CN113156129A

CN113156129A - High-sensitivity detection method and product of neutralizing antibody

Info

Publication number: CN113156129A
Application number: CN202110104780.4A
Authority: CN
Inventors: 夏良雨; 潘少丽; 覃丽芳; 岑静; 邓盛花; 范名俊
Original assignee: Guangdong Fapon Biotech Co Ltd
Current assignee: Guangdong Fapon Biotech Co Ltd
Priority date: 2021-01-13
Filing date: 2021-01-26
Publication date: 2021-07-23
Anticipated expiration: 2041-01-26
Also published as: WO2022151596A1; CN113156129B

Abstract

The invention relates to the field of antibody detection, in particular to an antibody detection method and a product, which have higher sensitivity when applied to detection of a neutralizing antibody. The detection method provided by the invention comprises the following steps: contacting the sample with a fragment containing a ligand, reagent 1, reagent 2; wherein one of the reagent 1 or the reagent 2 is linked with a label, and the other is immobilized on a solid phase carrier.

Description

High-sensitivity detection method and product of neutralizing antibody

Technical Field

The invention relates to the field of antibody detection, in particular to an antibody detection method and a product.

Background

Receptor ligand binding is one of the channels that enable signaling, and receptors are capable of recognizing and specifically binding to ligands. In the case of pathogen invasion into host cells, ligands on the pathogen surface can bind to receptors on the host cell, opening the door for host cell invasion.

However, in the face of brand new viruses, under the background that more relevant basic researches and more mechanism researches are not thorough, a large number of vaccine designs rapidly enter the clinic, and more contents still need to be further researched on the effectiveness, the protection period, the quality control and the accessibility of the vaccines.

The first vaccine in China is mainly an inactivated vaccine, however, BPL (BPL-based protein) inactivation adopted by the inactivated vaccine is adopted, and Spike protein has high proportion of post-fusion conformation and RBD (reduced beta-amyloid) conformation, so that the evidence that the total antibody level response is relatively low and the neutralizing antibody titer is not high is found in the primary immune population analysis.

In addition, from the current basic research of new crowns, not all individuals will produce sufficient titers of neutralizing antibodies after infection with new crowns, with the risk of secondary infection. The neutralizing antibody titer gradually decreases after reaching the peak value in 1 month, and a small part of low-titer convalescent people can decrease below the detection limit. With reference to other coronaviruses, new corona antibodies may exist for about 1-2 years and do not form long-lasting protection.

Neutralizing antibodies to SARS-CoV-2 can effectively control infection by blocking or inhibiting the interaction between SARS-CoV-2 and the host cell. The best studied mechanism is the interaction between the Receptor Binding Domain (RBD) on the S1 subunit of the SARS-CoV-2 Spike protein and the host cell Receptor ACE2, followed by conformational transition and membrane fusion. ACE2, also known as achh and known as angiotensin converting enzyme 2, is a metalloprotease with 805 amino acids in total length and is a type I transmembrane glycoprotein with a single extracellular catalytic domain.

Partial vaccines currently show evidence of low positive conversion and titers of neutralizing antibodies after immunization. In addition, SARS-CoV-2 as RNA virus has very high mutation frequency, and the neutralizing effect of serum and vaccine in convalescent period shows lower neutralizing capacity to partial mutation, and the accumulation of the partial mutation may result in immunological escape. Thus, higher demands are made on the sensitivity of detection of neutralizing antibodies.

The invention is therefore proposed.

Disclosure of Invention

The invention provides at least one of the following embodiments:

in some embodiments, the invention relates to a method of detection comprising the steps of:

(1) contacting the sample with a fragment containing a ligand, reagent 1, reagent 2;

reagent 1: a receptor comprising the ligand;

reagent 2: comprising an antibody that binds to the ligand, wherein the antibody competes with a receptor;

wherein one of the reagent 1 or the reagent 2 is connected with a marker, and the other is fixed on a solid phase carrier;

(2) and detecting the signal.

In some embodiments, the contacting comprises any one of:

(a) simultaneously contacting the sample with the fragment containing the ligand, the reagent 1 and the reagent 2;

(b) the sample is firstly contacted with the fragment containing the ligand, the reagent 1 and then contacted with the reagent 2;

(c) the sample is firstly contacted with the fragment containing the ligand and the reagent 2, and then contacted with the reagent 1;

(d) the sample is firstly contacted with the fragment containing the ligand, and then contacted with the reagent 1 and the reagent 2;

(e) contacting the sample with a fragment containing the ligand, then with reagent 1, and then with reagent 2;

(f) the sample is contacted with the ligand-containing fragment, then with reagent 2, and then with reagent 1.

