CN112710830A - Novel element mass spectrometry combined detection kit for coronavirus IgG and IgM antibodies - Google Patents

Novel element mass spectrometry combined detection kit for coronavirus IgG and IgM antibodies Download PDF

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CN112710830A
CN112710830A CN202011500900.4A CN202011500900A CN112710830A CN 112710830 A CN112710830 A CN 112710830A CN 202011500900 A CN202011500900 A CN 202011500900A CN 112710830 A CN112710830 A CN 112710830A
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lanthanide
kit
novel coronavirus
value
antibody
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盛琳峰
周东奇
杨文兴
黄一
刘华
焦梦荷
吕建材
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Beijing Qingfen Wentong Technology Co ltd
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Beijing Qingfen Wentong Technology Co ltd
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G01N27/626—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using heat to ionise a gas
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Abstract

The invention discloses an element mass spectrometry combined detection kit for novel coronavirus IgG and IgM antibodies. The kit provided by the invention comprises: an ELISA plate coated with a novel coronavirus antigen; an anti-human IgG antibody label labeled with lanthanide a 1; an anti-human IgM antibody label labelled with lanthanide a 2; wherein a1 and a2 are two different lanthanides. The method of the invention uses the element mass spectrometry, greatly shortens the reaction time, and has the advantages of high accuracy, strong specificity, high sensitivity, wide linear range, and stable and convenient detection. The kit can also realize large-flux detection and meet the requirement of novel coronavirus detection of the pedestrian flow in different cities.

Description

Novel element mass spectrometry combined detection kit for coronavirus IgG and IgM antibodies
Technical Field
The invention relates to the technical field of in-vitro diagnostic reagents, in particular to a novel element mass spectrometry joint detection kit for coronavirus IgG and IgM antibodies.
Background
2019A novel coronavirus (SARS-nCoV-2019), also called "new coronavirus", belongs to enveloped beta coronavirus, the genetic material of the coronavirus is a single positive sense RNA chain, the RNA is enveloped by a Protein shell with spike Protein (S Protein) distributed on the surface, and the spike Protein enables the new coronavirus to have high infectivity. The new coronavirus has the capability of human transmission, the transmission routes comprise respiratory droplet transmission and contact transmission, the transmission can be carried out through direct contact with secretion with the virus, and no specific treatment method for diseases caused by the new coronavirus exists at present.
Therefore, there is a need to develop detection systems for new coronaviruses to meet clinical needs. The diagnostic kits developed at present can be classified into two types according to the detection target, one is nucleic acid for detecting viruses, and the other is antibody for detecting viruses. However, these diagnostic kits have the following disadvantages that the requirements for epidemic prevention and clinical work, especially for epidemiological survey and general inspection, cannot be fully met: the specificity of the nucleic acid detection kit is close to 100%, but the sensitivity is low due to difficult sampling and the like, and is only 30% -50%, namely, more missed detections exist; the sensitivity of the antibody detection kit using SARS-nCoV-2019 constituent proteins such as S protein and N protein can reach more than 95%, but the specificity is low due to principle defects (particularly for some patients with diseases, non-SARS-nCoV-2019 antibodies in the bodies of the patients can be identified by the constituent proteins), namely more false detections exist.
Disclosure of Invention
In order to solve the above technical problems, the present invention aims to provide a novel coronavirus detection method which can reduce the detection cost, shorten the detection time, and improve the detection sensitivity and linear range.
In a first aspect, the invention claims a novel element mass spectrometry combined detection kit for coronavirus IgG and IgM antibodies.
The invention claims a novel element mass spectrometry combined detection kit of coronavirus IgG and IgM antibodies, which comprises:
(1) an ELISA plate coated with a novel coronavirus antigen;
(2) an anti-human IgG antibody label labeled with lanthanide a 1;
(3) an anti-human IgM antibody label labelled with lanthanide a 2;
the lanthanide a1 and the lanthanide a2 are two different lanthanides.
