BSA 5g、TritonX-100 0.1mL、Proclin300 1mL，pH 7.4；
The RBD + NTD protease conjugate is a horseradish peroxidase-labeled recombinant RBD + NTD protease conjugate; the enzyme-labeled enzyme conjugate has a working titer of 1:7000, the diluent is an enzyme conjugate diluent, and the formula of the enzyme conjugate diluent is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、BSA 3.5g、Proclin300 1mL；
The formula of the 20 multiplied concentrated washing solution is as follows: sterilized 50mL double distilled water containing 8.0g NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O2.9 g and Tween 200.5 mL;
the display liquid A is a Tetramethylbenzidine (TMB) solution with the concentration of 0.3 mg/mL; the display liquid B is a carbamide peroxide solution with the concentration of 0.74 mg/mL; mixing the display liquid A and the display liquid B at a volume ratio of 1: 1;
the stop solution is a sulfuric acid solution with the concentration of 2 mol/L.
In the novel coronavirus SARS-CoV-2 neutralizing antibody quantitative detection kit: each hole of the micro-porous plate 1 is preferably coated with the new coronavirus S1 protein with the concentration of 3 mug/ml; each well of the microplate 2 is preferably coated with an AXL protein, i.e. tyrosine protein kinase receptor UFO, at a concentration of 2 μ g/ml and an ACE2 protein, i.e. angiotensin converting enzyme 2, at a concentration of 4 μ g/ml, wherein the AXL protein and the ACE2 protein are preferably coated in a ratio of 1:2 per well.
In the novel coronavirus SARS-CoV-2 neutralizing antibody quantitative detection kit: the RBD neutralizing antibody is obtained by filtering inactivated novel coronavirus infector serum through an agarose microsphere chromatographic column provided with recombinant RBD protein coupling, wherein the RBD neutralizing antibody can be combined to an agarose microsphere, obtaining a purified RBD neutralizing antibody through impurity washing and elution, detecting and screening titer, fusing a determined RBD neutralizing antibody variable region sequence with humanized Fc, and performing recombinant expression through CHO cells; the NTD neutralizing antibody is obtained by filtering inactivated serum of a novel coronavirus infected person through an agarose microsphere chromatographic column provided with recombinant NTD protein coupling, combining the NTD neutralizing antibody on an agarose microsphere, washing, eluting to obtain a purified NTD neutralizing antibody, performing titer detection and screening, measuring an NTD neutralizing antibody variable region sequence, fusing with humanized Fc, and performing recombinant expression through CHO cells.
The invention relates to the application of the novel coronavirus SARS-CoV-2 neutralizing antibody quantitative detection kit in the detection of biological samples containing the novel coronavirus neutralizing antibody.
Further, the detection of the novel coronavirus neutralizing antibody comprises a content determination of the neutralizing antibody and an affinity determination of the neutralizing antibody; wherein the content of the first and second substances,
the content determination method of the neutralizing antibody comprises the following steps:
(1) respectively adding 25uL of sample diluent into each hole in the microporous plate 1 and the microporous plate 2, and then respectively adding 25uL of samples to be detected into the holes of the microporous plate containing the sample diluent; in the microplate 1, 7 standard solutions of 0AU/mL, 0.1563AU/mL, 0.3125AU/mL, 0.625AU/mL, 1.25AU/mL, 2.5AU/mL, 5AU/mL, respectively, were added to each well containing the sample diluent at 25 ℃ for 30 minutes;
(2) after the reaction is finished, washing the plate 5 +/-1 times by using washing liquid, soaking for 20-40 s each time, adding liquid in each hole of not less than 350 mu L, and drying by beating after washing;
(3) adding 100uL of RBD + NTD protease conjugate into each of the microporous plate 1 and the microporous plate 2, mixing, sealing the reaction plate with a self-adhesive sealing sheet, and bathing at 37 deg.C for 20 min;
(4) after the reaction is finished, washing the plate 5 +/-1 times by using washing liquid, soaking for 20-40 s each time, adding liquid in each hole of not less than 350 mu L, and drying by beating after washing;
(5) respectively adding 50 mu L of color development liquid A into the microporous plate 1 and 50 mu L of color development liquid B into the microporous plate 2, and carrying out light-proof warm bath at 37 ℃ for 10 minutes;
(6) respectively adding 50 mu L of stop solution into the microporous plate 1 and the microporous plate 2, stopping the reaction, and detecting the light absorption value of each hole at the dual wavelengths of 450nm and 630nm by using an enzyme-labeling instrument;