In some embodiments, the ligand is a ligand for pathogen invasion of the host cell.

In some embodiments, the pathogen is a coronavirus.

In some embodiments, the coronavirus is SARS-CoV-2 or SARS-CoV.

In some embodiments, the ligand is an RBD.

In some embodiments, the receptor is ACE 2.

In some embodiments, the ligand-containing fragment comprises a structure that is more than dimeric as a result of treatment of the fragment.

In some embodiments, the ligand-containing fragment comprises a fragment resulting from the expression of two or more ligand sequences in tandem.

In some embodiments, the detection method is used to detect the presence or absence of neutralizing antibodies in a sample.

In some embodiments, the sample is also contacted with reagent 3, which 3 rd reagent comprises a second antibody that is IgG directed against the species from which the sample is derived.

In some embodiments, the second antibody is anti-human IgG.

In some embodiments, the reagent 3 is linked to a label or solid support.

In some embodiments, if reagent 1 is linked to a label, reagent 3 is linked to a label.

In some embodiments, if reagent 1 is immobilized on a solid support, reagent 3 is immobilized on a solid support.

In some embodiments, the detection methods can also be used to detect the presence or absence of total antibodies in a sample.

In some embodiments, the invention relates to a detection assembly comprising:

(a) a ligand-containing fragment as described in any one of the embodiments above;

(b) the reagent 1 according to any of the above embodiments;

(c) the reagent 2 according to any of the above embodiments.

In some embodiments, the test assembly further comprises (d) a reagent 3 as described in any of the above embodiments.

In some embodiments, the invention relates to a chromatography assembly comprising: the device comprises a sample pad, a combination pad, a reaction membrane and an absorption pad, wherein a detection area is arranged on the reaction membrane; the chromatography assembly further comprises:

(b) the reagent 1 according to any of the above embodiments;

(c) the reagent 2 according to any of the above embodiments.

In some embodiments, the reagent 1 is immobilized in a detection zone and the reagent 2 is attached to a label and disposed on a conjugate pad; or; the reagent 1 is connected with a marker and is arranged on the bonding pad, and the reagent 2 is fixed on the detection area.

In some embodiments, the chromatography assembly further comprises (d) a reagent 3 as described in any of the above embodiments; the reagent 3 is immobilized in the detection zone.

In some embodiments, the ligand-containing fragment is disposed on a sample pad.

In some embodiments, the use of the detection method of any one of the above embodiments, the detection module of any one of the above embodiments, or the chromatography module of any one of the above embodiments in antibody detection or in the preparation of an antibody detection reagent.

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.

Herein, a "ligand" is understood to be any protein or polypeptide capable of binding to a receptor. "receptor" is understood to mean a molecule capable of recognizing and specifically binding to a ligand, the ligand receptor binding enabling signal transduction, and the receptor may include a co-receptor. Common cell surface receptors include, but are not limited to, G protein-coupled receptors, receptor tyrosine kinases, guanylate cyclase-coupled receptors, ion channels, adhesion receptors, and the like. The ease with which a ligand binds to a receptor and the strength after binding is called affinity. The easier the two bind, the greater the strength of the binding after binding, the stronger the affinity, and vice versa.

Herein, "antibody" is distinct from "receptor", and an antibody is understood to mean that immune cells secrete an immunological substance, which is used to identify and/or neutralize an antigenic substance. A "neutralizing antibody" is an antibody used to protect cells from an antigen or infectious agent.

Herein, a "sample" is understood to be any sample that may contain antibodies, in some embodiments, the sample is from a sample after infection or active immunization; in some embodiments, the sample is selected from the group consisting of bodily fluids, excreta, cells; such as, but not limited to, serum, plasma, whole blood, lymph fluid, cerebrospinal fluid, interstitial fluid, saliva, urine, lymphocytes, etc.

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.

As used herein, "solid support" is understood to mean a solid support capable of being immobilized, either directly or indirectly, to an object to be immobilized (e.g., a protein, polypeptide), such as is commonly used in immunoassays, including plastic, particulate, or membrane supports. The plastic may be, for example, polystyrene; the particles may be, for example, magnetic particles; the membrane support may be, for example, a nitrocellulose membrane, a glass cellulose membrane, or a nylon membrane.

