CN113219167A

CN113219167A - Dual competition detection method and product

Info

Publication number: CN113219167A
Application number: CN202110477332.9A
Authority: CN
Inventors: 夏良雨; 代双; 李俊; 谢洋洋; 刘江武; 黄慧; 向海军; 郭伟芸; 郝明; 邓盛花
Original assignee: Guangdong Fapon Biotech Co Ltd
Current assignee: Guangdong Fapon Biotech Co Ltd
Priority date: 2021-01-13
Filing date: 2021-04-29
Publication date: 2021-08-06

Abstract

The invention relates to the field of immunodetection, in particular to a detection method and a detection component adopting a dual competition mode and application. The scheme of the invention can be used for detecting the neutralizing antibody, for example, the scheme is applied to detecting the neutralizing antibody of the new coronavirus, and has higher sensitivity and/or detection rate, and particularly, the detection result can cover more complete neutralizing antibody.

Description

Dual competition detection method and product

Technical Field

The invention relates to the field of immunodetection, in particular to a 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.

In 2019, new coronavirus became abusive worldwide. By 2021, 9 days in Beijing, more than 8000 ten thousand of global cumulative infection new crown (COVID-19) cases and 190 ten thousand of death cases still exist in a pandemic stage at present. The frequent sporadic accidents in China and the quick approval of the vaccine on the market are particularly urgent. Some vaccines are currently marketed globally with authorization for emergency use or additional conditions.

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.

Many new neutralizing antibodies have also been isolated by current research, such as those that bind to the SARS-CoV-2 Spike protein RBD but do not compete for ACE2, and those that bind to the S1 subunit NTD region.

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 and sensitivity of detection of neutralizing antibodies.

The existing product mainly detects a neutralizing antibody blocking the interaction of RBD-ACE2 by an RBD-ACE2 competition method, and cannot detect the RBD neutralizing antibody of non-competitive ACE2, an NTD neutralizing antibody and the like.

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 and/or a first antibody that binds the ligand; wherein the first antibody competes with the receptor;

reagent 2: comprising a second antibody that binds to said ligand, wherein said second antibody does not compete with said 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, a method of detection, comprising the steps of:

(1) contacting the sample with a fragment comprising a first ligand and a second ligand, reagent A, reagent B;

reagent A: comprises A1 reagent and/or A2 reagent; wherein the a1 reagent comprises a receptor for the first ligand and/or a third antibody that binds the first ligand; wherein the third antibody competes with the receptor for the first ligand; the a2 reagent comprises a fourth antibody that binds the first ligand, wherein the fourth antibody does not compete with the receptor for the first ligand;

and (3) reagent B: comprises a B1 reagent and/or a B2 reagent; wherein the B1 reagent comprises a receptor for the second ligand and/or a fifth antibody that binds the second ligand; wherein the fifth antibody competes with a receptor for the second ligand; the B2 reagent comprises a sixth antibody that binds the second ligand, wherein the sixth antibody does not compete with the receptor for the second ligand;

wherein one of the reagent A or the reagent B is connected with a marker, and the other is fixed on a solid phase carrier; reagent a is different from reagent B;

(2) and detecting the signal.

In some embodiments, the contacting comprises any one of:

(a) simultaneously contacting the sample with a fragment containing a first ligand and a second ligand, a reagent A and a reagent B;

(b) contacting the sample with a fragment comprising a first ligand and a second ligand, reagent A, and then reagent B;

(c) contacting the sample with a fragment comprising a first ligand and a second ligand, reagent B, and then reagent A;

(d) contacting the sample with a fragment containing a first ligand and a second ligand, and then with a reagent A and a reagent B;

(e) contacting the sample with a fragment comprising a first ligand and a second ligand, followed by reagent A, and followed by reagent B;

(f) the sample is contacted with a fragment comprising the first ligand and the second ligand, followed by reagent B, and then reagent A.

In some embodiments, the antibody is a neutralizing antibody.

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.

In some embodiments, the ligand is selected from RBD, NTD.

In some embodiments, the ligand is selected from the group consisting of RBD of SARS-CoV-2S protein, NTD of SARS-CoV-2S protein.

In some embodiments, the receptor is ACE 2.

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

In some embodiments, the invention also 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, a detection assembly, comprising:

(a) a fragment comprising a first ligand and a second ligand as described in any one of the embodiments above;

(b) agent a according to any one of the embodiments above;

(c) agent B according to any of the preceding embodiments.

In some embodiments, the invention also relates to a detection method as described in any one of the above embodiments, or use of a detection module as described in any one of the above embodiments in antibody detection or preparation of an antibody detection reagent.