(7) the calculation method of the microplate 1 is as follows: drawing a standard curve on coordinate paper by taking each concentration of the standard substance as an abscissa and taking the OD value as an ordinate, and finding out the corresponding concentration of the sample to be detected from the standard curve according to the measured OD value; or calculating a linear regression equation of a standard curve by using the concentration and OD value of the standard substance, substituting the OD value of the sample to be measured into the equation, calculating the concentration of the sample, and multiplying the concentration by the dilution multiple to obtain the actual concentration of the sample;
the method for measuring the affinity of the neutralizing antibody comprises the following steps:
(1) adding 25uL of sample diluent into each hole in the microporous plate 2, respectively adding 25uL of the sample to be detected, the positive control and the negative control into the hole of the microporous plate containing the sample diluent, adding 100uL of RBD + NTD protease conjugate, and carrying out warm bath at 37 ℃ for 30 minutes;
(2) after the reaction is finished, washing the plate 5 +/-1 times by using washing liquid, soaking for 20-40 s each time, adding liquid in each hole of not less than 350 mu L, and drying by beating after washing;
(3) adding 100 mu L of color development liquid A into a microporous plate, then adding 100 mu L of color development liquid B, and carrying out light-proof warm bath at 37 ℃ for 10 minutes;
(4) adding 50 mu L of stop solution into the microporous plate, stopping the reaction, and detecting the light absorption value of each hole at the dual wavelengths of 450nm and 630nm by using an enzyme-labeling instrument;
(5) the calculation result method of the micro-porous plate 2 is as follows: and calculating the inhibition rate according to the light absorption value, and judging the result.
And (4) result judgment standard: S/CO is more than or equal to 30%: novel coronavirus neutralizing antibody positive, S/CO < 30%: the novel coronavirus neutralizing antibody is negative.
Where S is an abbreviation for sample and CO is an abbreviation for cut off.
The invention relates to application of a novel coronavirus neutralizing antibody quantitative detection kit and application of an index in neutralizing antibody evaluation of a new corona vaccine inoculated population and a new corona infected rehabilitation population.
The novel quantitative detection kit for the coronavirus neutralizing antibody provided by the invention has the advantages that the concentration of the new corona neutralizing antibody and the affinity of the new corona neutralizing antibody in the human body injected with the new corona vaccine can be intuitively and accurately reflected; the concentration of the new corona neutralizing antibody in the new corona vaccine injection population is directly measured through the detection result of the microporous plate 1, and the high and low affinity of the new corona neutralizing antibody is directly reflected through the detection result of the microporous plate 2; the two indexes complement each other, and are beneficial to epidemiological investigation of epidemic diseases, immune antibody monitoring and vaccine immunity efficacy evaluation.
The detection principle of the novel quantitative detection kit for the coronavirus neutralizing antibody is as follows: coating a polystyrene microporous plate with new crown S1 protein, adding a sample diluent and a sample, combining a neutralizing antibody in the sample with the new crown S1 protein to form a complex, washing, combining with horseradish peroxidase-labeled recombinant RBD + NTD protease conjugate (HRP-RBD-NTD) enzyme, forming an 'S1 protein-neutralizing antibody-RBD-NTD' complex if the sample contains a novel coronavirus neutralizing antibody, and not developing if the sample does not contain the novel coronavirus neutralizing antibody, and finally adding an enzyme substrate for developing, and reading OD values at 450nm and 630nm on an enzyme labeling instrument. The OD value is in direct proportion to the concentration of RBD neutralizing antibodies in the sample; coating a polystyrene microporous plate with AXL protein and ACE2 protein, adding sample diluent, a sample and a recombinant RBD + NTD protease conjugate (HRP-RBD-NTD), wherein a neutralizing antibody in the sample competes with the HRP-RBD-NTD to bind with the AXL protein and the ACE2 protein on the microporous plate, forming a 'neutralizing antibody-RBD-NTD' complex if the sample contains a novel coronavirus neutralizing antibody, forming an 'HRP-ACE 2-RBD' complex and an 'HRP-AXL-RBD' complex if the sample does not contain the novel coronavirus neutralizing antibody, finally adding an enzyme substrate for color development, and reading OD values at 450nm and 630nm on an enzyme labeling instrument. The OD value is inversely proportional to the activity of the neutralizing antibodies in the sample.