Herein, "detection signal" is understood to mean the acquisition or identification of the intensity or level of the detection signal in a manner that enables identification of the marker.

Herein, "ligand-containing fragment" is understood to mean a protein or polypeptide comprising the sequence of the ligand.

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, "agent" may be understood as a substance, a product, or the like, and is not limited to the form or state thereof, and may be a liquid or a solid.

The invention has higher sensitivity in the detection of the neutralizing antibody. The invention has universality, is not limited to a specific immunoassay platform and is not limited to a specific species.

The invention relates to a detection method of a neutralizing antibody, which comprises the following steps:

reagent 1: a receptor comprising the ligand;

(2) and detecting the signal.

In some embodiments, the contacting comprises: simultaneously contacting the sample with the fragment containing the ligand, the reagent 1 and the reagent 2; in some embodiments, the contacting comprises: the sample is firstly contacted with the fragment containing the ligand, the reagent 1 and then contacted with the reagent 2; in some embodiments, the contacting comprises: the sample is firstly contacted with the fragment containing the ligand and the reagent 2, and then contacted with the reagent 1; in some embodiments, the contacting comprises: the sample is firstly contacted with the fragment containing the ligand, and then contacted with the reagent 1 and the reagent 2; in some embodiments, the contacting comprises: contacting the sample with a fragment containing the ligand, then with reagent 1, and then with reagent 2; in some embodiments, the contacting comprises: the sample is contacted with the ligand-containing fragment, then with reagent 2, and then with reagent 1.

In some embodiments, reagent 1 is linked to a label and reagent 2 is immobilized on a solid support. In some embodiments, reagent 2 has a label attached thereto and reagent 1 is immobilized on a solid support.

In some embodiments, the ligand is a ligand for pathogen invasion of the host cell. In some embodiments, the pathogen is a coronavirus. Among them, common human-infecting coronaviruses include HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, MERS-CoV, or SARS-CoV-2. In some embodiments, the coronavirus is SARS-CoV-2 or SARS-CoV. Wherein, the SARS-CoV-2 comprises SARS-CoV-2 wild strain and variant strain thereof. The SARS-CoV includes SARS-CoV wild strain and its variant strain.

"pathogen" is understood herein to mean a microorganism (including bacteria, viruses, rickettsiae, fungi, etc.), parasite or other vector (e.g. recombinant microorganisms including hybrids or mutants) that can cause diseases of infection of humans or animals and plants.

In some embodiments, the ligand is an RBD, wherein the RBD comprises an RBD or a core region thereof, as long as its receptor binding function is achieved, and still be understood as an RBD according to the invention. Wherein RBD is Receptor Binding Domain. In some embodiments, the ligand is selected from the group consisting of RBD of SARS-CoV-2S protein. In some embodiments, the sequence of the RBD of the SARS-CoV-2S protein can be found in the Wrapp Daniel, Wang Nianshuan, Corbett Kizmekia S, et al Cryo-EM structure of the 2019-nCoV spike in the fusion formation, and amino acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in sequence thereto, and which still function as ligand-binding receptors and/or antibodies. In some embodiments, a fragment containing a ligand, such as an RBD protein, S1 protein, or S protein (see sequence Gene ID: 43740568).

In some embodiments, the Receptor is selected from ACE2(Angiotensin I Converting Enzyme 2), HDL (high density lipoprotein), hs (liver sulphase), SR-B1 (ballast Receptor Class B Member 1), apn (aminopeptidase n), DPP4(Dipeptidyl peptide 4), AGO4(Argonaute 4), IFITM3 (interstitial Induced Transmembrane Protein 3), egfr (epidermal Growth Factor Receptor), ICAM1 (interstitial addition Molecule 1), HSPA1B (Heat Shock Protein Family a (Hsp 1) Member 1B), ITGB6 (endogenous Receptor Family Member 6), Receptor mutation Family p (Receptor Family 3), WW 1(WW 1) Member 1B), WW 1(WW 3) Member 1a (WW 1 n 3), and WW 3 (WW 3). In some embodiments, the receptor is ACE2, and in some embodiments, the receptor is human ACE 2. In some embodiments, the sequence of human ACE2 can be referenced to Gene ID:59272, and amino acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in sequence thereto, and still function as receptor binding ligands.

