CN116413425A - SARS-CoV-2 immunogen detection kit and its application - Google Patents
SARS-CoV-2 immunogen detection kit and its application Download PDFInfo
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention discloses a kit and a method for detecting novel coronavirus (SARS-CoV-2) immunogen in a sample to be detected. The kit comprises a reaction plate coated with SARS-CoV-2 sheep polyclonal antibody, SARS-CoV-2 mouse polyclonal antibody, and a secondary antibody for the SARS-CoV-2 mouse polyclonal antibody. The kit and the method can detect intermediate samples and finished products of each working procedure in the preparation process of SARS-CoV-2 vaccine, meet the requirement of in-vitro effective quality evaluation, not only can be applied to preclinical research batches and detection of clinical batches of stage I, stage II and stage III, but also can be used for detecting the content and quality of immunogens of working procedure samples and finished products in the industrialization stage.
Description
Technical Field
The present disclosure relates to the field of vaccine immunogen detection, and in particular to a kit for detecting novel coronavirus (SARS-CoV-2) immunogens and methods of use thereof.
Background
The novel coronavirus pneumonia (COVID-19) is a disease taking novel coronavirus (SARS-CoV-2) as pathogen, and can cause acute respiratory infectious disease of human body. Coronaviruses are single-stranded RNA viruses, which have an envelope and particles on their surface, the particles being round or oval, often polymorphic, and 60-140 nm in diameter. SARS-CoV-2 has a typical coronavirus genomic structure, the 3' -end region of its genome encodes structural proteins, namely spike protein (S), envelope protein (E), membrane protein (M) and helical nucleocapsid (N), plus 8 accessory proteins. Most of the clinical symptoms of the patient with covd-19 are mainly represented in respiratory system, including fever, cough, hypodynamia and the like, and other symptoms of the patient, such as diarrhea, palpitation and the like, are easily infected by the old and the people with basic diseases and are easy to have serious consequences. For this reason, a safe and effective vaccine is the first choice for controlling spread of covd-19.
In the development and production process of vaccines, the content of active ingredients in intermediate samples of each process stage needs to be evaluated. The detection of antigen content is one of the most critical in vitro effective component evaluation means of viral vaccines. The antigen is the main immunogen for the in vitro effectiveness of SARS-CoV-2 virus, and the detection of the content thereof is particularly important in vaccine research and quality identification.
Disclosure of Invention
In order to solve one of the above technical problems in the prior art, the present disclosure provides a kit for detecting a novel coronavirus antigen, and also provides a method for constructing the kit for detecting a novel coronavirus antigen.
According to one aspect of the present disclosure, there is provided a kit for detecting a novel coronavirus (SARS-CoV-2) immunogen in a sample to be tested, and comprising: a reaction plate coated with SARS-CoV-2 sheep polyclonal antibody; SARS-CoV-2 mouse polyclonal antibody; a secondary antibody directed against said SARS-CoV-2 mouse polyclonal antibody.
According to an embodiment of the present disclosure, the kit further comprises a sample diluent, a chromogenic solution, a wash solution, and an antigen reference.
According to a preferred embodiment of the present disclosure, the secondary antibody of SARS-CoV-2 mouse polyclonal antibody is a horseradish peroxidase (HRP) or Alkaline Phosphatase (APL) labeled secondary antibody. According to an embodiment of the present disclosure, the color developing solution is o-phenylenediamine, tetramethyl benzidine, o-biphenylmethylamine, aminosalicylic acid, and the like. For example, the color-developing solution may be 3, 3-diaminobenzidine, 3', 5' -tetramethylbenzidine, or the like.
According to a preferred embodiment of the present disclosure, the washing solution is a phosphate buffer. The wash solution preferably comprises tween-20, for example a phosphate buffer comprising 0.05% tween-20. The pH of the washing solution may be in the range of 6.5 to 7.4.
According to another aspect of the present disclosure, there is provided a method for detecting the content of novel coronavirus (SARS-CoV-2) immunogen in a sample to be tested, characterized in that the method comprises: coating the reaction plate by using SARS-CoV-2 sheep polyclonal antibody to obtain a coated reaction plate; adding a sample to be detected into the reaction plate for incubation so as to combine the sample to be detected with the SARS-CoV-2 sheep polyclonal antibody; after washing the reaction plate, adding a sealing liquid for sealing; washing the reaction plate, and then adding SARS-CoV-2 mouse polyclonal antibody for incubation so as to enable the SARS-CoV-2 mouse polyclonal antibody to be combined with the sample to be detected; after washing the reaction plate, adding a color development liquid to develop color, and detecting the OD value at 450-630 nm.