The invention has the beneficial effects that:
(1) detecting a new crown neutralizing antibody by using the S1 protein and the recombinant RBD + NTD fusion protein in a double-antigen sandwich form, tracing a neutralizing antibody standard product according to a WHO neutralizing antibody standard disc, and obtaining a detection result which is an international unit and has international standardization;
(2) there are two main sites for neocorona neutralizing antibodies: RBD zone and NTD zone. The published or developed novel in vitro detection mode of the crown neutralizing antibody mainly aims at the RBD region for detection, and a kit for the combined detection of the RBD region neutralizing antibody and the NTD region neutralizing antibody is rarely reported. The method utilizes the AXL protein and the ACE2 protein as capture proteins to carry out activity identification of neutralizing antibodies by adopting a competitive inhibition method, and is an effective supplement and improvement to the prior art.
(3) The kit for detecting the novel coronavirus neutralizing antibody based on the enzyme-linked immunosorbent assay disclosed by the invention provides a more convenient detection method, and has the effects of intuitively and accurately reflecting the concentration of the novel corona neutralizing antibody and the affinity of the novel corona neutralizing antibody in a novel corona vaccine injection population.
Drawings
FIG. 1 is a graph showing the correlation between the concentrations of neutralizing antibodies in the standard.
FIG. 2 is a linear analysis of the novel quantitative detection kit for coronavirus neutralizing antibodies of the invention.
Detailed Description
The present invention will be described in detail with reference to the following detailed drawings and examples. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are only for explaining the present invention and not for limiting the present invention in any form, and any simple modifications, equivalent changes and modifications made to the embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.
In the following examples, materials, reagents and the like used were obtained commercially unless otherwise specified.
Example 1: preparation of novel coronavirus neutralizing antibody quantitative detection kit
1. Coating of the micro-porous plate:
the coating method of the micro-porous plate 1 comprises the following steps:
1.1) preparing a coating liquid: preparing carbonate buffer solution and adjusting the pH value to 9.6;
Na2CO3 1.59g
NaHCO3 2.94g
the purified water is fixed to the volume of 1000mL
Filtering for sterilization, and storing at 4 deg.C;
1.2) preparing a washing liquid, wherein the formula and the preparation method of the washing liquid are as follows:
filtering for sterilization, and storing at 4 deg.C;
1.3) preparing sealing liquid, wherein the formula and the preparation method of the sealing liquid are as follows:
filtering for sterilization, and storing at 4 deg.C;
1.4) diluting the new coronavirus S1 protein to the working concentration of 1-5 mug/mL by using a coating solution, uniformly mixing, and standing for 10 min;
1.5) taking a marked microporous plate, using an 8-pore row gun to spot the plate, enabling the protein solution of the new coronavirus S1 to reach 150 mu L/pore, and covering a cover plate film;
1.6) placing in a refrigerator with the temperature of 2-8 ℃ for coating for 4-16 hours;
1.7) taking out the coated plate, balancing for 30min at room temperature, throwing off protein liquid, beating the water-absorbing paper to be dry, adding 300 mu L of washing liquid into each hole, washing for 2 times, and beating the water-absorbing paper to be dry;
1.8) adding 250 mu L of sealing liquid into each hole, and sealing for 16 hours at 2-8 ℃ overnight;
1.9) taking out the coated plate, balancing the coated plate at room temperature for 30min, throwing off confining liquid, and patting the coated plate dry by absorbent paper; drying in a silica gel dryer at room temperature under humidity below 30% for 5 hr;
1.10) packaging the coated board in an aluminum foil bag in vacuum, marking, and storing at 2-8 ℃ for later use.
The coating method of the micro-porous plate 2 comprises the following steps:
1.11) preparing a coating liquid: preparing carbonate buffer solution and adjusting the pH value to 9.6;
Na2CO3 1.59g
NaHCO3 2.94g
the purified water is fixed to the volume of 1000mL
Filtering for sterilization, and storing at 4 deg.C;
1.12) preparing a washing liquid, wherein the formula and the preparation method of the washing liquid are as follows:
filtering for sterilization, and storing at 4 deg.C;
1.13) preparing sealing liquid, wherein the formula and the preparation method of the sealing liquid are as follows:
filtering for sterilization, and storing at 4 deg.C;
1.14) respectively diluting the AXL protein and the ACE2 protein by using coating liquid to working concentrations of 1-5 mu g/ml AXL protein and 2-5 mu g/ml ACE2, calculating and controlling the coating amount of the AXL protein and the ACE2 protein per micropore to be 1: 2-1: 4, uniformly mixing, and standing for 10 min;
1.15) taking the marked microporous plate, using an 8-pore row gun to spot the plate, enabling the protein solution to reach 150 mu L/pore, and covering a cover plate film;
1.16) placing in a refrigerator with the temperature of 2-8 ℃ for coating for 4-16 hours;
1.17) taking out the coated plate, balancing for 30min at room temperature, throwing off protein liquid, beating the water-absorbing paper to be dry, adding 300 mu L of washing liquid into each hole, washing for 2 times, and beating the water-absorbing paper to be dry;
1.18) adding 250 mu L of sealing liquid into each hole, sealing for 16 hours at 2-8 ℃ overnight;
1.19) taking out the coated plate, balancing the coated plate at room temperature for 30min, throwing off confining liquid, and patting the coated plate dry by absorbent paper; drying in a silica gel dryer at room temperature under humidity below 30% for 5 hr;
1.20) packaging the coated board in an aluminum foil bag in vacuum, marking, and storing at 2-8 ℃ for later use.
