WO2022121151A1 - 一种基于磁微粒化学发光的新型冠状病毒抗体检测试剂盒 - Google Patents

一种基于磁微粒化学发光的新型冠状病毒抗体检测试剂盒 Download PDF

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WO2022121151A1
WO2022121151A1 PCT/CN2021/082727 CN2021082727W WO2022121151A1 WO 2022121151 A1 WO2022121151 A1 WO 2022121151A1 CN 2021082727 W CN2021082727 W CN 2021082727W WO 2022121151 A1 WO2022121151 A1 WO 2022121151A1
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novel coronavirus
labeled
biotin
sample
antigen
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French (fr)
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刘春龙
张舟
马少华
王家顺
粟艳
周泽奇
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丹娜(天津)生物科技股份有限公司
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Publication of WO2022121151A1 publication Critical patent/WO2022121151A1/zh

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    • 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
    • G01N33/569—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
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • 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
    • G01N33/563—Immunoassay; Biospecific binding assay; Materials therefor involving antibody fragments
    • G—PHYSICS
    • 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
    • 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
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent

Definitions

  • the application belongs to the technical field of immunoassay detection, and relates to a novel coronavirus antibody detection kit based on magnetic particle chemiluminescence.
  • Coronaviruses are a class of single-stranded positive-stranded RNA viruses with an envelope, with a diameter of about 60-220 nm, and are widely present among humans and other mammals. Most coronavirus infections are mild, but two coronavirus outbreaks have occurred with serious consequences: severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV).
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • the new coronavirus (2019-nCoV) can be transmitted by respiratory droplets, contact, etc., and has strong human-to-human infectivity. Its basic reproduction number R0 is about 2.2 (90% high density range 1.4-3.8). The most common symptoms of this virus infection were fever, cough, myalgia, or fatigue, and all patients had complicated pneumonia and abnormal chest CT findings. Some patients develop dyspnea after a week, and in severe cases, the disease progresses rapidly, and acute respiratory distress syndrome, septic shock, difficult-to-correct metabolic acidosis, and coagulation dysfunction can occur within a few days. Overall, the novel coronavirus can cause severe respiratory illness.
  • the diagnosis methods of new coronavirus pneumonia mainly include imaging diagnosis, molecular diagnosis and serological diagnosis, and serological diagnosis methods include colloidal gold method, enzyme-linked immunosorbent assay and chemiluminescence method.
  • imaging diagnosis has the problem of different diseases and the same shadow, with low specificity and prone to false positive results.
  • Molecular diagnosis has high specificity but low sensitivity and is prone to false negative results.
  • the colloidal gold method has high sensitivity, but low specificity, and is prone to false positives; the enzyme-linked immunosorbent assay requires manual operation, which has biological safety risks.
  • the present application provides a novel coronavirus antibody detection kit based on magnetic particle chemiluminescence, and also provides a preparation method and use method of the detection kit.
  • the present application provides a novel coronavirus antibody detection kit based on magnetic particle chemiluminescence, including: streptavidin magnetic particles, biotin-labeled novel coronavirus antigen, acridinesulfonamide-labeled secondary antibody , sample diluents and quality control products;
  • the biotin-labeled novel coronavirus antigens include recombinant nucleocapsid protein and recombinant spike protein S1;
  • oligomeric proline residues are used to connect the dominant epitope of the original nucleocapsid protein, and oligomeric lysine residues are used to form the C-terminus;
  • an oligomeric proline residue is used to connect the dominant epitope of the original spike protein S1
  • an oligomeric lysine residue is used to form the C-terminus.
  • the detection principle of the detection kit is: based on the chemiluminescence of streptavidin magnetic particles, the sample to be tested, the biotin-labeled antigen and the streptavidin magnetic particles are mixed, incubated and washed, and then A signal-labeled secondary antibody was added, followed by a second incubation and washing. If there is a novel coronavirus IgM antibody in the sample, a magnetic particle-streptavidin-biotin-antigen-novel coronavirus IgM antibody-secondary antibody complex is formed, and the signal substance labeled on the secondary antibody is read to be tested. The luminous intensity value of the sample.
  • the signal substance can be acridine ester, acridine sulfonamide, alkaline phosphatase and horseradish peroxidase, and acridine sulfonamide is preferred in this application.
  • the present application uses oligoproline residues -(P)n- to connect these dominant epitopes to form a tandem predicted dominant epitope and easy to stretch and bend recombinant antigen, which is beneficial to the binding of antibodies to the dominant epitope and improves the detection sensitivity.
  • the C-terminus is formed by the oligomeric lysine residue -(K)n-. Since the lysine residue has an extra amino group, it is convenient for the coupling with labels such as biotin, acridine ester, and carboxyl magnetic particles.
  • the recombinant antigen can increase the binding probability of the recombinant antigen and the label, and at the same time, when the recombinant antigen is bound to the solid phase carrier through the C-terminal oligolysine residue, the N-terminal epitope is easier to contact with the antibody; on the other hand, it can Reduce the binding probability of the dominant epitope in the recombinant antigen and the label, and prevent the epitope from being blocked by the label, resulting in difficulty in antibody recognition.
  • the detection kit is capable of detecting novel coronavirus IgM and IgG antibodies, and the type of secondary antibody can be adjusted according to the type of antibody to be detected.
  • the type of secondary antibody can be adjusted according to the type of antibody to be detected.
  • detecting IgM goat anti-human IgM antibody can be selected as the secondary antibody to detect IgG.
  • goat anti-human IgG antibody can be used as the secondary antibody.
  • this application chooses to label the antigen with biotin and label the secondary antibody with acridine sulfonamide, and use the indirect method instead of the capture method (capture method: label the secondary antibody with biotin and label the antigen with acridine sulfonamide) , the method can improve the accuracy of the obtained test results of the kit.
  • the mass ratio of nucleocapsid protein and spike protein S1 is set to be (1-3):1, and 1.5:1 is the most preferred.
  • the ratio of nucleocapsid protein and spike protein S1 is 3 : 2, the discrimination between samples is larger.
  • the mass ratio of activated biotin (Sulfo-NHS-LC-Biotin) to the novel coronavirus antigen is (0.06-0.48):1; for example It can be 0.06:1, 0.1:1, 0.12:1, 0.15:1, 0.2:1, 0.25:1, 0.3:1, 0.4:1 or 0.48:1, etc., preferably 0.24:1.
  • the recombinant nucleocapsid protein comprises the amino acid sequence shown in SEQ ID NO.1.
  • SEQ ID NO.1 is:
  • underlined are the dominant epitopes of recombinant nucleocapsid protein.
  • sequence of the original nucleocapsid protein is shown in SEQ ID NO.2:
  • the original sequence was analyzed by DNAStar Protean software, and its amino acid sequences 18-49, 138-154, 170-216, 230-266, 273-301, 338-349, 362-392, 400-419 were mostly located in ⁇ -turns (mostly located in protein surface, easy to bind to antibodies), hydrophilic index, antigen index, and surface possibility are all high, and it is predicted to be a dominant epitope.
  • different dominant epitopes can also be randomly arranged and combined to form a new recombinant antigen. Since there is also a dominant epitope, its effect is similar to the amino acid sequence shown in SEQ ID NO.1.
  • the two ends of the recombinant nucleocapsid protein are connected with amino acid residues containing benzene rings (such as phenylalanine F/tryptophan W/tyrosine Y), which is beneficial to improve the stability of the recombinant antigen.
  • amino acid residues containing benzene rings such as phenylalanine F/tryptophan W/tyrosine Y
  • the recombinant spike protein S1 comprises the amino acid sequence shown in SEQ ID NO.3.
  • SEQ ID NO.3 is:
  • underlined are the dominant epitopes of recombinant spike protein S1.