In some embodiments, the ligand-containing fragment comprises a structure that is more than dimeric upon treatment of the fragment; the treatment method includes, but is not limited to, physical methods, chemical methods, and the like. In some embodiments, the segments containing a single ligand are treated, for example by photopolymerization, to obtain structures that are more dimeric. In some embodiments, the structure is more than dimeric, for example, by chemical crosslinking, for example, by crosslinking with amino and carboxyl groups on the fragment. In some embodiments, the ligand-containing fragment comprises a fragment resulting from the expression of two or more ligand sequences in tandem. Tandem expression of at least two ligand sequences is achieved, for example using recombinant techniques, and it will be appreciated by those skilled in the art that tandem expression results in more than two ligand regions being obtained for the fragment. In some embodiments, the ligand-containing fragment may be an RBD mer, an S1 protein mer, or an S protein mer of SARS-CoV-2S protein.

In some embodiments, the sample is also contacted with reagent 3, which 3 rd reagent comprises a second antibody that is IgG directed against the species from which the sample is derived; in some embodiments, when the sample object detected by the method of the invention is human, the second antibody is anti-human IgG. In some embodiments, the reagent 3 is linked to a label or solid support. In some embodiments, if reagent 1 is linked to a label, reagent 3 is linked to a label. In some embodiments, if reagent 1 is immobilized on a solid support, reagent 3 is immobilized on a solid support; in some embodiments, the detection methods can also be used to detect the presence or absence of total antibodies in a sample.

In some embodiments, the invention relates to a detection assembly comprising:

(b) the reagent 1 according to any of the above embodiments;

(c) the reagent 2 according to any of the above embodiments.

In some embodiments, the invention also relates to a chromatography assembly comprising: the device comprises a sample pad, a combination pad, a reaction membrane and an absorption pad, wherein a detection area is arranged on the reaction membrane; the chromatography assembly further comprises:

(b) the reagent 1 according to any of the above embodiments;

(c) the reagent 2 according to any of the above embodiments.

In some embodiments, the reagent 1 is immobilized in a detection zone and the reagent 2 is attached to a label and disposed on a conjugate pad. In some embodiments, the reagent 1 is linked to a label and disposed on a conjugate pad, and the reagent 2 is immobilized on a detection zone.

In some embodiments, the chromatography assembly further comprises (d) a reagent 3 as described in any of the above embodiments; the reagent 3 is fixed in the detection zone; in some embodiments, the ligand-containing fragment is disposed on a sample pad.

The invention also relates to the use of a detection method according to any of the above embodiments, a detection module according to any of the above embodiments, or a chromatographic module according to any of the above embodiments in antibody detection or in the preparation of an antibody detection reagent.

The principle of the scheme of the invention is described by taking the embodiment 1 of the invention as an example. Those skilled in the art will understand that the extended solution is still within the principle scope and is within the concept of the invention. And (2) contacting the RBD protein polymer with a positive sample of neutralizing antibody, wherein the RBD protein polymer can react with total RBD protein antibodies (including RBD neutralizing antibody) in the sample, further chromatographically combine with the RBD neutralizing antibody labeled with colloidal gold, and further chromatographically contact with ACE2 to generate achromatization.

Herein, 1 and 2 in the reagents 1 and 2 are not limited to the order, and are provided for convenience of description.

Aspects and embodiments of the present application will be discussed with reference to 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.

Example 1

1. Preparation of colloidal gold

The colloidal gold solution was prepared by the following method: preparing chloroauric acid into 1% solution, adding the solution with the final concentration of four parts per million into boiled purified water, continuously boiling for 3min, adding 580 μ L of 0.1M sodium citrate into each 100mL chloroauric acid solution, continuously stirring and heating for 10min, cooling to room temperature to obtain colloidal gold solution, and storing at 2-8 deg.C for later use.

2. Colloidal gold labeling

Using the SARS-CoV-2S protein RBD antibody (code No. G3, available from Guangdong Fengpo Bio Inc.) which is able to compete with human ACE2, the procedure for the attachment of colloidal gold was as follows: adding K to the colloidal gold solution₂CO₃Adjusting the pH to 8.0, adding G3 to the solution with the adjusted pH to ensure that the protein content is 10 mu G/mL, coupling for 5min, and adding 10% BSA to the solution to stop the coupling reaction; centrifuging, removing supernatant, dissolving precipitate again, and storing at 2-8 deg.C.