2. Preparation of a kit solution:
preparation of the base solution
The sample diluent formula is:
adjusting pH to 7.4, filtering for sterilization, and storing at 4 deg.C.
The formula of 20 × concentrated washing solution is: sterilized 50mL double distilled water containing 8.0g NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O2.9 g and Tween 200.5 mL.
The RBD neutralizing antibody is obtained by filtering inactivated novel coronavirus infector serum through an agarose microsphere chromatographic column provided with recombinant RBD protein coupling, wherein the RBD neutralizing antibody can be combined to an agarose microsphere, obtaining a purified RBD neutralizing antibody through impurity washing and elution, detecting and screening titer, fusing a determined RBD neutralizing antibody variable region sequence with humanized Fc, and performing recombinant expression through CHO cells;
the NTD neutralizing antibody is obtained by filtering inactivated serum of a novel coronavirus infected person through an agarose microsphere chromatographic column provided with recombinant NTD protein coupling, combining the NTD neutralizing antibody on an agarose microsphere, washing, eluting to obtain a purified NTD neutralizing antibody, performing titer detection and screening, fusing a determined NTD neutralizing antibody variable region sequence with humanized Fc, and performing recombinant expression through CHO (Chinese hamster ovary) cells;
the neutralizing antibody standard substance gradient solution is respectively as follows: 7 standard solutions with concentration gradient of 0AU/mL, 0.1563AU/mL, 0.3125AU/mL, 0.625AU/mL, 1.25AU/mL, 2.5AU/mL, 5AU/mL, wherein 0AU/mL is standard buffer solution; the standard buffer solution is as follows: the pH of the phosphate buffer solution is 7.4.
The negative control is a negative serum of a normal human new coronavirus SARS-CoV-2 antibody.
The positive control is a mixed solution of 3ug/ml RBD neutralizing antibody and 1ug/ml NTD neutralizing antibody;
in the above kit for quantitatively detecting a neutralizing antibody against a novel coronavirus, the formula of the diluent of the enzyme conjugate is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、BSA 3.5g、Proclin300 1mL。
The preparation method of the recombinant RBD + NTD enzyme conjugate comprises the following steps: the conjugate of the recombinant RBD + NDT protein marked by a sodium periodate method and horseradish peroxidase (HRP) is utilized. The preparation method is not limited to this method, and other protein labeling methods are also possible.
Optimization of the use amount of the recombinant RBD + NTD enzyme conjugate: and (3) selecting according to the sensitivity and the detection range as indexes by adopting a chessboard method. Preferred use titer concentrations are 1: 7000.
the formulation of the enzyme conjugate diluent was: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、BSA 3.5g、Proclin300 1mL；
The color developing agent is Tetramethylbenzidine (TMB) solution with the concentration of 0.3mg/mL and prepared by taking dimethyl sulfoxide (DMSO) as a solvent, named as solution A, and urea peroxide solution with the concentration of 0.74mg/mL and prepared by taking disodium hydrogen phosphate with the pH value of 5.5 and 0.2mol/L and citric acid buffer solution with the concentration of 0.1mol/L as a solvent, named as solution B, and when the color developing agent is used, the solution A and the solution B are mixed according to the volume ratio of 1: 1;
the stop solution is a sulfuric acid solution with the concentration of 2 mol/L.
Example 2: the invention discloses application of a novel coronavirus neutralizing antibody quantitative detection kit in detection of a biological sample containing the novel coronavirus neutralizing antibody.