  • SEQ ID NO.4 The sequence of the original spike protein S1 is shown in SEQ ID NO.4:
  • different dominant epitopes can also be randomly arranged and combined to form a new recombinant antigen. Since there is also a dominant epitope, its effect is similar to the amino acid sequence shown in SEQ ID NO.3.
  • the two ends of the recombinant spike protein S1 are connected to amino acid residues containing a benzene ring (eg, phenylalanine F/tryptophan W/tyrosine Y), which is beneficial to improve the stability of the recombinant antigen.
  • a benzene ring eg, phenylalanine F/tryptophan W/tyrosine Y
  • the biotin-labeled novel coronavirus antigen is dispersed in PBS buffer.
  • the concentration of the PBS buffer is 0.02-0.1M, such as 0.02M, 0.05M, 0.06M, 0.08M or 0.1M, etc., preferably 0.02M.
  • the pH of the PBS buffer is 7.2 to 9.0, such as 7.2, 7.3, 7.5, 7.6, 7.8, 8.0, 8.2, 8.5, 8.8 or 9.0, etc., preferably 7.2.
  • the buffer system is 0.02M PBS (pH7.2) buffer, compared with 0.1M PBS (pH7.2), 0.05M PBS (pH7.2), 0.1M CBS (pH9.0), 0.05 M CBS (pH 9.0), 0.02M CBS (pH 9.0) buffer, the best effect.
  • the acridinesulfonamide-labeled secondary antibody is dispersed in CBS buffer.
  • the concentration of the CBS buffer is 0.02-0.1M, such as 0.02M, 0.05M, 0.06M, 0.08M or 0.1M, etc., preferably 0.1M.
  • the pH of the CBS buffer is 7.2 to 9.0, such as 7.2, 7.3, 7.5, 7.6, 7.8, 8.0, 8.2, 8.5, 8.8 or 9.0, etc., preferably 9.0.
  • the buffer system is 0.1M CBS (pH 9.0) buffer, compared with 0.1M PBS (pH7.2), 0.05M PBS (pH7.2), 0.02M PBS (pH7.2), 0.1 M CBS (pH 9.0), 0.05M CBS (pH 9.0), 0.02M CBS (pH 9.0) buffer, the best effect.
  • the sample diluent contains any one or a combination of at least two of bovine serum albumin, rheumatoid factor adsorbent, bacteriostatic agent, urea or blocking agent.
  • the interference of heterophilic antibodies in blood samples can be eliminated; by using rheumatoid factor adsorbents, the interference of rheumatoid factors in blood samples can be eliminated; by using urea, the interference of blood samples on solid-phase carriers can be reduced nonspecific binding.
  • the sample diluent can be 0.01M PBS, wherein the added mass fraction is 0.4-0.6% (for example, it can be 0.42%, 0.44%, 0.45%, 0.48%, 0.5%, 0.55%, 0.58% or 0.6%, etc.), mass fraction of 0.01 to 0.05% (for example, can be 0.012%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04% or 0.05%, etc.) antibacterial ProClin300, mass fraction of 1 ⁇ 10% (for example, it can be 1%, 2%, 3%, 5%, 6%, 8% or 10%, etc.) urea and the mass fraction is 0.1 ⁇ 0.5% (for example, it can be 0.12%, 0.15%, 0.2% , 0.25%, 0.3%, 0.35%, 0.4% or 0.5%, etc.) blocker.
  • 0.4-0.6% for example, it can be 0.42%, 0.44%, 0.45%, 0.48%, 0.5%, 0.55%, 0.58% or 0.
  • the quality control product includes a positive quality control product and a negative quality control product.
  • the positive quality control product is a buffer containing novel coronavirus antibodies.
  • the novel coronavirus antibody detection kit also includes an excitation solution and a washing solution.
  • the present application provides a preparation method of the novel coronavirus antibody detection kit as described in the first aspect, the preparation method comprising the following steps:
  • biotin-labeled novel coronavirus antigen Mix the nucleocapsid protein and spike protein S1 of novel coronavirus as antigens with PBS buffer, add activated biotin for 1.5-2.5 hours, and then dialyze to obtain the Biotin-labeled novel coronavirus antigen;
  • the present application also provides a method of using the novel coronavirus antibody detection kit as described in the first aspect, the using method comprising the following steps:
  • a two-step method is used to detect the sample to be tested, that is, the sample, biotin-labeled antigen and streptavidin magnetic particles are mixed and incubated, and then acridine sulfonamide-labeled antibody is added to incubate; Mix and incubate with biotin-labeled antigen and sample, then add acridine sulfonamide-labeled antibody to incubate) and one-step method (directly combine sample, biotin-labeled antigen, streptavidin magnetic particles and acridine) sulfonamide-labeled antibody mixed incubation), the detection effect of the two-step method is better.
  • the sample to be tested is serum or plasma.
  • the absolute value of the relative deviation of the luminescence values detected by different sample types (plasma/serum) in the same sample is less than 10%, and the detection results of plasma and serum are considered to be the same.
  • the sample to be tested is diluted with a sample diluent.
  • the samples were diluted 25 times, 50 times, 100 times, 200 times, and 400 times with the sample diluent, respectively; the diluted samples were detected by chemiluminescence, and it was found that when the sample dilution was 100 times, the discrimination was the largest.
  • the titer ratio of the sample to be tested after dilution is 1:(103-110), for example, it can be 1:103, 1:104, 1:105, 1:106, 1:107, 1:108, 1:109 or 1:110, etc., preferably 1:103.
  • the liquid volume of the sample is 50, 75 and 100 ⁇ L
  • the volume of biotin-labeled antigen is 25, 50 and 75 ⁇ L
  • the volume of magnetic microparticles is 30, 40 and 50 ⁇ L
  • the reaction time of the first step is 4, 6, 8, 10min
  • the optimal concentration of acridinesulfonamide-labeled secondary antibody is 1, 0.5, 0.25, 0.125 ⁇ g/mL
  • the reaction time of the second step is 4, 6, 8, 10min
  • the number of washings is 1, 2, 3 or 4 times to test the best reaction conditions.
  • the volume ratio of the sample to be tested, the biotin-labeled novel coronavirus antigen and the streptavidin magnetic particles is (50-100):(25-75):(30-50), for example, it can be 50:25:30, 75:25:30, 100:25:30, 50:50:30, 50:75:30, 50:75:40, 100:50:40, 100:50:50, or 100: 75:50, etc., preferably 100:50:40.
  • the working concentration of the acridinesulfonamide-labeled secondary antibody is 0.125-1 ⁇ g/mL, such as 0.125 ⁇ g/mL, 0.25 ⁇ g/mL, 0.5 ⁇ g/mL, 0.75 ⁇ g/mL, 0.8 ⁇ g/mL, 0.9 ⁇ g/mL or 1 ⁇ g/mL, etc., preferably 0.25 ⁇ g/mL.
  • the time of the first incubation is 5-15 minutes, for example, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 12 minutes, 14 minutes or 15 minutes, preferably 8 minutes.
  • the second incubation time is 5 to 15 minutes, for example, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 12 minutes, 14 minutes, or 15 minutes, preferably 8 minutes.
  • the sample to be tested if the index I in the test result is greater than or equal to 1.0, the sample to be tested is positive; if the index I in the test result is less than 1.0, the sample to be tested is negative.