Drawings

FIG. 1: the schematic diagram of the present invention is described in example 2, wherein M represents a solid phase carrier, NTD-NAb represents an NTD neutralizing antibody, and RBD-NAb represents an RBD neutralizing antibody.

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, microspheres; 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", "first ligand-and second ligand-containing fragment" is understood to mean a protein or polypeptide comprising the corresponding ligand sequence.

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 and/or detection rate in antibody detection, especially in neutralizing antibody detection, and the detection result covers more complete neutralizing antibody. The invention has universality, is not limited to a specific immunoassay platform and is not limited to a specific species.

reagent 1: a receptor comprising the ligand and/or a first antibody that binds the ligand; wherein the first antibody competes with the receptor; reagent 2: comprising a second antibody that binds to said ligand, wherein said second antibody does not compete with said 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: 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, agent 1 comprises a receptor for the ligand, agent 2 comprises a second antibody that binds the ligand; wherein the receptor in reagent 1 is linked to a label and the second antibody in reagent 2 is immobilized on a solid support, or the receptor in reagent 1 is immobilized on a solid support and the second antibody in reagent 2 is linked to a label. In some embodiments, reagent 1 comprises a first antibody that binds to the ligand, and reagent 2 comprises a second antibody that binds to the ligand; wherein the first antibody in reagent 1 is linked to a label and the second antibody in reagent 2 is immobilized on a solid support, or the first antibody in reagent 1 is immobilized on a solid support and the second antibody in reagent 2 is linked to a label. In some embodiments, reagent 1 comprises a receptor for the ligand and a first antibody that binds the ligand, and reagent 2 comprises a second antibody that binds the ligand; wherein, the receptor and the first antibody in the reagent 1 are both connected with a label, and the second antibody in the reagent 2 is fixed on a solid phase carrier, or, the receptor and the first antibody in the reagent 1 are both fixed on a solid phase carrier, and the second antibody in the reagent 2 is connected with a label.

In some embodiments, a method of detection, comprising the steps of:

(2) and detecting the signal.

In some embodiments, the contacting comprises: simultaneously contacting the sample with a fragment containing a first ligand and a second ligand, a reagent A and a reagent B; in some embodiments, the contacting comprises: contacting the sample with a fragment comprising a first ligand and a second ligand, reagent A, and then reagent B; in some embodiments, the contacting comprises: contacting the sample with a fragment comprising a first ligand and a second ligand, reagent B, and then reagent A; in some embodiments, the contacting comprises: contacting the sample with a fragment containing a first ligand and a second ligand, and then with a reagent A and a reagent B; in some embodiments, the contacting comprises: contacting the sample with a fragment comprising a first ligand and a second ligand, followed by reagent A, and followed by reagent B; in some embodiments, the contacting comprises: the sample is contacted with a fragment comprising the first ligand and the second ligand, followed by reagent B, and then reagent A. In some embodiments, and so on, when more ligands are introduced, such as a third ligand, one skilled in the art will appreciate that the above steps or reagents may still be included in the methods or reagents. For example, an agent for a third ligand can be introduced into the A agent or the B agent.

In some embodiments, the fragment comprising the first ligand and the second ligand may be obtained recombinantly or by polymerization, e.g., by expressing the first ligand and the second ligand recombinantly in tandem. In some embodiments, the fragment containing the first ligand may also be polymerized with the fragment containing the second ligand, and the polymerization may be performed by physical or chemical means, such that one molecule thereof contains multiple ligand structures.

In some embodiments, the antibody is a neutralizing antibody.

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. The SARS-CoV-2 includes SARS-CoV-2 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 selected from RBD, NTD. Wherein, the RBD is a Receptor Binding Domain (Receptor Binding Domain) which includes the RBD and its core region, and as long as the function of Binding to the Receptor can be achieved, the RBD according to the present invention should still be understood. NTD is an N-terminal domain. In some embodiments, the ligand is selected from the group consisting of RBD of SARS-CoV-2S protein, NTD of SARS-CoV-2S protein. In some embodiments, the fragment containing the RBD of the SARS-CoV-2S protein can be the S protein and fragments thereof that encompass the RBD, e.g., the S protein reference Gene ID: 43740568. 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, the ligand-containing fragment can be the RBD, S1 protein, or the S protein of the SARS-CoV-2S protein. In some embodiments, the sequence of the NTD of the SARS-CoV-2S protein can be found in A neutral antibody binding to the N-terminal domain of the spike protein of SARS-CoV-2, 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 have the function of ligand binding to a receptor and/or antibody. In some embodiments, the fragments comprising the SARS-CoV-2S protein NTD and RBD may be the S1 protein or the S protein.