The novel coronavirus SARS-CoV-2 neutralizing antibody quantitative detection kit comprises a microporous plate 1 coated with a new coronavirus S1 protein, a microporous plate 2 coated with an AXL protein and an ACE2 protein, a neutralizing antibody standard solution, a positive control, a negative control, a sample diluent, a recombinant RBD + NTD protease conjugate, a 20 multiplied concentrated washing solution, a display solution A, a display solution B, a stop solution and an instruction book, wherein the microporous plate 1 is arranged in a box body;
specifically, the method comprises the following steps:
the microporous plate 1 is a polystyrene microporous plate, and each hole of the microporous plate 1 is coated with a new coronavirus S1 protein with the concentration of 3 mug/ml;
the microporous plate 2 is a polystyrene microporous plate, and each hole of the microporous plate 2 is coated with 2 mug/ml of AXL protein (tyrosine protein kinase receptor UFO) and 4 mug/ml of ACE2 protein (angiotensin converting enzyme 2), wherein the coating proportion of the AXL protein and the ACE2 protein in each hole is 1: 2;
the neutralizing antibody standard solution is a mixed solution containing an RBD neutralizing antibody and an NTD neutralizing antibody, wherein the molar ratio of the RBD neutralizing antibody to the NTD neutralizing antibody is 2: 1; the standard solution is 7 standard solutions with concentration gradients of 0AU/mL, 0.1563AU/mL, 0.3125AU/mL, 0.625AU/mL, 1.25AU/mL, 2.5AU/mL and 5AU/mL respectively, wherein 0AU/mL is standard buffer solution: phosphate buffer, pH 7.4;
the positive control is a mixed solution of 3ug/ml RBD neutralizing antibody and 1ug/ml NTD neutralizing antibody;
the negative control is a negative serum of a normal human new coronavirus SARS-CoV-2 antibody;
the formula of the sample diluent is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、CTAB 1g、BSA 5g、TritonX-100 0.1mL、Proclin300 1mL，pH 7.4；
The RBD + NTD protease conjugate is a horseradish peroxidase-labeled recombinant RBD + NTD protease conjugate; the enzyme-labeled enzyme conjugate has a working titer of 1:7000, the diluent is an enzyme conjugate diluent, and the formula of the enzyme conjugate diluent is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、BSA 3.5g、Proclin300 1mL；
The formula of the 20 multiplied concentrated washing solution is as follows: sterilized 50mL double distilled water containing 8.0g NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O2.9 g and Tween 200.5 mL;
the display liquid A is a Tetramethylbenzidine (TMB) solution with the concentration of 0.3 mg/mL; the display liquid B is a carbamide peroxide solution with the concentration of 0.74 mg/mL; mixing the display liquid A and the display liquid B at a volume ratio of 1: 1;
the stop solution is a sulfuric acid solution with the concentration of 2 mol/L.
The preferred method for detecting samples containing novel coronavirus neutralizing antibodies is:
a, content determination of neutralizing antibodies:
(1) respectively adding 25uL of sample diluent into each hole in the microporous plate 1 and the microporous plate 2, and then respectively adding 25uL of samples to be detected into the holes of the microporous plate containing the sample diluent; in the microplate 1, 7 standard solutions of 0AU/mL, 0.1563AU/mL, 0.3125AU/mL, 0.625AU/mL, 1.25AU/mL, 2.5AU/mL, 5AU/mL, respectively, were added to each well containing the sample diluent at 25 ℃ for 30 minutes;
(2) after the reaction is finished, washing the plate 5 +/-1 times by using washing liquid, soaking for 20-40 s each time, adding liquid in each hole of not less than 350 mu L, and drying by beating after washing;
(3) adding 100uL of RBD + NTD protease conjugate into each of the microporous plate 1 and the microporous plate 2, mixing, sealing the reaction plate with a self-adhesive sealing sheet, and bathing at 37 deg.C for 20 min;
(4) after the reaction is finished, washing the plate 5 +/-1 times by using washing liquid, soaking for 20-40 s each time, adding liquid in each hole of not less than 350 mu L, and drying by beating after washing;
(5) respectively adding 50 muL of color development liquid A into the microporous plate 1 and the microporous plate 2, then adding 50 muL of color development liquid B, and carrying out dark warm bath at 37 ℃ for 10 minutes;
(6) respectively adding 50 mu L of stop solution into the microporous plate 1 and the microporous plate 2, stopping the reaction, and detecting the light absorption value of each hole at the dual wavelengths of 450nm and 630nm by using an enzyme-labeling instrument;
(7) the calculation method of the microplate 1 is as follows: drawing a standard curve on coordinate paper by taking each concentration of the standard substance as an abscissa and taking the OD value as an ordinate, and finding out the corresponding concentration of the sample to be detected from the standard curve according to the measured OD value; or calculating a linear regression equation of the standard curve by using the concentration and OD value of the standard substance, substituting the OD value of the sample to be measured into the equation, calculating the concentration of the sample, and multiplying by the dilution factor to obtain the actual concentration of the sample.