  • the novel coronavirus antibody detection kit based on magnetic particle chemiluminescence provided in this application, selecting streptavidin magnetic particles, labeling the antigen with biotin and labeling the secondary antibody with acridine sulfonamide, on the one hand, by adjusting Magnetic particle coupling, blocking conditions and adjusting the composition of system stabilizers can effectively reduce false positive factors in blood samples;
  • the mass ratio of recombinant nucleocapsid protein and spike protein S1 is (1 ⁇ 3):1, which improves the specificity of detection;
  • the antibody detection kit provided by this application adopts recombinant nucleocapsid protein and recombinant spike protein as the antigen of the new coronavirus, and the recombinant protein has the dominant epitope of the original protein sequence, hydrophilic index, antigenic index, surface are highly likely to significantly increase the sensitivity of detection; and
  • the detection kit provided by the application has better sensitivity, the minimum detection limit is 1:103, the precision is higher, the repeatability is higher when repeated tests are repeated, the specificity is good, and the samples that may produce cross-reaction and normal human samples, the test results are all negative, and the relative deviation of the test results is controllable for the possible interfering substances in the samples; and through the supporting automated testing equipment, the risk of operator exposure can be effectively avoided, biological safety can be improved, and the Realize high-throughput detection and improve the detection speed of new coronavirus.
  • Figure 1 is a graph showing the fluorescence detection results obtained when the detection kit prepared with recombinant protein and original protein as antigens in Test Example 1 detects strong positive serum with different dilution times.
  • FIG. 2 is a scatter diagram of the detection results of IgM antibodies in Test Example 5.
  • FIG. 2 is a scatter diagram of the detection results of IgM antibodies in Test Example 5.
  • This embodiment provides a novel coronavirus IgM antibody detection kit based on magnetic particle chemiluminescence, which specifically includes:
  • streptavidin magnetic particles biotin-labeled novel coronavirus antigen, acridine sulfonamide-labeled goat anti-human IgM antibody, sample diluent, positive quality control substance, negative quality control substance, sample diluent and washing liquid.
  • sequence of the recombinant nucleocapsid protein is SEQ ID NO.1: the sequence of the recombinant spike protein S1 is SEQ ID NO.3:
  • the working concentration of biotin-labeled novel coronavirus antigen is 1 ⁇ g/mL
  • the working concentration of acridinesulfonamide-labeled goat anti-human IgM antibody was 1 ⁇ g/mL.
  • This example provides a novel coronavirus IgM antibody detection kit based on magnetic particle chemiluminescence.
  • the difference from Example 1 is that the mass ratio of recombinant nucleocapsid protein and recombinant spike protein S1 is set to 5:0 and 4:0, respectively. 1, 2:3, 1:4 and 0:5;
  • the reference sample is the standard sample, which is the sample to be tested used in the development process of the kit, numbered N01 ⁇ N15 (negative sample), P01 ⁇ 05 (positive sample) and L1 ⁇ L3 (quality control sample); It can be seen that when the ratio of nucleocapsid protein and spike protein S1 is 5:0, 4:1, 1:4, and 0:5, the negative coincidence rate (that is, the degree of discrimination is less than 1) is 14/15;
  • the discrimination degree of N10 samples is 2.08; when the ratio is 4:1, the discrimination degree of N10 samples is 1.16; when the ratio is 1:4, the discrimination degree of N06 samples is 1.10; the ratio is 0:5 , the N06 sample discrimination is 1.40;
  • the ratio of the two is 3:2 and 2:3, the negative coincidence rate is 15/15, and the positive coincidence rate is 5/5; and when the ratio is 2:3, the detection value of the negative reference product N06 is 0.71, which is higher than that of the negative reference product N06.
  • the N06 detection value of 0.41 is large and close to the critical value (for negative samples, the smaller the discrimination value, the better), so the detection effect is poor.
  • 3:2 is selected. The sample discrimination is good, so the ratio of nucleocapsid protein and spike protein S1 was selected as 3:2.
  • This example provides a novel coronavirus IgM antibody detection kit based on magnetic particle chemiluminescence.
  • the difference from Example 1 is that the addition amount of the Sulfo-NHS-LC-Biotin solution is set to 3, 6 and 24 ⁇ L, respectively.
  • This embodiment provides a novel coronavirus IgM antibody detection kit based on magnetic particle chemiluminescence.
  • the difference from Example 1 is that the buffer systems of the biotin-labeled novel coronavirus antigens are respectively set to 0.1M PBS (pH 7.2 ), 0.05M PBS (pH 7.2), 0.1M CBS (pH 9.0), 0.05M CBS (pH 9.0), 0.02M CBS (pH 9.0).
  • This example provides a novel coronavirus IgM antibody detection kit based on magnetic particle chemiluminescence.
  • the difference from Example 1 is that the buffer system of the acridinesulfonamide-labeled secondary antibody is set to 0.1M PBS (pH7.2 ), 0.05M PBS (pH7.2), 0.02M PBS (pH7.2), 0.05M CBS (pH9.0), 0.02M CBS (pH9.0).
  • the samples to be tested in Examples 3, 4 and 5 are standard samples, which are the samples to be tested used in the development process of the kit, numbered N01, P01 and P05;
  • the positive sample P01 has the highest degree of discrimination; when the antigen buffer system in Table 3 is 0.02M PBS, the positive sample P01 has the highest degree of discrimination. ;
  • the buffer system of the secondary antibody in Table 4 is 0.1M CBS, the discrimination of the positive sample P01 is the highest; therefore, the conditions provided in Example 1 include that the addition amount of the Sulfo-NHS-LC-Biotin solution is 12 ⁇ L, the antigen buffer system The highest degree of discrimination was obtained when the buffer system was 0.02M PBS and the secondary antibody was 0.1M CBS.
  • This embodiment provides three different detection steps, which specifically include:
  • Three-step method add 50 ⁇ L of streptavidin magnetic particles to 50 ⁇ L of biotin-labeled antigen, incubate for 5 min, and wash; add 100 ⁇ L of reference substance, incubate for 10 min, and wash; add 50 ⁇ L of acridinesulfonamide-labeled antibody, incubate for 10 min, and wash; add The excitation solution detects the luminescence value.
  • Two-step method add 50 ⁇ L of biotin-labeled antigen and 50 ⁇ L of streptavidin magnetic particles to 100 ⁇ L of reference substance, incubate for 10 min, and wash;
  • the reference sample is the standard sample, which is the sample to be tested used in the development process of the kit, numbered N01, P01 and P05; it can be seen from the above table that the two-step method has a large degree of discrimination, so the two-step method is selected as the operation step .
  • the 3 positive plasmas were diluted by multiples to obtain samples whose detection results covered the critical value; 3 samples were repeatedly diluted for each gradient, and each sample was repeatedly detected 20 times with the kit adopted in Example 1 (chemiluminescence). For detection, the sample needs to be diluted 100 times).
  • the positive detection rate of each sample was calculated, and the titer level of the antibody to be tested with the positive detection rate in the range of 90-95% was screened as the minimum detection limit.
  • the positive detection rate was in the range of 90-95%, so the titer of 1:103 was taken as the minimum detection limit.
  • Model 1 Fully automatic chemiluminescence analyzer of Chongqing Cosme Biotechnology Co., Ltd., model: SMART 6500;
  • Model 2 Fully automatic chemiluminescence immunoassay analyzer of Chongqing Cosmart Biotechnology Co., Ltd., model: SMART 500S.
  • SDR means: standard deviation caused by random errors
  • SDwi means standard deviation caused by kit batches, repeated tests during the day, operators and random errors in a laboratory
  • test results of the model SMART 500S also meet the requirements, and are not listed here for the sake of space and brevity.
  • novel coronavirus antibody detection kit provided in this application to detect samples that may have cross-reactions and normal human samples, and observe whether the test is positive; if it is positive, it means that such samples have an impact on the detection of novel coronavirus IgM antibodies.
  • Plasma samples of endemic human coronaviruses confirmed by Western blot and serological methods to contain specific IgM antibodies; H1N1 (novel H1N1 influenza virus (2009), seasonal H1N1 influenza virus ) IgM antibody positive plasma samples;
  • Test Example 4 Influence of Substances That May Cause Interference on Test Results
  • Hemoglobin in clinical samples is mainly due to specimen hemolysis, including pathological hemolysis and technical hemolysis.