3. Preparation of the conjugate pad

The G3 solution with the connected colloidal gold is diluted according to the dilution ratio of 10 percent, is spread on glass fiber and is placed in a drying room at 37 ℃ for drying overnight, thus obtaining the combined pad.

4. Preparation of reaction membranes

T-shaped wire wrapping: human ACE2 (from Guangdong Fengcong biological Co., Ltd., Ag2) was diluted to 0.8mg/mL, coated on a nitrocellulose membrane using a gold spraying synoviograph, and dried in an oven at 37 ℃ for more than 2h to obtain a reaction membrane.

5. Assembly

And overlapping and assembling the sample pad, the combination pad, the reaction membrane and the water absorption pad on a bottom plate in sequence, cutting into 2.7mm, and preparing into corresponding chromatography components.

6. Detection of

The sample was premixed with RBD poly antigen (accession number Ag29, from Guangdong Fengpo Bio Inc.) at a final concentration of 1. mu.g/mL for 30min and 60. mu.L of the sample was loaded.

7. Interpretation of results

The results are recorded as colloidal gold card readings:

when the reactivity of the T line of the sample to be detected is lower than the color development of the T line of the control sample by more than 1C, the sample to be detected contains the neutralizing antibody, and when the difference of the T line color card reading value is larger than that of the control sample, the content of the new crown neutralizing antibody in the sample to be detected is higher.

And when the reactivity of the T line of the sample to be detected is consistent with the color development depth of the T line of the control sample, indicating that no new crown neutralizing antibody exists in the sample to be detected.

The control sample is a sample without neutralizing antibody or quality control.

Example 2

1. Preparation of colloidal gold

2. Colloidal gold labeling

The method for connecting the SARS-CoV-2S protein RBD antibody G3 with the colloidal gold comprises the following steps: adding K to the colloidal gold solution₂CO₃Adjusting the pH to 8.0, adding G3 to the solution with the adjusted pH to ensure that the protein content is 10 mu G/mL, coupling for 5min, and adding 10% BSA to the solution to stop the coupling reaction; centrifuging, removing supernatant, dissolving precipitate again, and storing at 2-8 deg.C.

3. Preparation of the conjugate pad

4. Preparation of reaction membranes

T-shaped wire wrapping: diluting human ACE2 protein Ag2 to 1.0mg/mL, coating the diluted solution on a nitrocellulose membrane by using a gold spraying synovium instrument, and placing the nitrocellulose membrane in an oven at 37 ℃ for drying for more than 2h to obtain the reaction membrane.

5. Sample pad treatment

The RBD poly antigen Ag29 was diluted to 1. mu.g/mL with 1XPBST, spread on a sample pad, and dried at 37 ℃ for 1 h.

6. Assembly

7. Detection of

60 μ L of sample was applied.

8. Interpretation of results

The results are recorded as colloidal gold card readings:

Example 3

1. Preparation of colloidal gold

2. Colloidal gold labeling

The method adopts human ACE2 protein Ag2, and comprises the following steps of: adding K to the colloidal gold solution₂CO₃Adjusting the pH value to 8.0, adding Ag2 into the solution with the adjusted pH value to ensure that the protein content is 10 mu g/mL, coupling for 5min, and adding 10% BSA to stop the coupling reaction; centrifuging, removing supernatant, dissolving precipitate again, and storing at 2-8 deg.C.

3. Preparation of the conjugate pad

The Ag2 solution connected with the colloidal gold is diluted according to the dilution ratio of 10 percent, is spread on glass fiber, and is placed in a drying room at 37 ℃ for drying overnight, thus obtaining the bonding pad.

4. Preparation of reaction membranes

T-shaped wire wrapping: diluting the SARS-CoV-2S protein RBD antibody G3 to 0.8mg/mL, coating on a nitrocellulose membrane by using a gold spraying synoviograph, and placing in an oven at 37 ℃ for drying for more than 2h to obtain the reaction membrane.

5. Assembly

6. Detection of

The sample was premixed with RBD poly antigen Ag29 at a final concentration of 1. mu.g/mL for 30min, and 60. mu.L of sample was applied.