Wherein the lanthanide a 1-labeled anti-human IgG antibody label is derived from the reaction of a lanthanide a 1-chelator with an anti-human IgG antibody (i.e., the lanthanide a1 is labeled onto the anti-human IgG antibody by the chelator); the lanthanide A2 labeled anti-human IgM antibody label is obtained by reacting lanthanide A2-chelator with anti-human IgM antibody (i.e., by labeling the lanthanide A2 to the anti-human IgM antibody with chelator).
The lanthanide includes, but is not limited to, thulium (Tm), lutetium (Lu). The chelating agent includes, but is not limited to, diethyltriaminepentaacetic acid (DTPA).
In a particular embodiment of the invention, the lanthanide a1 is thulium (Tm); the lanthanide a2 is lutetium (Lu); the chelating agent is diethyltriaminepentaacetic acid (DTPA).
In a specific embodiment of the present invention, the lanthanide a1 labeled anti-human IgG antibody label is obtained by reacting the anti-human IgG antibody with DTPA-Tm. In the reaction process, the adding amount ratio of the anti-human IgG antibody to the DTPA-Tm is 1: 10 (molar ratio), the reaction temperature is 25 ℃, and the reaction time is 18 h.
In a specific embodiment of the present invention, the anti-human IgM antibody label labeled with lanthanide a2 is obtained by reacting the anti-human IgM antibody with DTPA-Lu. In the reaction process, the adding amount ratio of the anti-human IgM antibody to the DTPA-Lu is 1: 10 (molar ratio), the reaction temperature is 25 ℃, and the reaction time is 18 h.
Wherein, the DTPA-Tm and the DTPA-Lu can be prepared by a method comprising the following steps: separately reacting chelating agent (DTPA) with TmCl3、LuCl3According to a molar ratio of 1: 2 to obtain DTPA-Tm and DTPA-Lu. The reaction conditions may be: the reaction was carried out at 25 ℃ for 30 min.
Further, the kit may further comprise all or part of:
(4) an immune buffer;
the composition of the immune buffer is as follows: each liter of the immune buffer solution contains 4-6g (such as 5g) of sodium casein, 45-55g (such as 50g) of sucrose, 1.8-2.2g (such as 2g) of fish skin gelatin, 900-1100 mu L (such as 1000 mu L) of Proclin-300 liquid, 900-1100 mu L (such as 1000 mu L) of Tween 20 liquid, and the balance of Tris-HCl solution with the pH value of 7.8.
In a specific embodiment of the present invention, the Tris-HCl solution with pH 7.8 is prepared according to a method comprising the following steps: 56-66g (60.57 g) of Tris (hydroxymethyl) aminomethane (Tris) and 85-95g (90 g) of sodium chloride are added into 800ml of deionized water and fully stirred to be completely dissolved, a hydrochloric acid solution is added to adjust the pH value of the solution to 7.8, and deionized water is added to the solution to reach the constant volume of 1L, so that a Tris-HCl solution is obtained.
(5) Concentrating the cleaning solution (10 times the concentrated cleaning solution);
the solvent of the concentrated cleaning solution is water, and the solute and the concentration thereof are trihydroxymethyl aminomethane (tris)60.57g/L, sodium chloride 90g/L and Tween 20500 mu L/L; adding hydrochloric acid solution to adjust pH value to 7.8.
(6) A dissociation liquid;
the dissociation liquid is a solution with the Re concentration of 1ng/mL prepared by adopting a dilute nitric acid solution with the volume ratio of 1 percent. The rhenium (Re) is an internal standard element of inductively coupled plasma mass spectrometry (ICP-MS).
In the kit, the novel coronavirus antigen coated in the microplate may be a novel coronavirus surface protein (S protein) Receptor Binding Domain (RBD).
If necessary, the kit can also contain a confining liquid.
The solvent of the confining liquid can be water, and the solutes and the concentrations can be as follows: KCl 0.4g/L, KH2PO4 0.48g/L、Na2HPO42.88g/L, NaCl 16g/L, 10g/L BSA, 10g/L sodium casein, 50g/L sucrose, 0.1% (volume percentage) proclin-300.