  • Non-overt hemolysis means that the hemoglobin content is less than 0.5 mg/mL, and hemolysis cannot be observed with the naked eye.
  • the hemoglobin concentration of mild hemolysis is 0.5 to 3 mg/mL
  • the concentration of moderate hemolytic hemoglobin is 3.1 to 5 mg/mL
  • the concentration of severe hemolytic hemoglobin is more than 5 mg/mL.
  • the range of bilirubin in normal human serum is 2 to 8 mg/L (the range of bilirubin in infants within 1 week is 10 to 120 mg/L).
  • the bilirubin concentration in the sample is as high as 300 mg/L, the relative relative The absolute value of the deviation does not exceed 10%, indicating that there is no interference with the detection results.
  • the normal upper limit of triglyceride in clinical samples is 1.7mmol/L. From the experimental results, when the triglyceride content in the sample is 7.5mmol/L, the absolute value of the relative deviation of the test results does not exceed 10%, indicating that the test results have no effect. interference.
  • rheumatoid factor anti-nuclear antibody, anti-double-stranded DNA antibody, anti-mitochondrial antibody, HAMA positive samples, high-concentration new coronavirus IgG antibody samples, 50g/L total human IgG antibody (total IgG in healthy adults: 7-16.6 g/L), 10g/L human total IgM antibody (total IgM in healthy adults: 400-3450mg/L) and other possible interfering substances have no effect on the test results.
  • Test Example 5 Detecting samples after sampling at different time periods
  • This embodiment provides a novel coronavirus IgG antibody detection kit based on magnetic particle chemiluminescence, which specifically includes:
  • Streptavidin magnetic particles biotin-labeled novel coronavirus antigen, acridine sulfonamide-labeled goat anti-human IgG antibody, sample diluent, positive quality control substance, negative quality control substance, sample diluent and washing solution.
  • the working concentration of biotin-labeled novel coronavirus antigen is 1 ⁇ g/mL
  • the working concentration of acridinesulfonamide-labeled goat anti-human IgG antibody was 1 ⁇ g/mL.
  • the IgG antibody detection kit also has high accuracy in detecting the novel coronavirus.
  • the test results obtained after using it to detect different samples are shown in Table 10 below:
  • the IgG antibody detection kit provided in this example can accurately detect, with good sensitivity and high accuracy.
  • the detection kit provided by the present application has good sensitivity in detecting both IgM antibody and IgG antibody.
  • the minimum detection limit of the IgM antibody detection kit is 1:103, with high precision, high repeatability and good specificity when repeating the test multiple times. Negative, and for possible interfering substances in the sample, the relative deviation of the test results is controllable.

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Abstract

一种基于磁微粒化学发光的新型冠状病毒抗体检测试剂盒，包括链霉亲和素磁微粒、生物素标记的新型冠状病毒抗原、吖啶磺酰胺标记的二抗、样本稀释液和质控品；其中生物素标记的新型冠状病毒抗原包括重组核衣壳蛋白和重组棘突蛋白S1，重组核衣壳蛋白包括如SEQ ID NO.1所示的氨基酸序列，重组棘突蛋白S1包括如SEQ ID NO.3所示的氨基酸序列。试剂盒的使用方法包括：将待检样本、生物素标记的新型冠状病毒抗原与链霉亲和素磁微粒混合，孵育和洗涤，再加入吖啶磺酰胺标记的二抗，形成磁微粒‑链霉亲和素‑生物素‑新型冠状病毒抗原‑新型冠状病毒抗体‑二抗复合物，进而检测发光强度实现对待测样品的定性。

Description

一种基于磁微粒化学发光的新型冠状病毒抗体检测试剂盒 技术领域
本申请属于免疫分析检测技术领域，涉及一种基于磁微粒化学发光的新型冠状病毒抗体检测试剂盒。
背景技术