According to an embodiment of the present disclosure, the SARS-CoV-2 sheep polyclonal antibody coats the reaction plate in an amount of 50 to 150. Mu.l/well. According to an embodiment of the present disclosure, the SARS-CoV-2 sheep polyclonal antibody coats the reaction plate in an amount of 100 to 150. Mu.l/well.
According to an embodiment of the present disclosure, the sample to be tested is a SARS-CoV-2 vaccine or an intermediate sample during its production. According to the embodiment of the disclosure, the sample to be tested is SARS-CoV-2 inactivated vaccine or intermediate sample in the production process.
According to embodiments of the present disclosure, the sample to be tested is incubated with the coated reaction plate overnight at 2-8 ℃, or for 0.5-2 hours at 35-39 ℃.
According to an embodiment of the present disclosure, the SARS-CoV-2 mouse polyclonal antibody is added in an amount of 50 to 150. Mu.l/well. According to an embodiment of the present disclosure, the SARS-CoV-2 mouse polyclonal antibody is added in an amount of 100 to 150. Mu.l/well.
According to embodiments of the present disclosure, the SARS-CoV-2 mouse polyclonal antibody is incubated with the test sample at 35-39℃for 0.5-2 hours.
According to an embodiment of the present disclosure, the blocking solution is a buffer containing 5 to 15% calf serum or 1 to 5% Bovine Serum Albumin (BSA). According to a specific embodiment, the buffer is a phosphate buffer.
According to an embodiment of the present disclosure, the method further comprises: standard curves were made using gradient diluted antigen references. According to embodiments of the present disclosure, the immunogen of the test sample may be quantified based on the standard curve.
According to an embodiment of the present disclosure, the antigen reference is diluted to a concentration gradient of 12.5 to 400 SU/mL.
According to embodiments of the present disclosure, the methods employ a sample that does not contain SARS-CoV-2 immunogen as a negative control.
Preparing SARS-CoV-2 specific sheep polyclonal antibody and mouse polyclonal antibody, performing system matching, completing method establishment, and performing comprehensive verification and application on the established antigen system.
The method comprises the steps of pre-coating an anti-SARS-CoV-2 polyclonal antibody (also called a coating) into a 96-well ELISA plate, adding a sample to be detected and an antigen reference substance, combining the antigen and the polyclonal antibody to form an antibody-antigen complex, adding a specific primary antibody to form an antibody-antigen-antibody complex, adding an HRP (horseradish peroxidase) labeled anti-generic antibody, generating a blue compound through an HRP catalytic substrate, adding an acidic stop solution to form a yellow compound, and the yellow compound has a maximum absorption peak at a wavelength of 450nm (OD 450) (OD 630 is a reference wavelength). The antigen content and the number of the yellow compounds are in a linear relation, and according to a standard curve fitted by different concentrations of the antigen reference substance and corresponding absorbance OD450 values, absorbance OD450 values of the sample are substituted into the standard curve, so that the antigen content of the sample to be detected is calculated.
The present disclosure establishes the detection kit for in vitro detection of the immunogen content in SARS-CoV-2 inactivated vaccine. The kit can detect intermediate samples and finished products of each process section in the preparation process of SARS-CoV-2 vaccine, meets the requirement of in-vitro effective quality evaluation, is not only applied to preclinical research batches, and detection of clinical batches of I phase, II phase and III phase, but also can be used for monitoring antigen content and quality of process section samples and finished products in the industrialization stage.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present disclosure. Such structures and techniques are also described in a number of publications.
Definition of the definition
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate.
The terms "a" and "an" as used herein include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents thereof known to those skilled in the art, and so forth.
The term "about" as used herein means a range of + -20% of the numerical values thereafter. In some embodiments, the term "about" means a range of ±10% of the numerical value following that. In some embodiments, the term "about" means a range of ±5% of the numerical value following that.