The enzyme label plate coated with the novel coronavirus antigen can be prepared by the following steps: the novel coronavirus antigen is diluted to 1-3. mu.g/mL (e.g., 1. mu.g/mL) using 0.02-0.05M (e.g., 0.02M) carbonate buffer, added to an microplate, and coated at 4 ℃ for 20 hours. After the coating is finished, the method also comprises the step of sealing.
In the kit, the anti-human IgG antibody may be a murine anti-human IgG antibody; the anti-human IgM antibody may be a murine anti-human IgM antibody.
In a specific embodiment of the present invention, the murine anti-human IgG antibody is a product of beijing baiolaibo, a murine anti-human IgG (fc) antibody, having a cargo number of F010202; the mouse anti-human IgM antibody is specifically a product of Beijing Baiolaibo company, and the mouse anti-human IgM (mu chain) antibody has a cargo number of F010204.
In a second aspect, the invention claims a system for detecting whether a sample to be tested is infected with a novel coronavirus.
The system for detecting whether a sample to be detected is infected by the novel coronavirus or not, which is claimed by the invention, comprises the kit and the data processing device.
The data processing device may include the following modules:
(a1) a data acquisition module; the data acquisition module is configured to receive an S/CO value obtained by detecting novel coronavirus IgG antibodies and/or IgM antibodies by the kit through inductively coupled plasma mass spectrometry; the S/CO value is a detection concentration value/cutoff value of the sample to be detected; the cutoff value for detecting the novel coronavirus IgG antibody is 0.7, and the cutoff value for detecting the novel coronavirus IgM antibody is 0.9.
Wherein the detection concentration value is the ratio of the response value of lanthanide labeled with anti-human IgG antibody/anti-human IgM antibody to the response value of internal standard element.
(a2) A data comparison module; the data comparison module is configured to receive the S/CO value of the subject from the data acquisition module and compare it to a value of 1.
(a3) A result determination module; the structure determination module is configured to receive the comparison result from the data comparison module and then determine the result according to a predetermined condition.
Further, the predetermined condition is: if the S/CO value of the person to be detected is more than or equal to 1, judging that the sample to be detected is positive (namely the sample to be detected contains a novel coronavirus IgG antibody and/or IgM antibody, and the sample to be detected is infected by the novel coronavirus); and if the S/CO value of the person to be detected is less than 1, judging that the sample to be detected is negative (namely the sample to be detected does not contain a novel coronavirus IgG antibody and/or IgM antibody, and the sample to be detected is not infected by the novel coronavirus).
In a third aspect, the invention claims the use of a kit as hereinbefore described or a system as hereinbefore described in any of:
p1, detecting whether the sample to be detected contains a novel coronavirus IgG antibody and/or IgM antibody;
p2, preparation of products for detection of novel coronaviruses;
p3, product for preparing diagnostic COVID-19.
The application of P1 can be non-disease diagnosis and treatment application, such as simple detection of blood products (such as serum) containing novel coronavirus IgG antibody and/or IgM antibody.
In the above aspects, the sample to be tested may be an ex vivo human blood sample or a serum sample.
In each of the above aspects, the novel coronavirus is SARS-nCoV-2019.
The ELISA plate coated with the novel coronavirus antigen can be a 96-hole transparent microplate, the variation among the wells is not more than 10 percent, and the ELISA plate has the following functionality: matching other qualified components can ensure the coincidence rate of the negative reference substance, the coincidence rate of the positive reference substance, the precision and the detection limit of the novel coronavirus antibody. Store at 4 ℃.
Lutetium (Lu) -labeled mouse anti-human IgM antibody and thulium (Tm) -labeled mouse anti-human IgG antibody need to satisfy the requirements of clear and transparent appearance, no precipitation and no turbidity. Functionality: matching other qualified components can ensure the coincidence rate of the negative reference substance, the coincidence rate of the positive reference substance, the precision and the detection limit of the novel coronavirus antibody. Store at 4 ℃.
Negative reference substance: and (5) detecting 20 negative reference substances, and preventing false positives. Positive reference substance: and 5 positive reference substances are detected, and false negatives cannot occur.
Compared with the prior art, the invention has the beneficial effects that: the method uses the element mass spectrometry, greatly shortens the reaction time, and has the advantages of high accuracy, strong specificity, high sensitivity, wide linear range, and stable and convenient detection. The kit can also realize large-flux detection and meet the detection requirements of the pedestrian flow in different cities.