B neutralizing antibody affinity assay:
(1) adding 25uL of sample diluent into each hole in the microporous plate 2, respectively adding 25uL of the sample to be detected, the positive control and the negative control into the hole of the microporous plate containing the sample diluent, adding 100uL of RBD + NTD protease conjugate, and carrying out warm bath at 37 ℃ for 30 minutes;
(2) after the reaction is finished, washing the plate 5 +/-1 times by using washing liquid, soaking for 20-40 s each time, adding liquid in each hole of not less than 350 mu L, and drying by beating after washing;
(3) adding 100 mu L of color development liquid A into a microporous plate, then adding 100 mu L of color development liquid B, and carrying out light-proof warm bath at 37 ℃ for 10 minutes;
(4) adding 50 mu L of stop solution into the microporous plate, stopping the reaction, and detecting the light absorption value of each hole at the dual wavelengths of 450nm and 630nm by using an enzyme-labeling instrument;
(5) the calculation result method of the micro-porous plate 2 is as follows: and calculating the inhibition rate according to the light absorption value, and judging the result.
And (4) result judgment standard: S/CO is more than or equal to 30%: novel coronavirus neutralizing antibody positive, S/CO < 30%: the novel coronavirus neutralizing antibody is negative.
Example 3: the invention discloses a detection method for linearity, accuracy, recovery rate and precision of a novel coronavirus neutralizing antibody quantitative detection kit
1) Tracing and calibrating a neutralizing antibody standard product:
the neutralizing antibody standard solution is as follows: 7 standard solutions with a concentration gradient of 0AU/mL, 0.1563AU/mL, 0.3125AU/mL, 0.625AU/mL, 1.25AU/mL, 2.5AU/mL, 5AU/mL, comprising RBD neutralizing antibody and NTD neutralizing antibody at a molar ratio of 2: 1;
the first international standard (NIBSC code: 20/136) of the human anti-SARS-CoV-2 immunoglobulin of the world health organization is diluted according to the conversion relation of the neutralizing antibody (the international standard is the concentration of the neutralizing antibody/0.00798), and the corresponding international standard concentrations are respectively: 0AU/mL, 19.586AU/mL, 39.160AU/mL, 78.321AU/mL, 156.642AU/mL, 313.283AU/mL, 626.566AU/mL, the detection results of the international standard samples at each concentration are shown in Table 1, respectively, by performing the detection according to step A of example 2:
TABLE 1
Concentration correlation analysis: the correlation analysis of the concentration of neutralizing antibody in the standard is shown in FIG. 1.
2) Linearity of the kit
1) Preparing a standard neutralizing antibody: and 7 standard solutions with concentration gradient of 0AU/mL, 0.1563AU/mL, 0.3125AU/mL, 0.625AU/mL, 1.25AU/mL, 2.5AU/mL, 5AU/mL, wherein the RBD neutralizing antibody and the NTD neutralizing antibody are contained at a molar ratio of 2: 1.
2) And (3) testing a calibration sample: the OD value was obtained by testing the prepared calibrator by the method described in step a of example 2.
3) Establishment of a standard curve: and generating a calibration curve of the batch of reagents by adopting a proper fitting mode according to the prepared concentration and the OD value of the calibrator. See table 2 and figure 2.
Table 2: reagent kit linearity
| Concentration of | 0AU/mL | 0.1563AU/mL | 0.3125AU/mL | 0.625AU/mL | 1.25AU/mL | 2.5AU/mL | 5AU/mL |
| OD value | 0.012 | 0.150 | 0. | 0.412 | 0.441 | 1.089 | 2.357 |
Example 4 application of the novel coronavirus neutralizing antibody quantitative detection kit in screening clinical samples of injection vaccines
The immune samples of clinically injected new corona vaccines of 200 cases and the immune samples of not injected new corona vaccines of 400 cases are compared with the trace cell neutralization experiment, the statistical index of the coincidence or difference degree of the determination result of the kit of the invention and the trace cell neutralization experiment result by using the microporous plate 2 is calculated, and the evaluation method is shown in the table 3.
Table 3: the kit of the invention is compared with a trace cell neutralization test
And (3) sensitivity calculation: 194/194 × 100% ═ 100%.
And (3) calculating the specificity: 400/406X 100% ═ 98.52%
The total coincidence rate is as follows: (194+ 400)/600X 100% ═ 99%
The above tests show that the kit for detecting the novel coronavirus neutralizing antibody and the detection method thereof provided by the invention have good consistency with the gold standard for detecting the neutralizing antibody.