冠状病毒是一类具外套膜(envelope)的单股正链RNA病毒，直径约60～220nm，广泛存在与人和其它哺乳动物之间。大多数冠状病毒感染为轻症感染，但仍有两种冠状病毒曾爆发肆虐，引起严重后果：重症急性呼吸道综合征冠状病毒(SARS-CoV)和中东呼吸综合征冠状病毒(MERS-CoV)。
新型冠状病毒(2019-nCoV)可经呼吸道飞沫、接触等传播，具有较强的人与人传染能力，其基本再生数R0约为2.2(90％高密度区间1.4-3.8)。该病毒感染最常见的症状为发烧、咳嗽、肌痛或疲劳，所有患者均并发肺炎，胸部CT检查发现异常。部分患者在一周后出现呼吸困难，严重者病情进展迅速，数日内即可出现急性呼吸窘迫综合征、脓毒症休克、难以纠正的代谢性酸中毒和出凝血功能障碍。总体而言，新型冠状病毒可引起严重呼吸系统疾病。
目前，新型冠状病毒肺炎诊断方法主要包括影像学诊断、分子诊断和血清学诊断，血清学诊断方法包括胶体金法、酶联免疫法和化学发光法。然而，影像学诊断存在异病同影问题，特异度低，易出现假阳性结果；分子诊断特异度高，但灵敏度低，易出现假阴。对于血清学诊断方法，胶体金法灵敏度高，但特异度低，易出现假阳性；酶联免疫法需要手工操作，存在生物安全风险。
因此，利用化学发光法提供一种检测准确度高、特异性好且减少生物安全风险的检测试剂盒，对新型冠状病毒的检测和诊断具有重要的意义。
发明内容
本申请提供了一种基于磁微粒化学发光的新型冠状病毒抗体检测试剂盒，同时还提供了所述检测试剂盒的制备方法和使用方法。
第一方面，本申请提供一种基于磁微粒化学发光的新型冠状病毒抗体检测试剂盒，包括：链霉亲和素磁微粒、生物素标记的新型冠状病毒抗原、吖啶磺 酰胺标记的二抗、样本稀释液和质控品；所述生物素标记的新型冠状病毒抗原包括重组核衣壳蛋白和重组棘突蛋白S1；
所述重组核衣壳蛋白中利用寡聚脯氨酸残基连接原始核衣壳蛋白的优势表位，并利用寡聚赖氨酸残基形成C端；
所述重组棘突蛋白S1中利用寡聚脯氨酸残基连接原始棘突蛋白S1的优势表位，并利用寡聚赖氨酸残基形成C端。
本申请中，所述检测试剂盒的检测原理为：基于链霉亲和素磁微粒的化学发光，将待检样本、生物素标记抗原与链霉亲和素磁微粒混合，孵育和洗涤，再加入信号物标记二抗，进行二次孵育和洗涤。若如样本中存在新型冠状病毒IgM抗体，则形成磁微粒-链霉亲和素-生物素-抗原-新型冠状病毒IgM抗体-二抗复合物，通过二抗上标记的信号物读取待测样本的发光强度数值。所述信号物可以是吖啶酯、吖啶磺酰胺、碱性磷酸酶和辣根过氧化物酶，本申请中优选吖啶磺酰胺。
本申请使用寡聚脯氨酸残基-(P)n-连接这些优势表位，形成一条串联预测优势表位且易伸展弯曲的重组抗原，利于抗体对优势表位的结合，提高检测灵敏度。
以寡聚赖氨酸残基-(K)n-形成C端，由于赖氨酸残基有1个多余的氨基，便于与生物素、吖啶酯、羧基磁微粒等标记物的偶联，一方面能增大重组抗原与标记物的结合概率，同时当重组抗原通过C端寡聚赖氨酸残基与固相载体结合时，N端的抗原表位更容易与抗体接触；另一方面能够降低重组抗原中优势表位与标记物的结合概率，防止表位被标记物遮挡，导致抗体识别困难。
本申请中，所述检测试剂盒能够检测新型冠状病毒IgM和IgG抗体，可根据需要检测的抗体类型调整二抗的种类，例如检测IgM时，可选用羊抗人IgM抗体作为二抗，检测IgG时，可选用羊抗人IgG抗体作为二抗。
同时，本申请选择以生物素标记抗原、以吖啶磺酰胺标记二抗，检测时采用的是间接法而非捕获法(捕获法：以生物素标记二抗，以吖啶磺酰胺标记抗原)，该方法能提高所得试剂盒检测结果的准确度。
本申请中，将核衣壳蛋白和棘突蛋白S1的质量比设置为(1～3):1，其中最优选为1.5:1。相比于单独使用核衣壳蛋白或棘突蛋白S1，或使用其他比例，例如分别按照4:1、2:3、1:4的比例混合，核衣壳蛋白和棘突蛋白S1比例为3:2时样 本之间的区分度较大。
作为本申请优选的技术方案，所述生物素标记的新型冠状病毒抗原中，活化生物素(Sulfo-NHS-LC-Biotin)与新型冠状病毒抗原的质量比为(0.06～0.48):1；例如可以是0.06:1、0.1:1、0.12:1、0.15:1、0.2:1、0.25:1、0.3:1、0.4:1或0.48:1等，优选为0.24:1。
在制备过程中，取新型冠状病毒核衣壳蛋白和棘突蛋白S1置于玻璃器皿内，用0.02M PBS缓冲液将新型冠状病毒核衣壳蛋白和棘突蛋白S1稀释；取12μL的10mg/mL的Sulfo-NHS-LC-Biotin溶液加入到上述抗原缓冲液中，混匀，避光置于室温2小时；用0.02M PBS缓冲液在2～8℃环境中过夜透析，得到生物素标记抗原母液；添加12μL的样品相对于添加3、6和24μL的样品，其筛选区分度较大。
优选地，所述重组核衣壳蛋白包括如SEQ ID NO.1所示的氨基酸序列。
SEQ ID NO.1为：
Figure PCTCN2021082727-appb-000001
其中，下划线所示均为重组核衣壳蛋白的优势表位。
原始核衣壳蛋白的序列如SEQ ID NO.2所示：
Figure PCTCN2021082727-appb-000002
用DNAStar Protean软件分析原始序列，其氨基酸序列18～49、138～154、170～216、230～266、273～301、338～349、362～392、400～419多位于β转角(多 位于蛋白表面，易与抗体结合)，亲水指数、抗原指数、表面可能性均较高，预测为优势表位。
本申请中，还可将不同的优势表位随机排列组合，形成新的重组抗原。由于同样还有优势表位，其效果与SEQ ID NO.1所示的氨基酸序列相近。
优选地，所述重组核衣壳蛋白的两端连接含苯环的氨基酸残基(如苯丙氨酸F/色氨酸W/酪氨酸Y)，有利于提高重组抗原的稳定性。
优选地，所述重组棘突蛋白S1包括如SEQ ID NO.3所示的氨基酸序列。
SEQ ID NO.3为：
Figure PCTCN2021082727-appb-000003
其中，下划线所示均为重组棘突蛋白S1的优势表位。
原始棘突蛋白S1的序列如SEQ ID NO.4所示：
Figure PCTCN2021082727-appb-000004
Figure PCTCN2021082727-appb-000005
用DNAStar Protean软件分析原始序列结果，其氨基酸序列70～84、93～101、107～115、145～154、277～286、352～361、410～430、455～470、524～540、553～582、675～688、770～780、805～818、1034～1045、1068～1078、1104～1111、1138～1168、1180～1210、1255～1273多位于β转角(多位于蛋白表面，易与抗体结合)，亲水指数、抗原指数、表面可能性均较高，预测为优势表位。
本申请中，还可将不同的优势表位随机排列组合，形成新的重组抗原。由于同样还有优势表位，其效果与SEQ ID NO.3所示的氨基酸序列相近。
优选地，所述重组棘突蛋白S1的两端连接含苯环的氨基酸残基(如苯丙氨酸F/色氨酸W/酪氨酸Y)，有利于提高重组抗原的稳定性。
优选地，所述生物素标记的新型冠状病毒抗原分散于PBS缓冲液中。
优选地，所述PBS缓冲液的浓度为0.02～0.1M，例如可以是0.02M、0.05M、0.06M、0.08M或0.1M等，优选为0.02M。
优选地，所述PBS缓冲液的pH为7.2～9.0，例如可以是7.2、7.3、7.5、7.6、7.8、8.0、8.2、8.5、8.8或9.0等，优选为7.2。
本申请中，所述缓冲体系为0.02M PBS(pH7.2)缓冲液，相较于0.1M PBS(pH7.2)、0.05M PBS(pH7.2)、0.1M CBS(pH 9.0)、0.05M CBS(pH 9.0)、0.02M CBS(pH 9.0)缓冲液，其效果最佳。
作为本申请优选的技术方案，所述吖啶磺酰胺标记的二抗分散于CBS缓冲液中。
优选地，所述CBS缓冲液的浓度为0.02～0.1M，例如可以是0.02M、0.05M、0.06M、0.08M或0.1M等，优选为0.1M。