Compared with the prior art, the novel detection kit for the element mass spectrometry of the coronavirus IgG and IgM has the following advantages:
the effect comparison data is shown in table 1.
TABLE 1 Effect comparison data
Figure BDA0002843566640000041
Figure BDA0002843566640000051
Drawings
FIG. 1 is a ROC curve according to the present invention.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 preparation of element mass spectrometry joint detection kit for novel coronavirus IgG and IgM antibodies
The element mass spectrometry combined detection kit for the novel coronavirus IgG and IgM antibodies comprises an enzyme label plate coated with a novel coronavirus antigen, a mouse anti-human IgG antibody marker labeled by lanthanide thulium, a mouse anti-human IgM antibody marker labeled by lanthanide lutetium, an immune buffer solution, a concentrated cleaning solution, a dissociation solution and a confining solution.
First, preparation of various solutions
1. Immune buffer solution
60.57g of tris (hydroxymethyl) aminomethane (tris) and 90g of sodium chloride are weighed and added into 800ml of deionized water to be fully stirred so as to be completely dissolved to obtain a solution;
adding a hydrochloric acid solution to adjust the pH value of the solution to 7.8, adding deionized water to a constant volume of 1L to obtain a Tris-HCl solution;
thirdly, weighing 5g of sodium casein, 50g of cane sugar and 2g of fish skin gelatin, adding Tris-HCl, stirring and fully dissolving;
adding 1000 mul Proclin-300 liquid and 1000 mul Tween 20 liquid into the solution, and stirring uniformly to dissolve the solution fully;
adding Tris-HCl solution to constant volume of 1L.
2. Concentrated cleaning solution
60.57g of tris (hydroxymethyl) aminomethane (tris) and 90g of sodium chloride are weighed and added into 800ml of deionized water to be fully stirred so as to be completely dissolved to obtain a solution;
adding hydrochloric acid solution to adjust the pH value of the solution to 7.8;
adding 500 mu L of Tween 20 liquid into the solution obtained in the step II, and stirring uniformly to fully dissolve the Tween;
and fourthly, fixing the volume of the deionized water to 1L, and storing the deionized water at the normal temperature.
The preparation method is to prepare 10 times of concentrated cleaning solution, and when the cleaning solution is used, the 10 times of concentrated cleaning solution is mixed with purified water according to the weight ratio of 1: 10 dilutions (100mL concentrated wash plus 900mL purified water) gave 1 × wash. If the washing liquid has crystals, the washing liquid is placed at room temperature or is diluted after the crystals are dissolved at 37 ℃.
3. Dissociation liquid
The dissociation liquid is a solution with the Re concentration of 1ng/mL, and the solution is prepared by adopting a 1% (volume ratio) dilute nitric acid solution. Rhenium (Re) is an internal standard element for inductively coupled plasma mass spectrometry (ICP-MS).
PBS blocking solution
Weighing KCl 0.4g and KH2PO4 0.48g、Na2HPO42.88g and 16g of NaCl, and adding 800mL of deionized water for dissolution;
adding 10g BSA, 10g sodium casein, 50g sucrose and 0.1% (volume percentage) proclin-300;
and adding deionized water to a constant volume of 1L.
5. Preparation of Probe
Separately reacting chelating agent (DTPA) with TmCl3、LuCl3According to a molar ratio of 1: 2, and reacting at 25 ℃ for 30min to obtain DTPA-Tm and DTPA-Lu.
Second, preparation of enzyme label plate coated with novel coronavirus antigen
The solid phase carrier of the ELISA plate is a transparent micropore plate with 96 holes, the variation among the holes is not more than 10 percent, and the ELISA plate has the following functionality: matching other qualified components can ensure the coincidence rate of the negative reference substance, the coincidence rate of the positive reference substance, the precision and the detection limit of the novel coronavirus antibody. Store at 4 ℃.
Novel coronavirus antigens (Ginnapo, 2019-nCov-S-RBDC-mFC-Ag, AG0016) are diluted to 1 mu g/mL by using 0.02M carbonate buffer solution, added into an enzyme label plate, and coated for 20 hours at 4 ℃.