7. Interpretation of results

The results are recorded as colloidal gold card readings:

Comparative example 1

1. Colloidal gold labeling

Colloidal gold was prepared as in example 1. Labeling RBD antigen (number Ag26, from Guangdong Fengpo biology, Inc.), adding K2CO3 into colloidal gold solution, adjusting pH to 7.5, adding Ag26 into the solution with adjusted pH to enable the protein content to be 10 mu g/mL, coupling for 5min, and adding 10% BSA to stop the coupling reaction; centrifuging, removing supernatant, dissolving precipitate again, and storing at 2-8 deg.C.

2. Preparation of the conjugate pad

The Ag26 solution connected with the colloidal gold is diluted according to the dilution ratio of 10 percent, is spread on glass fiber, and is placed in a drying room at 37 ℃ for drying overnight, thus obtaining the bonding pad.

3. Preparation of reaction membranes

T-shaped wire wrapping: diluting human ACE2 protein Ag2 to 0.8mg/mL, coating the diluted solution on a nitrocellulose membrane by using a gold spraying synovium instrument, and placing the nitrocellulose membrane in an oven at 37 ℃ for drying for more than 2h to obtain the reaction membrane.

4. Assembly

5. Detection of

60 μ L of sample was applied.

6. Interpretation of results

The results are recorded as colloidal gold card readings:

Example 4

1. Preparation of colloidal gold

2. Colloidal gold labeling

Using a SARS-CoV-2S protein RBD antibody (accession number Ab7, available from Guangdong Fengpo Bio Inc.) capable of competing with human ACE2, the procedure for attaching colloidal gold was as follows: adding K to the colloidal gold solution₂CO₃Adjusting the pH to 8.0, adding Ab7 to the solution with the adjusted pH to ensure that the protein content is 10 mu g/mL, coupling for 5min, and adding 10% BSA to the solution to stop the coupling reaction; centrifuging, removing supernatant, dissolving precipitate again, and storing at 2-8 deg.C.

3. Preparation of the conjugate pad

The Ab7 solution connected with the colloidal gold is diluted according to the dilution ratio of 10 percent, is spread on glass fiber, and is placed in a drying room at 37 ℃ to be dried overnight, thus obtaining the bonding pad.

4. Preparation of reaction membranes

T1 coil coating: diluting the anti-human IgG antibody to 1.0mg/mL, and coating the anti-human IgG antibody on a nitrocellulose membrane by using a gold spraying synoviograph; t2 coil coating: human ACE2 (code ACE2-Ag13, from Guangdong Fengcong biological Co., Ltd.) was diluted to 1.0mg/mL, coated on a nitrocellulose membrane using a gold spraying synoviograph, and dried in an oven at 37 ℃ for more than 2 hours to obtain a reaction membrane. The T1 wire is arranged below the T2 wire, and the T1 wire is close to the combination pad after assembly.

5. Assembly

6. Detection of

The sample was premixed with a final concentration of 1. mu.g/mL of protein S poly antigen (accession number S-Ag21, from Guangdong Fengpo Bio Inc.) for 30min, and 60. mu.L of the sample was loaded.

7. Interpretation of results

The results are recorded as colloidal gold card readings:

when the reactivity of the T2 line of the sample to be detected is lower than the color development of the T2 line of the control sample by more than 1C, the sample to be detected contains the neutralizing antibody, and when the difference of the T2 line color card reading value is larger than that of the control sample, the content of the new crown neutralizing antibody in the sample to be detected is higher.

When the reactivity of the T2 line of the sample to be detected is consistent with the color development depth of the T2 line of the control sample, the fact that no new crown neutralizing antibody exists in the sample to be detected is indicated.

When the T1 line of the sample to be detected is colored, the new corona total antibody exists in the sample to be detected.

The control sample is a sample or quality control without neutralizing antibody and total antibody.

This example enables simultaneous detection of total antibodies and neutralizing antibodies, and the detection sensitivity and detection rate of the neutralizing antibodies are comparable to those of example 1.

Example 5

1. Magnetic particle immobilization

Washing the magnetic particles, suspending the magnetic particles in MES buffer solution, adding carbodiimide (EDAC) for reaction, and placing the mixture on a blood mixing instrument for medium-speed mixing; adding SARS-CoV-2S protein RBD antibody G3, and reacting at room temperature in dark to obtain a coating product; the blocking reaction was terminated by adding a quenching buffer at room temperature with exclusion of light, and G3 immobilized on magnetic particles was collected.