优选地，所述CBS缓冲液的pH为7.2～9.0，例如可以是7.2、7.3、7.5、7.6、7.8、8.0、8.2、8.5、8.8或9.0等，优选为9.0。
本申请中，所述缓冲体系为0.1M CBS(pH 9.0)缓冲液，相较于0.1M PBS(pH7.2)、0.05M PBS(pH7.2)、0.02M PBS(pH7.2)、0.1M CBS(pH 9.0)、0.05M CBS(pH 9.0)、0.02M CBS(pH 9.0)缓冲液，其效果最佳。
作为本申请优选的技术方案，所述样本稀释液中包含牛血清白蛋白、类风湿因子吸附剂、抑菌剂、尿素或阻断剂中的任意一种或至少两种的组合。
通过使用阻断剂，可消除血液样本中异嗜性抗体的干扰；通过采用类风湿因子吸附剂，可以消除血液样本中类风湿因子的干扰；通过使用尿素，可以降低血液样本对固相载体的非特异性结合。
本申请中，所述样本稀释液可以为0.01M的PBS，其中添加质量分数为0.4～0.6％(例如可以是0.42％、0.44％、0.45％、0.48％、0.5％、0.55％、0.58％或0.6％等)、质量分数为0.01～0.05％(例如可以是0.012％、0.015％、0.02％、0.025％、0.03％、0.035％、0.04％或0.05％等)抑菌剂ProClin300、质量分数为1～10％(例如可以是1％、2％、3％、5％、6％、8％或10％等)尿素和质量分数为0.1～0.5％(例如可以是0.12％、0.15％、0.2％、0.25％、0.3％、0.35％、0.4％或0.5％等)阻断剂。
作为本申请优选的技术方案，所述质控品包括阳性质控品和阴性质控品。
在一个优选的实施方案中，所述阳性质控品为含新型冠状病毒抗体的缓冲液。
作为本申请优选的技术方案，所述新型冠状病毒抗体检测试剂盒中还包括激发液和洗涤液。
第二方面，本申请提供一种如第一方面所述的新型冠状病毒抗体检测试剂盒的制备方法，所述制备方法包括如下步骤：
(1)制备生物素标记的新型冠状病毒抗原：将新型冠状病毒的核衣壳蛋白和棘突蛋白S1作为抗原与PBS缓冲液混合，加入活化生物素标记1.5～2.5h，而后透析得到所述生物素标记的新型冠状病毒抗原；
(2)制备吖啶磺酰胺标记的二抗：将二抗与CBS缓冲液混合，加入NSP-SA-NHS标记0.5～1.5h，再加入赖氨酸溶液，透析得到所述吖啶磺酰胺标记的二抗；以及
(3)将所述生物素标记的新型冠状病毒抗原、吖啶磺酰胺标记的二抗、链霉亲和素磁微粒、样本稀释液和质控品分别包装，得到所述新型冠状病毒抗体检测试剂盒。
第三方面，本申请还提供一种如第一方面所述的新型冠状病毒抗体检测试剂盒的使用方法，所述使用方法包括如下步骤：
在待测样品中加入生物素标记的新型冠状病毒抗原和链霉亲和素磁微粒，进行第一次孵育，洗涤，再加吖啶磺酰胺标记的二抗进行第二次孵育，洗涤，而后检测得到检测结果。
本申请中采用两步法检测待测样品，即将样品、生物素标记抗原和链霉亲和素磁微粒混合孵育，再加入吖啶磺酰胺标记抗体孵育；相对于三步法(将链霉亲和素磁微粒与生物素标记抗原混合孵育，再加入样品孵育，再加入吖啶磺酰胺标记抗体孵育)和一步法(直接将样品、生物素标记抗原、链霉亲和素磁微粒和吖啶磺酰胺标记抗体混合孵育)，两步法的检测效果较好。
优选地，所述待测样品为血清或血浆。同一样本不同样本类型(血浆/血清)检测发光值的相对偏差绝对值小于10％，认为血浆和血清检测结果相同。
优选地，所述待测样品经过样品稀释液稀释。用样本稀释液将样本分别稀释25倍、50倍、100倍、200倍、400倍；用化学发光法检测经稀释的样本，可知样品稀释倍数为100倍时，区分度最大。
优选地，所述待测样品稀释后的效价比为1:(103～110)，例如可以是1:103、1:104、1:105、1:106、1:107、1:108、1:109或1:110等，优选为1:103。
本申请中，分别以样品的加液量为50、75和100μL、生物素标记抗原加液量为25、50和75μL、磁微粒加液量为30、40和50μL、第一步反应时间为4、6、8、10min、最佳吖啶磺酰胺标记二抗浓度1、0.5、0.25、0.125μg/mL、第二步反应时间为4、6、8、10min、洗涤次数为1、2、3、4次，测试最佳的反应条件。
优选地，所述待测样品、生物素标记的新型冠状病毒抗原与链霉亲和素磁微粒的体积比为(50～100):(25～75):(30～50)，例如可以是50:25:30、75:25:30、100:25:30、50:50:30、50:75:30、50:75:40、100:50:40、100:50:50或100:75:50等，优选为100:50:40。
优选地，所述吖啶磺酰胺标记二抗的工作浓度为0.125～1μg/mL，例如可以 是0.125μg/mL、0.25μg/mL、0.5μg/mL、0.75μg/mL、0.8μg/mL、0.9μg/mL或1μg/mL等，优选为0.25μg/mL。
优选地，所述第一次孵育的时间为5～15min，例如可以是5min、6min、7min、8min、9min、10min、12min、14min或15min等，优选为8min。
优选地，所述第二次孵育的时间为5～15min，例如可以是5min、6min、7min、8min、9min、10min、12min、14min或15min等，优选为8min。
在一些具体实施方案中，所述检测结果中指数I≥1.0，则待测样本为阳性；所述检测结果中指数I<1.0，则待测样本为阴性。
本申请所述的数值范围不仅包括上述例举的点值，还包括没有例举出的上述数值范围之间的任意的点值，限于篇幅及出于简明的考虑，本申请不再穷尽列举所述范围包括的具体点值。
与现有技术相比，本申请的有益效果为：
(1)本申请提供的基于磁微粒化学发光的新型冠状病毒抗体检测试剂盒，选择链霉亲和素磁微粒，以生物素标记抗原、以吖啶磺酰胺标记二抗，一方面，通过调整磁微粒偶联、封闭条件以及调整体系稳定剂组成，有效地降低血液样本中假阳性因素；另一方面，通过采用新型冠状病毒两种抗原(棘突蛋白和核衣壳蛋白)混合的手段，重组核衣壳蛋白和棘突蛋白S1的质量比为(1～3):1，提高检测的特异度；
(2)本申请提供的抗体检测试剂盒采用重组核衣壳蛋白和重组棘突蛋白作为新型冠状病毒的抗原，所述重组蛋白具有原始蛋白序列的优势表位，亲水指数、抗原指数、表面可能性均较高，能够显著提高检测的灵敏度；以及
(3)本申请提供的检测试剂盒的灵敏度较好，其最低检测限为1:103，精密度较高，多次重复试验时重复度较高，特异性好，对可能产生交叉反应的样本及正常人样本，检测结果均为阴性，且对于样本中可能存在的干扰物质，检测结果的相对偏差可控；且通过配套自动化检测仪器，有效避免操作人员暴露风险，提高生物安全性，还能实现高通量检测，提高新型冠状病毒的检测速度。
附图说明
图1为试验例1中以重组蛋白和原始蛋白为抗原制备的检测试剂盒在检测不同稀释倍数的强阳性血清时得到的荧光检测结果曲线图。
图2为试验例5中IgM抗体检测结果散点图。
具体实施方式
下面结合附图并通过具体实施方式来进一步说明本申请的技术方案，但下述的实例仅仅是本申请的简易例子，并不代表或限制本申请的权利保护范围，本申请的保护范围以权利要求书为准。
以下实施例中，若无特殊说明，均采用本领域常见的技术手段进行实验和检测。
实施例1
本实施例提供一种基于磁微粒化学发光的新型冠状病毒IgM抗体检测试剂盒，具体包括：
0.3mg/mL链霉亲和素磁微粒、生物素标记的新型冠状病毒抗原、吖啶磺酰胺标记的羊抗人IgM抗体、样本稀释液，阳性质控品、阴性质控品、样本稀释液和洗涤液。
其中，(1)生物素标记的新型冠状病毒抗原的制备方法为：
将0.3mg新型冠状病毒的重组核衣壳蛋白和0.2mg重组棘突蛋白S1使用0.02M PBS(pH7.2)缓冲液稀释，最终混合抗原的浓度为1mg/mL；