② after coating, discarding the liquid in the hole, washing the plate with diluted 1 × cleaning solution, adding PBS sealing solution, sealing for 2 hours at 37 ℃.
Thirdly, filling the mixture into an aluminum foil bag, putting a proper drying agent into the aluminum foil bag, labeling the aluminum foil bag, and sealing and storing the aluminum foil bag at the temperature of 4 ℃.
Preparation of mouse anti-human IgG antibody marker labeled by lanthanide thulium (Tm)
Putting a mouse anti-human IgG antibody (Beijing Baiolaibobo mouse anti-human IgG (Fc) antibody F010202) into a dialysis bag with a cut-off amount of 25KD, replacing two thirds of the dialysis solution by using 0.05M carbonate buffer solution as dialysis solution for 2 hours, 4 hours and 8 hours respectively, and collecting the dialyzed antibody after 24 hours.
Mixing the dialyzed antibody with 0.1M carbonate buffer solution and DTPA-Tm, wherein the adding amount ratio of the new crown antibody to thulium is 1: 10 (molar ratio) and at 25 ℃ for 18 hours.
③ after the marking solution stays overnight, the solution is purified by gel column chromatography balanced by 0.05M Tris-HCl buffer solution, and the first peak is collected and subpackaged by A280 detection.
Preparation of mouse anti-human IgM antibody marker labeled by lanthanide lutetium (Lu)
Putting a mouse anti-human IgM antibody (a Beijing Baiolaibobo mouse anti-human IgM (mu chain) antibody F010204) into a dialysis bag with a cut-off amount of 25KD, replacing two thirds of dialysate in 2 hours, 4 hours and 8 hours respectively by using 0.05M carbonate buffer as dialysate, and collecting the dialyzed antibody after 24 hours.
Mixing the dialyzed antibody with 0.1M carbonate buffer solution and DTPA-Lu, wherein the addition ratio of the neocoronal antibody to lutetium is 1: 10 (molar ratio) and at 25 ℃ for 18 hours.
③ after the marking solution stays overnight, the solution is purified by gel column chromatography balanced by 0.05M Tris-HCl buffer solution, and the first peak is collected and subpackaged by A280 detection.
Fifthly, spraying codes and labeling, and assembling into a finished product kit
Example 2 method for using element mass spectrometry combined detection kit for novel coronavirus IgG and IgM antibodies
First, the kit components were equilibrated at room temperature (18-25 ℃).
II, preparing liquid: the 10-fold concentrated washing solution was washed with purified water in a ratio of 1: 10 dilutions (100mL of concentrated wash plus 900mL of purified water) gave 1 × washes. If the washing liquid has crystals, the washing liquid is placed at room temperature or is diluted after the crystals are dissolved at 37 ℃.
Third, sample analysis Process
1. And (3) using immune buffer solution for a sample to be detected according to the ratio of 1: diluting at 200 volume ratio, mixing, and mixing for 3 seconds by a vortex mixer.
2. Taking out a proper amount of the ELISA plate coated with the novel coronavirus antigen according to the experimental requirements. Two positive control holes and two negative control holes are respectively arranged, and the rest are sample holes to be detected.
3. 50 μ L of diluted sample, plus negative and positive control samples, was added to each well.
Positive control sample: xinguan serum quality control, Wanbo biology, cat number ABNCOVN 001.
Negative control samples: normal human serum.
4. The sample wells were sealed with a sealing membrane by shaking gently and shaking up manually, and reacted in an incubator at 37 ℃ for 30 minutes.
5. After the reaction is finished, the sealing plate membrane is uncovered, the reaction solution is discarded and is dried by beating, 1 Xwashing solution is manually added, the washing solution in each hole is not less than 200 mu L, the washing solution is discarded, and the plate washing is repeated for five times. Washing can also be carried out using a plate washer.
6. After the plate washing was completed, 50. mu.L of the element-labeled mixture of mouse anti-human IgG and IgM antibodies, each having a concentration of 0.1mg/mL, was added to each well.
