CN112881706B - Method for simultaneously detecting total antibody and neutralizing antibody - Google Patents
Method for simultaneously detecting total antibody and neutralizing antibody Download PDFInfo
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Abstract
The invention relates to the field of antibody detection, in particular to a detection method and a detection assembly.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an antibody detection method.
Background
In the process that pathogenic microorganisms infect cells, the virus surface receptor binding protein is a key ' for the pathogenic microorganisms to enter the cells, and can unlock the ' lock ' of the cell receptor protein, so that the pathogenic microorganisms enter the cells and start the replication process of the pathogenic microorganisms.
Neutralizing antibodies are certain antibodies produced by B lymphocytes and capable of binding to an antigen (ligand) on the surface of a pathogenic microorganism, thereby preventing the pathogenic microorganism from adhering to a target cell receptor and invading the cell.
Current detection of neutralizing antibodies is mainly performed in the biosafety tertiary laboratory (BSL 3). The neutralization test detection by using live virus cell culture has high requirements on laboratory grade, and seriously limits the clinical large-scale popularization and application. The development of a rapid neutralizing antibody detection method which can be developed in a biosafety secondary laboratory (BSL2) or a common laboratory is of great significance. Currently, some research groups develop ELISA tests to meet the requirements of BSL2 detection, but the detection reagents can only detect the neutralizing antibodies alone, and cannot detect the total antibodies at the same time. The detection of the total antibody can reflect whether the antibody is generated after the vaccination, and the detection of the neutralizing antibody can reflect whether the neutralizing antibody immune effect is obtained after the vaccination, so as to guide the adjustment of the individual immunization scheme and judge whether the immunization strategy needs to be adjusted. Thereby promoting the auxiliary support of the immunity protection of the population and having great significance. The detection of the total antibody and the neutralizing antibody not only can objectively reflect the current immune condition of the organism, but also can be used for analyzing the past or future immune judgment and prediction of the organism on pathogenic microorganisms.
SARS-CoV-2 surface spinous process S protein (spike protein) is important receptor binding site on coronavirus surface, which can bind with cell surface virus specific receptor, mediate virus outer membrane and cell fusion, virus adsorption and membrane penetration; the coronavirus S protein receptor binding domain (RBM) can make the virus bind with angiotensin-converting enzyme2 (ACE 2) on the surface of host cell and make the virus enter the host cell. When SARS-CoV-2 invades the body, it can stimulate the body to produce neutralizing antibody with protective action, specially prevent pathogenic microorganism from entering into cell, and prevent infection. The amount of the neutralizing antibody is an important index of the immune protection effect of the vaccine, and is an important basis for the evaluation and quality control of the vaccine.
After individual is inoculated with vaccine, protective antibody, namely neutralizing antibody, can be generated through immune response, and the titer of the neutralizing antibody can be measured, so that the clinical curative effect of the vaccine can be judged. Therefore, the detection of the neutralizing antibody can be applied to research and development evaluation of vaccines and evaluation of the autoimmune effect of individuals after vaccination. In addition, the detection of the new crown patient after healing can judge whether the risk of reinfection exists.
The current vaccine development concept is based on the RBD-ACE2 invasion mechanism, the core fragment of which is designed for RBD, and some treatment regimens are also based on the development of antibodies that block the RBD-ACE2 pathway.
With the popularization and inoculation of the new corona vaccine, the development of a reagent which is convenient and quick and does not need to detect total antibodies and neutralizing antibodies under the condition of a BSL3 laboratory is urgently needed.
The existing novel crown antibody detection reagent can only detect total antibodies or neutralizing antibodies individually, and the total antibodies and the neutralizing antibodies cannot be detected simultaneously. The present invention has the advantage of being able to detect both total and neutralizing antibodies in a sample. The method can bring convenience in data interpretation, and can correct the test result by detecting the total antibody and the neutralizing antibody, for example, the abnormal detection result can be fed back when the total antibody is detected to be negative and the neutralizing antibody is detected to be positive, so that the repeated detection is needed.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention provides at least one of the following embodiments:
the invention provides a detection method, which comprises the following steps:
a method of detecting an antibody, the method comprising:
step 1: contacting the sample with the ligand or a fragment containing the ligand, and detecting a reaction signal;
step 2: and then contacted with a receptor of the ligand or a fragment thereof, and a reaction signal is detected.