2. Linking acridine labels

Taking human ACE2 protein Ag2 to be resuspended in MES buffer solution, then adding acridinium ester, uniformly mixing, marking, and adding 10% BSA for sealing after the marking reaction is finished; the unconjugated acridinium ester was removed by centrifugation and the ACE2 acridinium label was collected.

3. Detection of

Taking a sample to react with RBD poly antigen Ag29 at 37 ℃, adding G3 and ACE2 acridine markers fixed on magnetic particles to react at 37 ℃, finally cleaning a reaction system, and measuring a luminescent signal. And (4) calculating a result: inhibition rate is 1-sample detection signal/negative quality control detection signal.

Example 6

1. Magnetic particle immobilization

Washing the magnetic particles, suspending the magnetic particles in MES buffer solution, adding carbodiimide (EDAC) for reaction, and placing the mixture on a blood mixing instrument for medium-speed mixing; adding human ACE2 protein Ag2, and reacting at room temperature in a dark place to obtain a coated product; the blocking reaction was terminated by adding a quenching buffer at room temperature with the exclusion of light, and Ag2 immobilized on magnetic particles was collected.

2. Linking acridine labels

Taking a SARS-CoV-2S protein RBD antibody (a serial number G3, can be purchased from Fipeng organisms and competes with ACE 2) and suspending in MES buffer solution, then adding acridine ester, uniformly mixing, then marking, and after the marking reaction is finished, adding 10% BSA for sealing; and centrifuging to remove the unconjugated acridinium ester, and collecting the G3 acridinium marker.

3. Detection of

And (3) taking a sample to react with an RBD poly antigen Ag29, Ag2 fixed on magnetic particles and a G3 acridine marker at 37 ℃, finally cleaning a reaction system, and measuring a luminescent signal. And (4) calculating a result: inhibition rate is 1-sample detection signal/negative quality control detection signal.

Comparative example 2

1. Magnetic particle immobilization

2. Linking acridine labels

Taking RBD antigen Ag26 to be resuspended in MES buffer solution, then adding acridinium ester, uniformly mixing, labeling, and adding 10% BSA for blocking after the labeling reaction is finished; and centrifuging to remove the unconjugated acridinium ester, and collecting the Ag26 acridinium marker.

3. Detection of

Taking a sample to react with an Ag26 acridine marker at 37 ℃, adding Ag2 fixed on magnetic particles to react at 37 ℃, finally cleaning a reaction system, and measuring a luminescent signal. And (4) calculating a result: inhibition rate is 1-sample detection signal/negative quality control detection signal.

According to experimental evaluation, the clinical vaccine specimens can be effectively detected in the examples 5 and 6, and the inhibition efficiency sensitivity is higher than that of the comparative example 2.

Example 7

1. Latex cross-linked RBD neutralizing antibody or ACE2

Suspending polystyrene latex microspheres in MES buffer solution, adding EDC and NHS to activate at 37 ℃, adding CB buffer solution and RBD neutralizing antibody (G3) or human ACE2(Ag2), mixing uniformly, reacting at 37 ℃ for 2-3 hours, sealing with 10% BSA, centrifuging, removing supernatant, washing for 1-2 times, and suspending to obtain the cross-linked latex G3 or Ag 2.

2. Detection of

Uniformly mixing the sample with RBD poly antigen Ag29, reacting for 7.5min, adding crosslinked latex G3, reacting for 6min, reading A1, adding crosslinked latex Ag2, reacting for 10min, reading A2, and calculating delta A ═ A2-A1.

By adopting the method of the embodiment, the negative and positive samples can be effectively distinguished. When a 50ng/mLG3 sample (simulated positive sample) is tested, the inhibition rate can reach 15% -20%, and when a 8000ng/mLG3 sample (simulated positive sample) is tested, the inhibition rate can reach more than 80%.

To facilitate understanding of the present invention, some experimental results are shown below:

experimental results 1

The protocols of examples 1-3 and comparative example 1 were used to simulate the content of neutralizing antibodies in vaccine specimens with RBD neutralizing antibodies, and the results are shown in the following table. In the table, the smaller the color chart reading, the darker the color developed, + the darker the color developed at the same reading, and B the no color developed.

As can be seen from the table, examples 1-3 are all superior to comparative example 1, indicating that the inventive protocol has higher detection sensitivity.