7. The sample wells were sealed with a sealing membrane by shaking gently manually and reacted for 15 minutes in a 37 ℃ incubator.
8. After the reaction is finished, the sealing plate membrane is uncovered, the reaction solution is discarded and is dried by beating, 1 Xwashing solution is manually added, the washing solution in each hole is not less than 200 mu L, the washing solution is discarded, and the plate washing is repeated for five times. Washing can also be carried out using a plate washer.
9. Adding 100 μ L dissociation solution into each well, and dissociating for 5 min
10. And after the dissociation is finished, the sample is tested on a machine. And (3) introducing the sample treated in the step (9) into inductively coupled plasma mass spectrometry (ICP-MS) for detection, and collecting response values of the lanthanide Tm, Lu and the internal standard element Re, namely a count value (cps) of corresponding ions per second to obtain response value ratios Tm/Re and Lu/Re of the Tm, Lu and the internal standard element Re, namely detection concentration values. S/CO is further calculated according to the ratio of Tm/Re and Lu/Re (namely a detection concentration value), and the S/CO refers to the ratio of Tm/Re or Lu/Re and corresponds to a cutoff value.
Example 3 application example of element mass spectrometry joint detection kit for novel coronavirus IgG and IgM antibodies
1. Supplying a sample book: serum from 5 clinically confirmed patients positive for the novel coronavirus (SARS-nCoV-2019); 20 clinically confirmed sera of patients negative for the novel coronavirus (SARS-nCoV-2019).
The following instrumentation was used: inductively coupled plasma mass spectrometers. The model is as follows: iCAP Q. The manufacturer: thermo Scientific.
2. The precision design requirement is as follows:
precision reference product N: the negative detection rate is 100 percent, (n is 20);
precision reference L: the positive detection rate is more than or equal to 95 percent (n is 20);
precision reference CV value: the positive detection rate is 100%, and the CV value is less than or equal to 10% (n is 20).
3. Test method
Internal precision: detecting the precision reference substance, performing parallel determination for 20 times, and calculating the negative detection rate and the positive detection rate. The result should meet the design requirement of precision.
Coefficient of variation: CV is SD/AV 100%;
wherein CV is a coefficient of variation, SD is a standard deviation of the test results, and AV is an average value of the test results.
4. Results
The results of the test were analyzed by using the SPSS software to perform ROC curve analysis, which is shown in Table 2 and FIG. 1. The S/CO of the sample is more than or equal to 1, and the detection result is judged to be positive for the novel coronavirus (namely the sample to be detected contains a novel coronavirus IgG antibody/IgM antibody and is infected by the novel coronavirus); and (3) judging the sample to be negative for the novel coronavirus (namely the sample to be detected does not contain the novel coronavirus IgG antibody/IgM antibody, and the sample to be detected is not infected by the novel coronavirus) by the detection result when the S/CO of the sample is less than 1. And determining the embodying of the precision of the kit under the condition of different cutoff values through an ROC curve, and screening out the optimal cutoff value of 0.7 for IgG and the optimal cutoff value of 0.9 for IgM.
The ROC curve analysis result shows that the IgG area under the curve is 0.915 and the IgM area under the curve is 0.947, which indicates that the kit has higher accuracy in clinical diagnosis.
TABLE 2 ROC Curve analysis results
Figure BDA0002843566640000091
The data of the negative and positive coincidence rates and the precision detection of each sample under the optimal cutoff value (0.7 for IgG and 0.9 for IgM) are shown in Table 3.
TABLE 3 negative and positive coincidence rates and precision detection data (optimal cutoff value) for each sample
Figure BDA0002843566640000092
Figure BDA0002843566640000101
Figure BDA0002843566640000111
Note: in the table, P1-P5 indicates the serum of 5 clinically confirmed patients positive for the novel coronavirus (SARS-nCoV-2019); N1-N20 shows the sera of 20 clinically confirmed patients who were negative for the novel coronavirus (SARS-nCoV-2019).
At the optimal cutoff values (IgG of 0.7 and IgM of 0.9), the results of the novel coronavirus infection detection (parts) are shown in tables 4 and 5. It can be seen that the detection result of the kit of the invention is consistent with the detection result of nucleic acid.