Characterised in that the detection method allows the detection of neutralising and/or total antibodies, the total antibodies capable of binding to the ligand being detected by step 1 and the neutralising antibodies binding to the ligand being detected by step 2.
In some embodiments, the ligand or ligand-containing fragment may be conjugated to a label; the selectable marker is latex microspheres.
In some embodiments, the receptor or fragment thereof can be conjugated to a label; the selectable marker is latex microspheres.
In some embodiments, the ligand or ligand-containing fragment is selected from the group consisting of SARSr-CoV; a particular ligand or ligand-containing fragment is a ligand selected from the group consisting of a ligand containing the SARS-CoV-2RBM region.
In some embodiments, the receptor or fragment thereof is a cell surface receptor or fragment thereof; the specific receptor is ACE2 or the receptor fragment is an ACE 2-containing fragment.
In some embodiments, the sample can be a blood sample, a lymph sample, a saliva sample, a urine sample. In one embodiment, the sample may be serum.
The present invention also provides an assembly for detecting an antibody, comprising:
(i) a ligand or a fragment containing a ligand, and
(ii) (ii) a receptor or fragment thereof that binds to the ligand of (i).
In some embodiments, the antibody detection assembly has a ligand or ligand-containing fragment conjugated to a label, which in some embodiments is a latex microsphere.
In some embodiments, the receptor or fragment thereof that binds to the ligand of (i) is conjugated to a label, in some embodiments the label is a latex microsphere.
In some embodiments, the antibody detection component wherein the ligand or ligand-containing fragment is selected from SARSr-CoV, and in particular from a ligand comprising the SARS-CoV-2RBM region.
SARS-CoV-2 includes SARS-CoV-2 and variants thereof.
In some embodiments, the antibody detection module wherein the receptor or fragment thereof is a cell surface receptor or fragment thereof, and the particular receptor is ACE2 or the receptor fragment is an ACE 2-containing fragment.
Drawings
FIG. 1 shows the crystal structure and the electron density of the interaction interface of SARS-CoV-2 spike protein receptor binding region RBD (core + RBM) and ACE2 complex.
FIG. 2 shows the correlation of total antibodies in the simultaneous detection and neutralization of total antibodies with the luminescence platform total antibody detection according to the present invention.
FIG. 3 shows the correlation of simultaneous detection of total and neutralizing antibody neutralizing antibodies with detection of neutralizing antibodies by a luminescent platform according to the present invention.
FIG. 4 shows a scatter plot of the inhibition rate distribution of the negative and positive samples tested according to the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
The embodiment of the invention can detect the levels of the total antibody and the neutralizing antibody in the sample to be detected, and has higher sensitivity and/or specificity. The methods and assemblies of the invention are versatile and are not limited to a particular immunoassay platform, nor to a particular species.
As used herein, the term "receptor" refers to a specific protein present in the cell membrane or intracellular environment that binds to a specific signal molecule outside the cell and activates a series of biochemical reactions in the cell, which in turn causes the cell to respond to external stimuli.
As used herein, the term "ligand" refers to a protein or protein fragment that is capable of binding to a "receptor".
Herein, the term "total antibody" refers to the collection of all antibodies capable of binding to the "ligand", and the term "neutralizing antibody" refers to an antibody capable of competitively binding to the "ligand" with the "receptor".
Herein, "label" is understood to be capable of directly generating a signal; or directly or indirectly trigger the specific substance to generate a signal, and the label may be directly or indirectly linked to the labeled substance. For example, labels commonly used for immunodetection include, but are not limited to, metal particles, fluorescent labels, chromophore labels, electron dense labels, chemiluminescent labels, electrochemiluminescent labels, radioactive labels, nucleic acid labels, polypeptide labels, or enzymes. In some embodiments, the label can be colloidal gold, fluorescein, fluorescent microspheres, acridinium ester, horseradish peroxidase, alkaline phosphatase, latex microspheres, ruthenium triad, luminols, Eu chelates.