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 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 detection method is used to detect the presence or absence of neutralizing antibodies in a sample. In some embodiments, a method of detecting a neutralizing antibody, comprising the steps of: (1) contacting the sample with a fragment containing an RBD, ACE2, and an RBD neutralizing antibody that does not compete with ACE2, wherein ACE2 is linked to a label, and the RBD neutralizing antibody that does not compete with ACE2 is immobilized on a solid support, and (2) detecting a signal. In some embodiments, a method of detecting a neutralizing antibody, comprising the steps of: (1) contacting the sample with a fragment containing an RBD, an RBD neutralizing antibody that competes with ACE2, and an RBD neutralizing antibody that does not compete with ACE2, wherein the RBD antibody that competes with ACE2 is immobilized on a solid support, and the RBD antibody that does not compete with ACE2 is linked to a label, and (2) detecting a signal. In some embodiments, a method of detecting a neutralizing antibody, comprising the steps of: (1) contacting the sample with a fragment containing RBD and NTD (such as S1 fragment or S fragment), ACE2, and an NTD neutralizing antibody competing with a receptor of NTD, wherein ACE2 is linked to a label, and the NTD antibody competing with the receptor of NTD is immobilized on a solid phase carrier, and (2) detecting a signal. In some embodiments, a method of detecting a neutralizing antibody, comprising the steps of: (1) contacting the sample with a fragment containing RBD and NTD (e.g., S1 fragment or S fragment), ACE2, an NTD neutralizing antibody that competes with a receptor for NTD, and an RBD neutralizing antibody that does not compete with ACE2, wherein the ACE2 is linked to a label, and the NTD antibody that competes with a receptor for NTD and the RBD neutralizing antibody that does not compete with ACE2 are immobilized on a solid support, respectively, (2) detecting a signal.

(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, a detection assembly, comprising:

(b) agent a according to any one of the embodiments above;

(c) agent B according to any of the preceding embodiments.

By "module" is herein understood any form of an immunological product, such as a kit, chromatographic strip, reagent card, chip, etc. conventional forms of immunological products.

The invention also relates to the detection method of any one of the above embodiments, or the application of the detection assembly of any one of the above embodiments in antibody detection or preparation of an antibody detection reagent.

The principle of the scheme of the present invention is described by taking the embodiment 2 of the present invention as an example (see fig. 1). 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. Contacting the S1 protein with a positive sample, reacting the S1 protein with various antibodies in the sample, such as total antibodies and neutralizing antibodies including NTD, total antibodies and neutralizing antibodies of RBD, and other total antibodies and neutralizing antibodies, further contacting with NTD neutralizing antibodies in a reagent, generating an equivalent competition relationship with the level of the neutralizing antibodies of NTD in the sample, further contacting with ACE2 in the reagent, generating an equivalent competition relationship with the level of the neutralizing antibodies of RBD in the sample, and finally generating a signal, thereby realizing the addition competition of double neutralizing epitopes, and identifying the sum of the level of the neutralizing antibodies of NTD and the level of the neutralizing antibodies of RBD in the sample.

The principle of the scheme of the invention is described by taking the embodiment 1 of the invention as an example. The RBD protein is contacted with a positive sample, the RBD protein reacts with a plurality of antibodies in the sample, such as total antibodies including RBD, RBD neutralizing antibodies competing for ACE2, RBD neutralizing antibodies competing for ACE2, RBD neutralizing antibodies further contacting with RBD neutralizing antibodies competing for ACE2 in a reagent, generating equivalent competition with the RBD neutralizing antibodies of competing for ACE2 in the sample, further contacting with ACE2, generating equivalent competition with the level of the RBD neutralizing antibodies competing for ACE2 in the sample, and finally generating signals, thereby realizing double competition and detecting the full RBD neutralizing antibodies.

The numerical descriptions of the first, second, … …, reagent 1, reagent 2, etc. in the present invention are not limited to the order, but are provided for convenience of description.

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.

Example 1

1. Magnetic particle immobilization

Mixing 1 μ M magnetic particles with 0.1M MES buffer solution with pH of 5.0, mixing, placing on a magnetic separator until the supernatant is not turbid, removing the supernatant, and collecting magnetic particles; adding MES buffer solution into the magnetic particles to fully suspend the magnetic beads, separating and re-suspending according to the method; adding 10mg/ml carbodiimide (EDAC) into the washed magnetic particles, reacting at 25 deg.C for 30min, placing on a blood mixing machine, and mixing at medium speed; adding RBD neutralizing antibody (No. S452, obtained by screening recovered peripheral blood of SARS-CoV-2 infected person by single cell PCR technology and available from Guangdong Fengpo biology Co., Ltd.) which does not compete with ACE2, and reacting at 25 deg.C in dark for 3h to obtain coated product; quenching buffer (10mM PBS, 1g/100ml BSA, 40mM glycine, pH 7.4) was added to stop the blocking reaction by keeping away from light at 25 ℃ for 1h, and S452 immobilized on magnetic microparticles was collected.