其中，重组核衣壳蛋白的序列为SEQ ID NO.1：重组棘突蛋白S1的序列为SEQ ID NO.3：
取12μL的10mg/mL的活化生物素加入到上述抗原缓冲液中，混匀，避光置于室温2小时；用0.02M PBS缓冲液在4℃环境中过夜透析，得到生物素标记抗原母液；
生物素标记的新型冠状病毒抗原的工作浓度为1μg/mL；
(2)吖啶磺酰胺标记的羊抗人IgM抗体的制备方法为：
取0.2mg羊抗人IgM抗体置于玻璃器皿内，用0.1M CBS(pH9.0)缓冲液将羊抗人IgM抗体稀释到1mg/mL；取9.1μL 2mg/mL的NSP-SA-NHS溶液加入到上述抗体稀释液中，混匀，避光置于室温60min；
再加入1.33μL的10％赖氨酸溶液，混匀，避光置于室温30分钟；用0.02M PBS缓冲液在4℃环境中过夜透析，得到叮吖啶磺酰胺标记的抗体母液；
吖啶磺酰胺标记的羊抗人IgM抗体的工作浓度为1μg/mL。
实施例2
本实施例提供基于磁微粒化学发光的新型冠状病毒IgM抗体检测试剂盒，与实施例1的区别在于，重组核衣壳蛋白和重组棘突蛋白S1的质量比分别设置为5:0、4:1、2:3、1:4和0:5；
检测区分度，结果如表1所示：
表1
Figure PCTCN2021082727-appb-000006
其中，参考品为标准样品，是试剂盒研发过程中使用的待测样品，编号为N01～N15(阴性样本)、P01～05(阳性样本)和L1～L3(质控品)；由上表可知，核衣壳蛋白和棘突蛋白S1比例为5:0、4:1、1:4、0:5时，阴性符合率(即区分 度小于1)均为14/15；
具体为：比例为5:0时，N10样本区分度为2.08；比例为4:1时，N10样本区分度为1.16；比例为1:4时，N06样本区分度为1.10；比例为0:5时，N06样本区分度为1.40；
而当二者比例为3:2和2:3时阴性符合率均为15/15，阳性符合率为5/5；且比例为2:3时，阴性参考品N06的检测值为0.71，较比例为3:2时N06检测值的0.41较大且接近临界值(对于阴性样本而言区分度数值越小越好)，因此检测效果较差，出于高特异性考虑，选择3:2时样本区分度较好，故选择核衣壳蛋白和棘突蛋白S1比例为3:2。
实施例3
本实施例提供一种基于磁微粒化学发光的新型冠状病毒IgM抗体检测试剂盒，与实施例1的区别在于，Sulfo-NHS-LC-Biotin溶液的添加量分别设置为3、6和24μL。
具体结果如下表2所示；
表2
Figure PCTCN2021082727-appb-000007
实施例4
本实施例提供一种基于磁微粒化学发光的新型冠状病毒IgM抗体检测试剂盒，与实施例1的区别在于，生物素标记的新型冠状病毒抗原的缓冲体系分别设置为0.1M PBS(pH7.2)、0.05M PBS(pH7.2)、0.1M CBS(pH 9.0)、0.05M CBS(pH 9.0)、0.02M CBS(pH 9.0)。
具体结果如下表3所示；
表3
Figure PCTCN2021082727-appb-000008
实施例5
本实施例提供一种基于磁微粒化学发光的新型冠状病毒IgM抗体检测试剂盒，与实施例1的区别在于，吖啶磺酰胺标记的二抗的缓冲体系分别设置为0.1M PBS(pH7.2)、0.05M PBS(pH7.2)、0.02M PBS(pH7.2)、0.05M CBS(pH 9.0)、0.02M CBS(pH 9.0)。
具体结果如下表4所示；
表4
Figure PCTCN2021082727-appb-000009
实施例3、4和5中的待测样品为标准样品，是试剂盒研发过程中使用的待测样品，编号为N01、P01和P05；
由实验结果可知，表2中Sulfo-NHS-LC-Biotin溶液的添加量为12μL时，阳性样本P01的区分度最高；表3中抗原缓冲体系为0.02M PBS时，阳性样本P01的区分度最高；表4中二抗的缓冲体系为0.1M CBS时，阳性样本P01的区分度最高；因此，实施例1中提供的条件包括Sulfo-NHS-LC-Biotin溶液的添加 量为12μL、抗原缓冲体系为0.02M PBS和二抗的缓冲体系为0.1M CBS时，所得到的区分度最大。
实施例6
本实施例提供三种不同的检测步骤，具体包括：
(1)三步法：50μL链霉亲和素磁微粒加入50μL生物素标记抗原孵育5min，洗涤；加100μL参考品，孵育10min，洗涤；加入50μL吖啶磺酰胺标记抗体孵育10min，洗涤；加激发液检测发光值。
(2)两步法：100μL参考品加入50μL生物素标记抗原、50μL链霉亲和素磁微粒，孵育10min，洗涤；
加50μL吖啶磺酰胺标记抗体孵育10min，洗涤；加激发液检测发光值。以及
(3)一步法：100μL参考品加入50μL生物素标记抗原、50μL吖啶磺酰胺标记抗体、50μL链霉亲和素一磁微粒孵育20min，洗涤；加激发液检测发光值。
检测区分度，结果如表5所示：
表5
Figure PCTCN2021082727-appb-000010
其中，参考品为标准样品，是试剂盒研发过程中使用的待测样品，编号为N01、P01和P05；从上表可知，两步法的区分度较大，故选择两步法作为操作步骤。
试验例1：最低检测限评估
将3份阳性血浆分别按倍数进行稀释，得到检测结果覆盖临界值附近的样品；每个梯度重复稀释3份样品，每份样品用实施例1中采用的试剂盒各重复检测20次(化学发光法检测时，样本需稀释100倍)。
计算每份样品的阳性检出率，筛选阳性检出率在90～95％范围内的待测抗体效价水平作为最低检测限。
检测结果如下表6所示：
表6
Figure PCTCN2021082727-appb-000011
当梯度稀释样品的效价为1:103时，阳性检出率在90～95％范围内，故将效价为1:103作为最低检测限。
此外，以强阳性样本为待测样本，在以重组蛋白和原始蛋白为抗原制备的检测试剂盒在检测时，针对不同稀释倍数的强阳性血清所得到的荧光检测结果如图1所示；
由图1可知，重组蛋白在检测相同效价的时，所得到的荧光强度明显高于原始蛋白，这说明，本申请中提供的重组蛋白在检测时具有更高的灵敏度。
试验例2：精密度评估
将4个临床样本(1例阴性样本、1例临界阳性样本、1例中阳性样本、1例强阳性样本)和2个质控品用2个机型分别检测，每种机型2个操作人员，共4个操作人员，每种机型分别使用3个批次的实施例1提供的试剂盒，检测5天，每天每个样品做5个重复(2个机型×3个试剂盒批次×5天×5个重复/天＝150个结果/样品)；
机型1：重庆科斯迈生物科技有限公司的全自动化学发光测定仪，型号： SMART 6500；
机型2：重庆科斯迈生物科技有限公司的全自动化学发光免疫分析仪，型号：SMART 500S。
检测结果如下表7所示：
表7
Figure PCTCN2021082727-appb-000012
其中，SDR表示：随机误差引起的标准差；SDwi表示在一个实验室内由试剂盒批次、日间重复检测、操作人员及随机误差引起的标准差；
从上表数据分析结果可知，机型SMART 6500检测中阳性样本、强阳性样本和阳性质控品的重复性、室内精密度和批间精密度CV均小于等于10％，符合要求(重复性CV<10％，批间CV<15％)；临界阳性样本的阳性检出率≥95％，符合要求；阴性样本及阴性质控品的阴性检出率应为100％，符合要求；
同样的，机型SMART 500S的检测结果同样符合要求，此处出于篇幅和简明的考虑，不再列出。
试验例3：特异性评价
使用本申请提供的新型冠状病毒抗体检测试剂盒检测可能产生交叉反应的样本及正常人样本，观察是否检测为阳性；若为阳性，说明该类样本对新型冠状病毒IgM抗体检测存在影响。
地方性人类冠状病毒(HKU1，OC43，NL63和229E)血浆样本，用蛋白 印迹和血清学方法确认血浆中含有特异性IgM抗体；H1N1(新型甲型H1N1流感病毒(2009)、季节性H1N1流感病毒)IgM抗体阳性血浆样本；
检测结果如表8所示：
表8
Figure PCTCN2021082727-appb-000013