The term "monoclonal antibody" as used herein refers to an antibody that is highly homogeneous and directed against only a specific epitope produced by a single B cell clone. The term "polyclonal antibody" as used herein refers to a mixture of a plurality of monoclonal antibodies directed against a plurality of epitopes by immunizing an animal with an antigen comprising the plurality of epitopes and stimulating a plurality of B cell clones of the animal organism.
The term "enzyme-linked immunosorbent assay" or "ELISA" as used herein refers to a qualitative and quantitative detection method in which soluble antigen or antibody is bound to a solid support, and an antigen-antibody specific binding is used to perform an immune reaction.
As used herein, the term "horseradish peroxidase" or "HRP" is a glycoprotein having a molecular weight of 44000 and is formed by combining colorless enzyme protein and dark brown iron porphyrin, with neutral sugar and amino sugar accounting for about 18%, and mainly mannose, xylose, arabinose, hexosamine, etc. Each HRP molecule contains a hemin IX as a prosthetic group with a maximum absorbance peak at 403nm, whereas the apo-enzyme protein has a maximum absorbance at 275 nm. In theory, compounds that are capable of reacting with HRP and producing colored products can be used as the color developing solution.
The term "inactivated vaccine" as used herein refers to a virus that is cultured to be infectious by physical or chemical means to lose its pathogenicity while retaining its antigenicity. Inactivated vaccines may consist of whole viruses or may consist of split fragments of them as split vaccines.
The following examples are provided to aid in the understanding of the present invention. It is to be understood that these examples are illustrative of the present invention only and are not to be construed as limiting in any way. The actual scope of the invention is set forth in the following claims. It will be understood that any modifications and variations may be made without departing from the spirit of the invention.
Example 1: preparation of raw materials
(1) Preparation of sheep polyclonal antibody serum
1 sheep was immunized with SARS-CoV-2 prototype strain immunogen. Briefly, the first needle of immunized sheep is intramuscular injection immunization after Freund's complete adjuvant is added, the immunization dose is 50-500 mug/dose, freund's incomplete adjuvant is added for enhancing immunization, and the intramuscular injection is 1-100 mug/dose. Blood collection is carried out after immunization, serum is centrifugally separated, and sheep serum titer is detected by an indirect enzyme-linked immunosorbent assay.
(2) Preparation of mouse polyclonal antibody serum
The SARS-CoV-2 prototype strain immunogen was intraperitoneally injected into Balb/C mice. Briefly, freund's complete adjuvant was added to the first needle of immunized mice, freund's incomplete adjuvant was used for booster immunization, the amount of immunization was 1-10. Mu.g/mouse, blood was collected, and serum was centrifuged.
(3) Purification of SARS-CoV-2 sheep polyclonal antiserum and mouse polyclonal antiserum
Purifying by protein G affinity chromatography, and respectively detecting ELISA titers of the purified sheep polyclonal antibody and the mouse polyclonal antibody, wherein the results are 10 5 。
Example 2: establishment of virus antigen detection kit
In this example, goat polyclonal antibody is used to coat the ELISA plate, and mouse polyclonal antibody is used as primary antibody, and square titration is performed.
Specifically, the sheep polyclonal antibody obtained in example 1 was diluted with a coating solution at 1:1000, 1:2000, 1:4000 or 1:8000, respectively, and then 100. Mu.l/well of the diluted antibody was applied to an ELISA plate, and incubated at 2 to 8℃overnight (12 to 18 hours) or 37℃for 2 to 4 hours. The plates were washed three times with PBST wash, then 150-300. Mu.l/well of PBS blocking solution containing 10% calf serum (Minhai) or 1% BSA (BOEOGEN Co.) was added, and blocked at 37℃for 1-3 hours. And (3) removing the sealing liquid after the reaction is finished, beating, adding 100 mu l/hole antigen reference after serial dilution, reacting for 0.5 to 2 hours at 37 ℃, and washing the plate for 3 to 5 times by using washing liquid. After which 1:2000, 1:3000 or 1:40 is addedThe mouse polyclonal antibody obtained in example 1 after 00 dilutions was reacted at 37℃for 0.5 to 2 hours at 100. Mu.l/well, washed 3 times and patted dry. HRP-labeled goat anti-mouse IgG antibody (KPL) was added at a dilution of 1:12000, reacted at 37℃for 0.5-2 hours, and washed. Finally, adding color development A/B solution (Beijing Bubang Biotechnology Co., ltd.) and developing at 37 ℃ for 10-20 minutes, adopting 2M H 2 SO 4 The reaction was terminated and then read at a wavelength of 450 to 630 nm. The results are shown in Table 1 below.