Herein, "detection signal" is understood to mean the acquisition or identification of the intensity or level of a detection signal in a manner that enables identification of the signal substance.
As used herein, the term "fragment" refers to a fragment of amino acids that contains at least a binding segment.
Herein, "contacting" is understood to allow binding thereof to occur. The contact time is not particularly limited, and may vary from embodiment to embodiment and from platform to platform, but is within the purview of one skilled in the art.
Herein, ACE2 is not limited to species, and different species of ACE2 may be selected depending on the species source of the sample to be tested, e.g., mammals, non-cold flying animals, bats and birds, etc., and in some embodiments, human ACE2 is used for coronavirus neutralizing antibody detection in human samples.
Herein, the RBM region is a region of the RBD to which ACE2 can bind, and the RBD-neutralizing antibody and ACE2 can compete for binding to the RBM region.
Herein, "SARSr-CoV" refers to severe acute respiratory syndrome-associated coronavirus.
In some embodiments, the invention provides an antibody detection method comprising:
step 1: contacting the sample with the ligand or a fragment containing the ligand, and detecting a reaction signal;
step 2: and then contacted with a receptor of the ligand or a fragment thereof, and a reaction signal is detected.
Characterised in that the detection method allows the detection of neutralising and/or total antibodies, the total antibodies capable of binding to the ligand being detected by step 1 and the neutralising antibodies binding to the ligand being detected by step 2.
Herein, the sample to be tested is understood to be a sample which may contain antibodies, and the sample may be serum, plasma, whole blood, oral mucosa exudate, lymph fluid, saliva, urine, but is not limited thereto.
Herein, the detection of the reaction signal may be a detection of a change in the signal value by known detection means such as turbidimetry, absorbance, dispersion ratio, nuclear magnetic resonance, and the like.
In some embodiments, the reaction signal is further amplified by coupling latex microspheres to receptors and/or ligands, and total and neutralizing antibodies are detected by immunoturbidimetric detection.
Aspects and embodiments of the present application will be discussed with reference to the figures and the following examples. Other aspects and embodiments will be apparent to those skilled in the art. Although the present application has been described in conjunction with exemplary embodiments, many equivalent modifications and variations will be apparent to those skilled in the art in light of the present application. Accordingly, the exemplary embodiments of the present application are intended to be illustrative, not limiting. Various changes may be made to the embodiments described without departing from the spirit and scope of the present application.
Examples
Example 1 antibody detection
In this example, the inventors propose a method and assembly for simultaneous detection of SARS-CoV-2 total antibodies and neutralizing antibodies.
1.1 materials and methods
Recombinant human ACE2, recombinant RBD antigen, neocorona neutralizing antibody standard (G3), and latex microspheres were purchased from Guangdong Fenpeng biological Co., Ltd. The instrument comprises the following steps: full-automatic biochemical analyzer.
1.2ACE2 coupled latex microspheres
1)390ul of cross-linking solution (50mM HEPES, pH6.1), adding 100ul of latex microspheres (JSR), and shaking and mixing uniformly on a shaking table to obtain latex microsphere solution;
2) adding 13ul of EDAC solution into the latex microsphere solution, and oscillating and activating for 1 hour at 37 ℃;
3) adding 50ul of ACE2(2mg/ml) into the latex microsphere solution, and performing oscillation crosslinking at 37 ℃ for 4 hours;
4) adding 50ul TW-20 solution, shaking at 37 ℃ and sealing for 1 hour;
5) adding the crosslinking liquid into a centrifuge tube, centrifuging, and carefully discarding the supernatant by using a pipette;
6) adding 2mL of cleaning solution (50mM glycine + 0.1% NaN3, pH8.0), vortex shaking, centrifuging again, and carefully discarding the supernatant;
7) 5mL of latex microsphere suspension (50mM glycine + 0.1% BSA + 10% sucrose + 0.1% NaN3, pH8.0) was added and sonicated.