The results of the experiments of neutralizing the trace cells of the immune samples after completing the clinical injection of the new corona vaccine in 200 cases and the results of the assay of the kit of the invention by using the microplate 1 are counted, and the relationship of the antibody titer is shown in Table 4:
TABLE 4
Neutralization assays with live viruses cultured in vitro reflect the titer of total neutralizing antibodies in human blood samples that block viral infection. Due to the source of the live virus strain and the limitation of operation in a high-grade biosafety laboratory, the method has certain limitations on popularization and operability. The invention provides a method for quantitatively detecting a novel coronavirus neutralizing antibody and application thereof, which can effectively presume the neutralizing antibody titer in a trace cell neutralizing test according to the concentration of the detected neutralizing antibody, can be used for evaluating the generation of the neutralizing antibody after the inoculation of a novel coronavirus vaccine, has good sensitivity and specificity, and is favorable for accurately evaluating the vaccine effect clinically.
Claims (5)
1. A novel coronavirus SARS-CoV-2 neutralizing antibody quantitative detection kit comprises a microporous plate 1 coated with new coronavirus S1 protein, a microporous plate 2 coated with AXL protein and ACE2 protein, a neutralizing antibody standard solution, a positive control, a negative control, a sample diluent, a recombinant RBD + NTD protease conjugate, 20 Xconcentrated washing solution, a display solution A, a display solution B, a stop solution and an instruction, which are arranged in a box body;
the method is characterized in that:
the microporous plate 1 is a polystyrene microporous plate, and each hole of the microporous plate is coated with a new coronavirus S1 protein with the concentration of 1-5 mug/ml;
the microporous plate 2 is a polystyrene microporous plate, and each hole of the microporous plate is coated with an AXL protein (tyrosine protein kinase receptor UFO) with the concentration of 1-5 mug/ml and an ACE2 protein (angiotensin converting enzyme 2) with the concentration of 2-5 mug/ml, wherein the coating proportion of the AXL protein and the ACE2 protein in each micropore is 1: 2-1: 4;
the neutralizing antibody standard solution is a mixed solution containing an RBD neutralizing antibody and an NTD neutralizing antibody, wherein the molar ratio of the RBD neutralizing antibody to the NTD neutralizing antibody is 2: 1; the standard solution is 7 standard solutions with concentration gradients of 0AU/mL, 0.1563AU/mL, 0.3125AU/mL, 0.625AU/mL, 1.25AU/mL, 2.5AU/mL and 5AU/mL respectively, wherein 0AU/mL is standard buffer solution: phosphate buffer, pH 7.4;
the positive control is a mixed solution of 3ug/ml RBD neutralizing antibody and 1ug/ml NTD neutralizing antibody;
the negative control is a negative serum of a normal human new coronavirus SARS-CoV-2 antibody;
the formula of the sample diluent is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、CTAB 1g、BSA 5g、TritonX-100 0.1mL、Proclin300 1mL，pH 7.4；
The RBD + NTD protease conjugate is a horseradish peroxidase-labeled recombinant RBD + NTD protease conjugate; the enzyme-labeled enzyme conjugate has a working titer of 1:7000, the diluent is enzyme conjugate diluentThe formula is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、BSA 3.5g、Proclin300 1mL；
The formula of the 20 multiplied concentrated washing solution is as follows: sterilized 50mL double distilled water containing 8.0g NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O2.9 g and Tween 200.5 mL;
the display liquid A is a Tetramethylbenzidine (TMB) solution with the concentration of 0.3 mg/mL; the display liquid B is a carbamide peroxide solution with the concentration of 0.74 mg/mL; mixing the display liquid A and the display liquid B at a volume ratio of 1: 1;
the stop solution is a sulfuric acid solution with the concentration of 2 mol/L.
2. The kit for quantitatively detecting a neutralizing antibody against SARS-CoV-2 as a coronavirus according to claim 1, wherein: each hole of the micropore plate 1 is coated with a new coronavirus S1 protein with the concentration of 3 mug/ml; the microporous plate 2 is coated with 2 mug/ml AXL protein (tyrosine protein kinase receptor UFO) and 4 mug/ml ACE2 protein (angiotensin converting enzyme 2) in each hole, wherein the ratio of the AXL protein to the ACE2 protein in each hole is 1: 2.