从上述试验结果可知，用本试剂盒检测上述可能产生交叉反应的样本及正常人样本，检测结果均为阴性，可见上述病原体感染样本及正常人样本与本试剂盒不存在交叉反应。
试验例4：可能产生干扰的物质对检测结果的影响
临床样本中的血红蛋白主要是由于标本溶血，包括病理性溶血与技术性溶血。非显性溶血是指血红蛋白含量小于0.5mg/mL，此时肉眼观察不到溶血。轻度溶血血红蛋白浓度为0.5～3mg/mL，中度溶血血红蛋白浓度为3.1～5mg/mL，重度溶血血红蛋白浓度>5mg/mL。实验证明，血红蛋白浓度达到7mg/mL时，检测结果的相对偏差绝对值未超过10％，说明对检测结果无干扰。
正常人血清中胆红素范围为2～8mg/L(1周内的婴儿的胆红素范围为10～120mg/L)，样本中的胆红素浓度高达300mg/L时，检测结果的相对偏差绝对值未超过10％，说明对检测结果无干扰。
临床样本中甘油三酯正常高限为1.7mmol/L，从实验结果可知，样本中甘油三酯含量为7.5mmoL/L时，检测结果的相对偏差绝对值未超过10％，说明对检测结果无干扰。
此外，类风湿因子、抗核抗体、抗双链DNA抗体、抗线粒体抗体、HAMA阳性样本，高浓度新型冠状病毒IgG抗体样本，50g/L人总IgG抗体(健康成人总IgG量：7～16.6g/L)、10g/L人总IgM抗体(健康成人总IgM量：400-3450mg/L)等可能的干扰物质对本检测结果均无影响。
试验例5：对不同时间段采样后的样本进行检测
对临床收集到的有明确发病日期和采样日期的65份确诊样本进行检测，分别求出不同采样时间段的阳性检出率，再以发病天数(采样日期和发病日期的间隔天数)为横坐标，以对应发病天数样本检测结果I值为纵坐标，绘制IgM抗体随发病天数变化的散点图，如图2所示。
且结果如下表9所示：
表9
Figure PCTCN2021082727-appb-000014
从表9和图1可知，IgM抗体在发病早期检出率为62.5％，随着发病时间的推移，IgM抗体持续升高，在2周时IgM抗体浓度达到峰值，随着发病时间推移，逐渐出现了下降。
实施例7
本实施例提供一种基于磁微粒化学发光的新型冠状病毒IgG抗体检测试剂盒，具体包括：
链霉亲和素磁微粒、生物素标记的新型冠状病毒抗原、吖啶磺酰胺标记的羊抗人IgG抗体、样本稀释液，阳性质控品、阴性质控品、样本稀释液和洗涤液。
其中，(1)生物素标记的新型冠状病毒抗原的制备方法为：
将0.3mg新型冠状病毒的核衣壳蛋白和0.2mg棘突蛋白S1使用0.02M PBS(pH7.2)缓冲液稀释，最终混合抗原的浓度为1mg/mL；
取12μL的10mg/mL的活化生物素加入到上述抗原缓冲液中，混匀，避光置于室温2小时；用0.02M PBS缓冲液在4℃环境中过夜透析，得到生物素标记抗原母液；
生物素标记的新型冠状病毒抗原的工作浓度为1μg/mL；
(2)吖啶磺酰胺标记的羊抗人IgG抗体的制备方法为：
取0.2mg羊抗人IgG抗体置于玻璃器皿内，用0.1M CBS(pH9.0)缓冲液将羊抗人IgG稀释到1mg/mL；
取9.1μL 2mg/mL的NSP-SA-NHS溶液加入到上述抗体稀释液中，混匀，避光置于室温60min；
再加入1.33μL的10％赖氨酸溶液，混匀，避光置于室温30分钟；用0.02M PBS缓冲液在4℃环境中过夜透析，得到吖啶磺酰胺标记的抗体母液；
吖啶磺酰胺标记的羊抗人IgG抗体的工作浓度为1μg/mL。
同样的，所述IgG抗体检测试剂盒在检测新型冠状病毒时也具有较高的准确度。使用其检测不同样本后所得检测结果如下表10所示：
表10
Figure PCTCN2021082727-appb-000015
由上表可知，本实施例提供的IgG抗体检测试剂盒能够精确检测，灵敏度好，准确性高。
综上所述，本申请提供的检测试剂盒在检测IgM抗体和IgG抗体时均就具有较好的灵敏度。其中，IgM抗体检测试剂盒最低检测限为1:103，精密度较高，多次重复试验时重复度较高，特异性好，对可能产生交叉反应的样本及正常人样本，检测结果均为阴性，且对于样本中可能存在的干扰物质，检测结果的相 对偏差可控。
申请人声明，以上所述仅为本申请的具体实施方式，但本申请的保护范围并不局限于此，所属技术领域的技术人员应该明了，任何属于本技术领域的技术人员在本申请揭露的技术范围内，可轻易想到的变化或替换，均落在本申请的保护范围和公开范围之内。

Claims (10)

  1. 一种基于磁微粒化学发光的新型冠状病毒抗体检测试剂盒，其包括：
    链霉亲和素磁微粒、生物素标记的新型冠状病毒抗原、吖啶磺酰胺标记的二抗、样本稀释液和质控品，所述生物素标记的新型冠状病毒抗原中包括重组核衣壳蛋白和重组棘突蛋白S1；
    其中，所述重组核衣壳蛋白中利用寡聚脯氨酸残基连接原始核衣壳蛋白的优势表位，并利用寡聚赖氨酸残基形成C端；
    所述重组棘突蛋白S1中利用寡聚脯氨酸残基连接原始棘突蛋白S1的优势表位，并利用寡聚赖氨酸残基形成C端；
    所述重组核衣壳蛋白包括如SEQ ID NO.1所示的氨基酸序列；并且
    所述重组棘突蛋白S1包括如SEQ ID NO.3所示的氨基酸序列。
  2. 根据权利要求1所述的新型冠状病毒抗体检测试剂盒，其中，所述重组核衣壳蛋白与重组棘突蛋白S1的质量比为(1～3):1。
  3. 根据权利要求1或2所述的新型冠状病毒抗体检测试剂盒，其中，所述生物素标记的新型冠状病毒抗原中，生物素与新型冠状病毒抗原的质量比为(0.06～0.48):1。
  4. 根据权利要求1所述的新型冠状病毒抗体检测试剂盒，其中，所述样本稀释液中包含牛血清白蛋白、类风湿因子吸附剂、抑菌剂、尿素或阻断剂中的任意一种或至少两种的组合。
  5. 根据权利要求1所述的新型冠状病毒抗体检测试剂盒，其中，所述质控品包括阳性质控品和阴性质控品；
    所述阳性质控品为含新型冠状病毒抗体的缓冲液。
  6. 根据权利要求1所述的新型冠状病毒抗体检测试剂盒，其中，所述新型冠状病毒抗体检测试剂盒中还包括激发液和洗涤液。
  7. 一种如权利要求1～6任一项所述的新型冠状病毒抗体检测试剂盒的制备方法，其包括：
    (1)制备生物素标记的新型冠状病毒抗原：将新型冠状病毒的核衣壳蛋白和棘突蛋白S1作为抗原与PBS缓冲液混合，加入活化生物素标记1.5～2.5h，而后透析得到所述生物素标记的新型冠状病毒抗原；
    (2)制备吖啶磺酰胺标记的二抗：将二抗与CBS缓冲液混合，加入NSP-SA-NHS标记0.5～1.5h，再加入赖氨酸溶液，透析得到所述吖啶磺酰胺标 记的二抗；以及
    (3)将所述生物素标记的新型冠状病毒抗原、吖啶磺酰胺标记的二抗、链霉亲和素磁微粒、样本稀释液和质控品分别包装，得到所述新型冠状病毒抗体检测试剂盒。
  8. 一种如权利要求1～6任一项所述的新型冠状病毒抗体检测试剂盒的使用方法，其包括如下步骤：
    在待测样品中加入生物素标记的新型冠状病毒抗原和链霉亲和素磁微粒，进行第一次孵育，洗涤，再加吖啶磺酰胺标记的二抗进行第二次孵育，洗涤，而后检测得到检测结果。
  9. 根据权利要求8所述的使用方法，其中，所述待测样品为血清或血浆；
    所述待测样品经过样品稀释液稀释；
    所述待测样品稀释后的效价比为1:(103～110)；
    所述待测样品、生物素标记的新型冠状病毒抗原与链霉亲和素磁微粒的体积比为(50～100):(25～75):(30～50)；
    所述吖啶磺酰胺标记二抗的工作浓度为0.125～1μg/mL；
    所述第一次孵育的时间为5～15min；并且
    所述第二次孵育的时间为5～15min。
  10. 根据权利要求8所述的使用方法，其中，所述检测结果中指数I≥1.0，则待测样本为阳性；所述检测结果中指数I<1.0，则待测样本为阴性。
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