Experimental results 2

The results of the measurement at different temperatures and times with the protocol of example 1, using RBD neutralizing antibodies to simulate the content of neutralizing antibodies in the vaccine specimen, are shown in the table below. In the table, the smaller the color chart reading, the darker the color developed, + the darker the color developed at the same reading, and B the no color developed.

As can be seen from the table, the protocol of the present invention has high stability and wide detection time.

Experimental results 3

The results of the detection of neutralizing antibodies using the protocol of example 1 on 5 vaccinated specimens are shown in the table below. In the table, the smaller the color chart reading, the darker the color developed, + the darker the color developed at the same reading, B the no color developed, P the positive, N the negative. And comparing with a certain neutralizing antibody detection kit of the EUA.

As can be seen from the table, the scheme of the invention has high detection rate on clinical vaccine samples and has more sensitive inhibition effect.

Experimental results 4

The test using the protocols of example 5 and comparative example 2 using negative blood, 2 parts of mixed vaccine serum, and 2 parts of single vaccine serum, respectively, results are shown in the following table:

experimental results 5

The protocol of example 5 and comparative example 2 was used to test with negative dilutions, different concentrations (0.0625. mu.g/ml, 0.3125. mu.g/ml) of neutralizing antibody G3, respectively, with the results shown in the following table:

experimental results 6

The protocol of example 6 and comparative example 2 was used to test the neutralizing antibody G3 at different concentrations (0.0625. mu.g/ml, 0.3125. mu.g/ml) using negative blood, 2 pooled vaccine sera, 1 single vaccine serum, respectively, and the results are shown in the following table:

the technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. The detection method comprises the following steps:

reagent 1: a receptor comprising the ligand;

(2) and detecting the signal.

2. The detection method according to claim 1, wherein the contact manner includes any one of:

3. The detection method according to claim 1, wherein the ligand is a ligand for invasion of a pathogen into a host cell;

optionally, the pathogen is a coronavirus;

optionally, the coronavirus is SARS-CoV-2 or SARS-CoV;

optionally, the ligand is RBD;

optionally, the receptor is ACE 2.

4. The detection method according to claim 1, wherein the ligand-containing fragment comprises a structure which is more than dimeric by treating the fragment;

alternatively, the ligand-containing fragment includes a fragment obtained by expressing a ligand sequence in two or more tandem.

5. The assay of claim 1, wherein the assay is used to detect the presence or absence of neutralizing antibodies in a sample.

6. The detection method according to any one of claims 1 to 5, wherein the sample is further contacted with a reagent 3, wherein the 3 rd reagent comprises a second antibody, and wherein the second antibody is an IgG antibody against the species from which the sample is derived;

optionally, the second antibody is anti-human IgG;

optionally, the reagent 3 is linked to a label or solid support;

alternatively, if reagent 1 is linked to the label, reagent 3 is linked to the label;

alternatively, if reagent 1 is immobilized on a solid support, reagent 3 is immobilized on a solid support;

optionally, the detection method may also be used to detect the presence or absence of total antibodies in a sample.

7. A detection assembly, comprising:

(a) a ligand-containing fragment according to any one of claims 1 to 6;

(b) the reagent 1 of any one of claims 1 to 6;

(c) the reagent 2 according to any one of claims 1 to 6.

8. The test assembly of claim 7, further comprising (d) the reagent 3 of claim 6.

9. A chromatography assembly comprising: the device comprises a sample pad, a combination pad, a reaction membrane and an absorption pad, wherein a detection area is arranged on the reaction membrane; the chromatography assembly further comprises:

(a) a ligand-containing fragment according to any one of claims 1 to 6;

(b) the reagent 1 of any one of claims 1 to 6;

(c) the reagent 2 of any one of claims 1-6;

optionally, the reagent 1 is fixed on the detection zone, and the reagent 2 is connected with a marker and arranged on the binding pad; or; the reagent 1 is connected with a marker and is arranged on the bonding pad, and the reagent 2 is fixed on the detection area;

optionally, the chromatography assembly further comprises (d) the reagent 3 of claim 6; the reagent 3 is fixed in the detection zone;

optionally, the ligand-containing fragment is disposed on a sample pad.

10. Use of the detection method of any one of claims 1 to 6, the detection module of claim 7 or 8, or the chromatographic module of claim 9 for antibody detection or for the preparation of an antibody detection reagent.