TABLE 1 goat polyclonal antibody coating and mouse polyclonal antibody matrix titration OD 450-630 Value of
Example 3: determination of sensitivity and Linear Range of kit
The method comprises the following steps: serial dilution of antigen reference to 400-6.25 SU/ml in multiple ratio and detection of OD in 7 points by ELISA 450-630 Values. The test was repeated twice (test 1 and test 2), two parallel samples at a time.
If antigen reference OD 450-630 Value not less than negative control OD 450-630 The value x 1.5, the detection sensitivity is judged to be high. If negative control OD 450-630 The value is less than 0.05, calculated as 0.05.
Linear range: OD of antigen references at 6 concentrations 450-630 Four-parameter regression is carried out on the value and the antigen content of the sample, and the correlation coefficient R 2 Not less than 0.99, the concentration range is the linear range of the kit of the embodiment.
TABLE 2 sensitivity of SARS-CoV-2 antigen detection kit and Linear Range assay OD 450-630 Value results
As can be seen from the results in Table 2, the OD of the negative control 450-630 On average 0.0692, the OD of the negative control X1.5 450-630 0.1038. Thus, when the antigen containsOD obtained in runs 1 and 2 at a dose of 12.5SU/ml 450-630 Average value of 0.1183, range of 0.1144-0.1240, OD in the range 450-630 The lowest value was already close to 0.1038 (negative control x 1.5). As a result, the detection sensitivity of the detection kit of the present invention was 12.5SU/ml.
R is R when the linear range is 12.5-400 SU/ml 2 =1, conform to R 2 Not less than 0.99.
Example 4: verification of specificity of the kit
The method comprises the following steps: and (3) respectively detecting the antigen content of the diluent for the sample, the hepatitis A antigen reference, the Vero cell culture, the Coxsackie virus (Coxsackie virus) antigen reference, the enterovirus 71 (EV 71) antigen reference and the SARS-CoV-2 antigen reference by adopting the established detection system.
Standard: if the detection OD for the sample 450-630 Values below the negative control OD 450-630 And judging the sample as negative if the average value is multiplied by 1.5.
The results of the specificity verification of the kit are shown in Table 3.
TABLE 3 specificity test results of SARS-CoV-2 antigen detection kit
The sample dilutions, hepatitis A antigen reference, 1% BSA blocking solution, vero cell culture, coxsackie antigen reference, EV71 antigen reference used in Table 3 were all from vitamin technologies Inc. in Beijing.
As can be seen from the results in Table 3, the kit of the present invention did not cross-react with the sample diluent, the hepatitis A antigen reference, the 1% BSA blocking solution, the Vero cell culture, the Coxsackie antigen reference, and the EV71 antigen reference, and only SARS-CoV-2 antigen was specifically detected.
Example 5: verification of the Linear Range of the kit
The method comprises the following steps: according to the linear range of the antigen detection method which is preliminarily determinedDiluting antigen reference substance to proper concentration gradient of 12.5-400 SU/ml, and measuring OD by each concentration 450-630 Four-parameter regression was performed on the values and antigen concentrations, and the measurement was repeated 3 times.
The results of the linear validation of the antigen detection system are shown in table 4:
TABLE 4 Linear validation results of antigen detection systems
As can be seen from the results of Table 4, the linear correlation coefficient R of the three tests was found when the antigen content was in the range of 12.5 to 400SU/ml 2 All are 1, and all conform to the correlation coefficient R of the four-parameter regression equation 2 Linear requirement of 0.99 or more.
Example 6: verification of the suitability of the kit
The method comprises the following steps: 3 batches of SARS-CoV-2 vaccine stock solution or finished product are selected as verification samples, each batch of test sample is diluted to be within the linear range of the reference sample, antigen reference sample with a certain concentration (marked theoretical value in tables 5 and 6) is added in an equal volume, the antigen content of the test sample and the antigen content of the test sample after marked are measured, and the recovery rate of the antigen reference sample is calculated.
Recovery = (sample concentration after labeling-sample concentration)/added reference sample concentration x 100%.