3. The kit for quantitatively detecting a neutralizing antibody against SARS-CoV-2 as a coronavirus according to claim 1, wherein: the RBD neutralizing antibody is obtained by filtering inactivated novel coronavirus infector serum through an agarose microsphere chromatographic column provided with recombinant RBD protein coupling, wherein the RBD neutralizing antibody can be combined to an agarose microsphere, obtaining a purified RBD neutralizing antibody through impurity washing and elution, detecting and screening titer, fusing a determined RBD neutralizing antibody variable region sequence with humanized Fc, and performing recombinant expression through CHO cells; the NTD neutralizing antibody is obtained by filtering inactivated serum of a novel coronavirus infected person through an agarose microsphere chromatographic column provided with recombinant NTD protein coupling, combining the NTD neutralizing antibody on an agarose microsphere, washing, eluting to obtain a purified NTD neutralizing antibody, performing titer detection and screening, measuring an NTD neutralizing antibody variable region sequence, fusing with humanized Fc, and performing recombinant expression through CHO cells.
4. Use of the novel coronavirus SARS-CoV-2 neutralizing antibody quantitative detection kit as claimed in claim 1 in the detection of biological samples containing the novel coronavirus neutralizing antibody.
5. Use according to claim 4, characterized in that: the detection of the novel coronavirus neutralizing antibody comprises the content determination of the neutralizing antibody and the affinity determination of the neutralizing antibody; wherein the content of the first and second substances,
the content determination method of the neutralizing antibody comprises the following steps:
(1) respectively adding 25uL of sample diluent into each hole in the microporous plate 1 and the microporous plate 2, and then respectively adding 25uL of samples to be detected into the holes of the microporous plate containing the sample diluent; in the microplate 1, 7 standard solutions of 0AU/mL, 0.1563AU/mL, 0.3125AU/mL, 0.625AU/mL, 1.25AU/mL, 2.5AU/mL, 5AU/mL, respectively, were added to each well containing the sample diluent at 25 ℃ for 30 minutes;
(2) after the reaction is finished, washing the plate 5 +/-1 times by using washing liquid, soaking for 20-40 s each time, adding liquid in each hole of not less than 350 mu L, and drying by beating after washing;
(3) adding 100uL of RBD + NTD protease conjugate into each of the microporous plate 1 and the microporous plate 2, mixing, sealing the reaction plate with a self-adhesive sealing sheet, and bathing at 37 deg.C for 20 min;
(4) after the reaction is finished, washing the plate 5 +/-1 times by using washing liquid, soaking for 20-40 s each time, adding liquid in each hole of not less than 350 mu L, and drying by beating after washing;
(5) respectively adding 50 mu L of color development liquid A into the microporous plate 1 and 50 mu L of color development liquid B into the microporous plate 2, and carrying out light-proof warm bath at 37 ℃ for 10 minutes;
(6) respectively adding 50 mu L of stop solution into the microporous plate 1 and the microporous plate 2, stopping the reaction, and detecting the light absorption value of each hole at the dual wavelengths of 450nm and 630nm by using an enzyme-labeling instrument;
(7) the calculation method of the microplate 1 is as follows: drawing a standard curve on coordinate paper by taking each concentration of the standard substance as an abscissa and taking the OD value as an ordinate, and finding out the corresponding concentration of the sample to be detected from the standard curve according to the measured OD value; or calculating a linear regression equation of a standard curve by using the concentration and OD value of the standard substance, substituting the OD value of the sample to be measured into the equation, calculating the concentration of the sample, and multiplying the concentration by the dilution multiple to obtain the actual concentration of the sample;
the method for measuring the affinity of the neutralizing antibody comprises the following steps:
(1) adding 25uL of sample diluent into each hole in the microporous plate 2, respectively adding 25uL of the sample to be detected, the positive control and the negative control into the hole of the microporous plate containing the sample diluent, adding 100uL of RBD + NTD protease conjugate, and carrying out warm bath at 37 ℃ for 30 minutes;
(2) after the reaction is finished, washing the plate 5 +/-1 times by using washing liquid, soaking for 20-40 s each time, adding liquid in each hole of not less than 350 mu L, and drying by beating after washing;
(3) adding 100 mu L of color development liquid A into a microporous plate, then adding 100 mu L of color development liquid B, and carrying out light-proof warm bath at 37 ℃ for 10 minutes;
(4) adding 50 mu L of stop solution into the microporous plate, stopping the reaction, and detecting the light absorption value of each hole at the dual wavelengths of 450nm and 630nm by using an enzyme-labeling instrument;
(5) the calculation result method of the micro-porous plate 2 is as follows: calculating the inhibition rate according to the light absorption value, and judging the result;
and (4) result judgment standard: S/CO is more than or equal to 30%: novel coronavirus neutralizing antibody positive, S/CO < 30%: the novel coronavirus neutralizing antibody is negative.
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