1.3RBD coupled latex microspheres
1)390ul of cross-linking solution (50mM HEPES, pH6.1), adding 100ul of latex microspheres (JSR), and shaking and mixing uniformly on a shaking table to obtain latex microsphere solution;
2) adding 13ul of EDAC solution into the latex microsphere solution, and oscillating and activating for 1 hour at 37 ℃;
3) adding 50ul RBD (2mg/ml) into the latex microsphere solution, and oscillating and crosslinking for 4 hours at 37 ℃;
4) adding 50ul TW-20 solution, shaking at 37 ℃ and sealing for 1 hour;
5) adding the crosslinking liquid into a centrifuge tube, centrifuging, and carefully discarding the supernatant by using a pipette;
6) adding 2mL of cleaning solution (50mM glycine + 0.1% NaN3, pH8.0), vortex shaking, centrifuging again, and carefully discarding the supernatant;
7) 5mL of latex microsphere suspension (50mM glycine + 0.1% BSA + 10% sucrose + 0.1% NaN3, pH8.0) was added and sonicated.
1.4 Standard preparation
The neutralizing antibody G3 standard (purchased from philippine organisms) was diluted with matrix serum to the following different concentrations: 0.25 ng/ml, 50ng/ml, 100ng/ml, 200ng/ml, 500ng/ml, 1000ng/ml, 2000 ng/ml.
1.5 on-machine testing
The instrument comprises the following steps: full-automatic biochemical analyzer
And (3) detection flow: sucking 15ul of the sample on the sample plate, sucking R1135 ul at the same time, adding into a cuvette, mechanically stirring, and incubating at 37 ℃ for 5 minutes; adding R215 ul into the cuvette, and mechanically stirring; the instrument records the absorbance values of 7 minutes 30 seconds and 13 minutes 30 seconds from the start of sample addition, the difference is the sample's reactivity, which is directly proportional to the total antibody concentration in the sample. After the reaction is continued for 10 minutes, adding R350 ul into the cuvette, and mechanically stirring; the instrument records the reactivity values at 15 minutes, 30 seconds and 26 minutes from the start of sample addition, the difference being the reactivity of the sample, which is inversely related to the concentration of neutralizing antibody in the sample.
The sample adding ratio of S, R1, R2, R3, 15, 135, 15 and 50 is
Wavelength: 570nm
Blank time 1: 51-51
Reaction time 1: 90-90
Blank time 2: 104-104
Reaction time 2: 172-172
R1: tris buffer
R2: RBD coupled latex microsphere reagent
R3: ACE2 coupled latex microsphere reagent
Table 1: detecting total and neutralizing antibodies in standard
Note: inhibition rate 1-sample detection signal/negative detection signal
Analyzing the data in Table 1, the test sample is a standard substance of neutralizing antibody, and can still detect 17% inhibition rate at a standard concentration of 25ng/ml, that is, the detection sensitivity of the reagent prepared according to the invention can reach at least 25 ng/ml.
Example 2 clinical sample antibody detection
2.1 reagent preparation following the procedure in example 1, the following reagents were prepared:
r1: tris buffer
R2: RBD coupled latex microsphere reagent
R3: ACE2 coupled latex microsphere reagent
2.2 serum samples were collected after inoculation of New crown vaccine 23 cases
2.3 on-machine testing
The instrument comprises: merrill BS480
And (3) detection flow: sucking 15ul of the sample on the sample plate, sucking R1135 ul at the same time, adding into a cuvette, mechanically stirring, and incubating at 37 ℃ for 5 minutes; adding R215 ul into the cuvette, and mechanically stirring; the instrument records the absorbance values of 7 minutes 30 seconds and 13 minutes 30 seconds from the start of adding the sample, the difference is the reaction absorbance of the sample, and the absorbance and the total antibody concentration in the sample are in a direct relation under specific conditions. After the reaction is continued for 10 minutes, adding R350 ul into the cuvette, and mechanically stirring; the instrument records the absorbance values at 15 minutes, 30 seconds and 26 minutes from the start of sample addition, the difference is the reaction absorbance of the sample, and the absorbance and the concentration of neutralizing antibody in the sample are in direct proportion under specific conditions.