TABLE 4 negative test results (optimal cutoff values) for part of the novel coronavirus infection
Figure BDA0002843566640000112
Figure BDA0002843566640000121
TABLE 5 positive test results (optimal cutoff values) for part of the novel coronavirus infection
Figure BDA0002843566640000122
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Claims (10)

1. A novel element mass spectrometry joint detection kit for coronavirus IgG and IgM antibodies, comprising:
(1) an ELISA plate coated with a novel coronavirus antigen;
(2) an anti-human IgG antibody label labeled with lanthanide a 1;
(3) an anti-human IgM antibody label labelled with lanthanide a 2;
the lanthanide a1 and the lanthanide a2 are two different lanthanides.
2. The kit of claim 1, wherein: the lanthanide A1 labeled anti-human IgG antibody marker is obtained by reacting lanthanide A1-chelator with anti-human IgG antibody;
the lanthanide A2 labeled anti-human IgM antibody label is obtained by reacting lanthanide A2-chelating agent with anti-human IgM antibody.
3. The kit of claim 2, wherein: the lanthanide a1 and the lanthanide a2 are selected from thulium and lutetium.
4. The kit according to claim 2 or 3, characterized in that: the chelating agent is diethyl triaminepentaacetic acid.
5. The kit according to any one of claims 1 to 4, wherein: the kit also comprises all or part of the following components:
(4) an immune buffer;
the composition of the immune buffer is as follows: each liter of the immune buffer solution contains 4-6g of sodium casein, 45-55g of sucrose, 1.8-2.2g of fish skin gelatin, 1100 mu L of Proclin-300 liquid 900-;
(5) concentrating the cleaning solution;
the solvent of the concentrated cleaning solution is water, and the solute and the concentration thereof are 60.57g/L of trihydroxymethyl aminomethane, 90g/L of sodium chloride and 20500 mu L/L of tween; the pH value is 7.8;
(6) a dissociation liquid;
the dissociation liquid is a solution with the Re concentration of 1ng/mL, which is prepared by a dilute nitric acid solution with the volume percentage content of 1%.
6. The kit according to any one of claims 1 to 5, wherein: the novel coronavirus antigen coated in the enzyme label plate is a novel coronavirus surface protein receptor binding area.
7. The kit according to any one of claims 1 to 6, wherein: the anti-human IgG antibody is a murine anti-human IgG antibody; the anti-human IgM antibody is a murine anti-human IgM antibody.
8. A system for detecting whether a sample to be tested is infected with a novel coronavirus, comprising the kit of any one of claims 1 to 7 and a data processing device;
the data processing device comprises the following modules:
(a1) a data acquisition module; the data acquisition module is configured to receive an S/CO value obtained by detecting novel coronavirus IgG antibodies and/or IgM antibodies by the kit through inductively coupled plasma mass spectrometry; the S/CO value is a detection concentration value/cutoff value of the sample to be detected; the cutoff value for detecting the novel coronavirus IgG antibody is 0.7, and the cutoff value for detecting the novel coronavirus IgM antibody is 0.9;
(a2) a data comparison module; the data comparison module is configured to receive the S/CO value of the subject from the data acquisition module and compare it to a value of 1;
(a3) a result determination module; the structure determination module is configured to receive the comparison result from the data comparison module and then determine the result according to a predetermined condition.
9. The system of claim 8, wherein: (a3) wherein the predetermined condition is: if the S/CO value of the person to be tested is more than or equal to 1, judging that the sample to be tested is positive; and if the S/CO value of the person to be detected is less than 1, judging that the sample to be detected is negative.
10. Use of a kit according to any one of claims 1 to 7 or a system according to claim 8 or 9 in any one of:
p1, detecting whether the sample to be detected contains a novel coronavirus IgG antibody and/or IgM antibody;
p2, preparation of products for detection of novel coronaviruses;
p3, product for preparing diagnostic COVID-19.
CN202011500900.4A 2020-12-18 2020-12-18 Novel element mass spectrometry combined detection kit for coronavirus IgG and IgM antibodies Pending CN112710830A (en)

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