Standard: the sample standard recovery rate of each batch should be 80-120%.
TABLE 5 stock solution labeling recovery results
TABLE 6 recovery results of finished product with standard
The results in Table 5 show that the statistics of the three batches of stock solutions are carried out, the results are 98-104%, the requirements of the variation range of 80% -120% of the ELISA method are met, and the antigen reference substances added into the stock solutions can be accurately detected.
The results in Table 6 show that the statistics of the labeling recovery results of three batches of finished products show that the results are between 81 and 108 percent, and meet the requirements of 80 to 120 percent of variation range of the ELISA method, which shows that antigen reference substances added into samples after the dissociation of vaccine finished products can be accurately detected.
Example 7: practical application of kit
Using the kit of the present invention, the amount of antigen after dissociation of SARS-CoV-2 vaccine was measured, and the statistics of the results are shown in Table 7.
TABLE 7 application of the kit of the invention to SARS-CoV-2 antigen content detection
As can be seen from the results in Table 7, the statistics of the three finished product labeled recovery results are 94-117%, and the requirements of 80% -120% variation range of the ELISA methodology are met. This further shows that the antigen reference substances added into the sample after the dissociation of the vaccine finished product can be accurately detected, and the kit can be used for detecting the vaccine finished product.
The technical scheme of the invention is not limited to the specific embodiment, and all technical modifications made according to the technical scheme of the invention fall within the protection scope of the invention.
Claims (10)
1. A kit for detecting a novel coronavirus (SARS-CoV-2) immunogen in a sample to be tested, said kit comprising: a reaction plate coated with SARS-CoV-2 sheep polyclonal antibody; SARS-CoV-2 mouse polyclonal antibody; and a secondary antibody against said SARS-CoV-2 mouse polyclonal antibody,
preferably, the sample to be tested is SARS-CoV-2 vaccine or intermediate sample in its production process, preferably SARS-CoV-2 inactivated vaccine or intermediate sample in its production process.
2. The kit of claim 1, further comprising a sample diluent, a chromogenic solution, a wash solution and an antigen reference,
preferably, the secondary antibody of SARS-CoV-2 mouse polyclonal antibody is horseradish peroxidase or alkaline phosphatase labeled secondary antibody;
preferably, the washing solution is a phosphate buffer, more preferably a Tween-20-containing phosphate buffer.
3. A method for detecting a novel coronavirus (SARS-CoV-2) immunogen in a sample to be tested, said method comprising:
coating the reaction plate by using SARS-CoV-2 sheep polyclonal antibody to obtain a coated reaction plate;
adding a sample to be detected into the reaction plate for incubation so as to combine the sample to be detected with the SARS-CoV-2 sheep polyclonal antibody;
after washing the reaction plate, adding a sealing liquid for sealing;
washing the reaction plate, and then adding SARS-CoV-2 mouse polyclonal antibody for incubation so as to enable the SARS-CoV-2 mouse polyclonal antibody to be combined with the sample to be detected;
after washing the reaction plate, adding a color development liquid to develop color, and detecting the OD value at 450-630 nm.
4. A method according to claim 3, wherein the sample to be tested is a SARS-CoV-2 vaccine or an intermediate sample in its production, preferably a SARS-CoV-2 inactivated vaccine or an intermediate sample in its production.
5. The method according to claim 3 or 4, wherein the sample to be tested is incubated with the coated reaction plate overnight at 2-8 ℃ or for 0.5-2 hours at 35-39 ℃.
6. The method according to any one of claims 3 to 5, wherein the SARS-CoV-2 mouse polyclonal antibody is incubated with the test sample at 35-39 ℃ for 0.5-2 hours.
7. The method according to any one of claims 3 to 6, further comprising: a standard curve was made using a gradient diluted antigen reference,
preferably, the immunogen of the sample to be tested is quantified based on the standard curve.
8. The method of claim 7, wherein the antigen reference is diluted to a concentration gradient of 12.5 to 400 SU/mL.
9. The method according to any one of claims 3 to 8, wherein the method employs a sample free of SARS-CoV-2 immunogen as a negative control.
10. The method according to any one of claims 3 to 9, wherein the reaction plate is washed with a phosphate buffer, preferably a tween-20 containing phosphate buffer.
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