The sample adding ratio of S: R1: R2: R3: 15:135:15:50
Wavelength: 570nm
Blank time 1: 51-51
Reaction time 1: 90-90
Blank time 2: 104-104
Reaction time 2: 172-172
R1: tris buffer
R2: RBD coupled latex microsphere reagent
R3: ACE2 coupled latex microsphere reagent
Comparative example 1 detection of total antibodies in clinical samples Using existing luminescence platform
3.1 chemiluminescence detection of total antibody: 50ul of the sample was added to the reaction cuvette, 50ul of RBD (0.25mg/ml) coated with magnetic beads and 40ul of Anti-human IgG labeling solution (0.1ug/ml) labeled with acridinium ester were added thereto, and the reaction was carried out at 37 ℃ for 15min to detect the luminescence value.
3.2 detection of chemiluminescent neutralizing antibody: 50ul of the sample was added to the reaction cuvette, 50ul of ACE2(0.25mg/ml) coated with magnetic beads and 40ul of acridinium ester-labeled RBD labeling solution (0.1ug/ml) were added thereto, and the reaction was carried out at 37 ℃ for 15min to detect the luminescence value.
As a result:
table one: comparison of Total antibody detection data
| Sample(s) | Example 2Au/ml | Comparative example 1Au/ml |
| V-01 | 6.0 | 6.24 |
| V-02 | 5.8 | 2.28 |
| V-03 | 6.5 | 5.55 |
| V-04 | 6.7 | 2.97 |
| V-05 | 16.4 | 14.46 |
| V-06 | 5.9 | 2.61 |
| V-07 | 5.4 | 3.15 |
| V-08 | 9.1 | 7.08 |
| V-09 | 3.0 | 1.89 |
| V-10 | 12.2 | 7.05 |
| V-11 | 3.3 | 2.01 |
| V-12 | 1.7 | 1.77 |
| V-13 | 2.2 | 1.44 |
| V-14 | 2.0 | 1.24 |
| V-15 | 4.7 | 1.95 |
| V-16 | 3.8 | 1.89 |
| V-17 | 15.3 | 7.77 |
| V-18 | 13.7 | 13.32 |
| V-19 | 8.7 | 4.92 |
| V-20 | 6.7 | 3.81 |
| V-21 | 10.4 | 3.15 |
| V-22 | 31.3 | 17.07 |
| V-23 | 12.2 | 6.72 |
TABLE 2 comparison of neutralizing antibody detection data
Note: inhibition rate 1-sample reactivity/reactivity in PBS
And (3) analysis: the antibody detection reagent prepared by the invention can simultaneously detect total antibodies and neutralizing antibodies in a sample, and can independently detect the correlation R of the total antibodies with a high-sensitivity chemiluminescence platform2Correlation R to reach 0.81 and neutralizing antibodies2Reaching 0.78, the conformity is consistent.
Example the reagents prepared in example 1 were tested using 50 negative samples and 23 positive samples (vaccinated).
As a result: as can be seen from FIG. 4, the reagent prepared by the present invention can distinguish negative and sample samples well, and has high reaction specificity. And the method has higher discrimination on different positive samples, and the linear range of reaction detection is wide.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. A method of detecting an antibody to SARSr-CoV, the method comprising:
step 1: contacting the sample with an RBD or a fragment containing an RBD, detecting a reaction signal, and calculating the amount of total antibodies bound to the RBD;
step 2: contacting the reaction mixture obtained in the step 1 with ACE2 or ACE2 fragment, detecting reaction signals, and calculating the amount of neutralizing antibodies;
the RBD or the fragment containing the RBD is coupled with a marker, and the ACE2 or ACE2 fragment is coupled with a marker.
2. The method of claim 1, wherein the SARSr-CoV is further SARS-CoV-2.
3. The method of claim 1, wherein the RBD or RBD-containing fragment-conjugated label is a latex microsphere.
4. The detection method according to claim 1, wherein the label conjugated with ACE2 or ACE2 fragment is latex microsphere.
5. The method of claim 1, wherein the RBD or RBD-containing fragment is selected from the group consisting of ligands containing the RBM region of SARS-CoV-2.
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| CN111812336A (en) * | 2020-08-10 | 2020-10-23 | 苏州康和顺医疗技术有限公司 | Detection kit for detecting coronavirus antibody and preparation method thereof |
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