2. Linking acridine labels

Taking human ACE2 (purchased from Guangdong Fengcong biological limited company, number Ag2), ultrafiltering and centrifuging, replacing with 0.1M MES buffer solution with pH of 5.0, centrifuging, adding acridine ester, mixing uniformly, labeling, and labeling at 37 ℃ for 4 h; after the labeling reaction is finished, adding a blocking buffer (10% BSA buffer with the pH of 8.0 is used as a blocking buffer), and blocking for 1 h; after blocking, the solution was centrifuged by ultrafiltration to remove the unconjugated acridinium ester and collect the ACE2 acridinium label.

3. Detection of

50 mu L of sample is taken to react with 50 mu L of SARS-COV-2S protein RBD (40ng/mL, purchased from Guangdong Fengpo biology Co., Ltd., number Ag27) at 37 ℃ for 30min, then S4520.25mg/mL fixed on magnetic particles and ACE2 acridine marker 1 mu g/mL are added to react at 37 ℃ for 15min, and finally the reaction system is cleaned and the luminescent signal is measured. And (4) calculating a result: inhibition rate is 1-sample detection signal/negative quality control detection signal.

Example 2

1. Magnetic particle immobilization

Mixing 1 μ M magnetic particles with 0.1M MES buffer solution with pH of 5.0, mixing, placing on a magnetic separator until the supernatant is not turbid, removing the supernatant, and collecting magnetic particles; adding MES buffer solution into the magnetic particles to fully suspend the magnetic beads, separating and re-suspending according to the method; adding 10mg/ml carbodiimide (EDAC) into the washed magnetic particles, reacting at 25 deg.C for 30min, placing on a blood mixing machine, and mixing at medium speed; adding NTD neutralizing antibody (number 3F6), and reacting at 25 deg.C in dark for 3h to obtain coated product; the blocking reaction was terminated by adding a quenching buffer (10mM PBS, 1g/100ml BSA, 40mM glycine, pH 7.4) and reacting at 25 ℃ with light for 1h, and 3F6 immobilized on magnetic microparticles was collected. Wherein, the NTD neutralizing antibody can be obtained by screening peripheral blood of SARS-CoV-2 infected person by single cell PCR technology, can be purchased from Guangdong Fengcong biological Limited company, and can also be prepared by referring to the sequences of GenBank: MT712309.1(L chain) and GenBank: MT712296.1(H chain).

2. Linking acridine labels

Taking human ACE2 (serial number Ag2), ultrafiltering and centrifuging, replacing with 0.1M MES buffer solution with pH of 5.0, centrifuging, adding acridine ester, uniformly mixing, labeling, and labeling at 37 ℃ for 4 h; after the labeling reaction is finished, adding a blocking buffer (10% BSA buffer with the pH of 8.0 is used as a blocking buffer), and blocking for 1 h; after blocking, the solution was centrifuged by ultrafiltration to remove the unconjugated acridinium ester and collect the ACE2 acridinium label.

3. Detection of

50 mu L of sample is taken to react with 50 mu L of SARS-COV-2S1 protein (40ng/mL, purchased from Guangdong Fengpeng biological Co., Ltd., Ag40) at 37 ℃ for 30min, then 3 F60.25mg/mL fixed on magnetic particles and 1 mu g/mL of ACE2 acridine marker are added to react for 15min at 37 ℃, and finally the reaction system is cleaned and the luminescent signal is measured. And (4) calculating a result: inhibition rate is 1-sample detection signal/negative quality control detection signal.

Example 3

1. Magnetic particle immobilization

Mixing 1 μ M magnetic particles with 0.1M MES buffer solution with pH of 5.0, mixing, placing on a magnetic separator until the supernatant is not turbid, removing the supernatant, and collecting magnetic particles; adding MES buffer solution into the magnetic particles to fully suspend the magnetic beads, separating and re-suspending according to the method; adding 10mg/ml carbodiimide (EDAC) into the washed magnetic particles, reacting at 25 deg.C for 30min, placing on a blood mixing machine, and mixing at medium speed; adding an NTD neutralizing antibody (number 3F6) and an RBD neutralizing antibody (number S452) which does not compete with ACE2 in an equal molar ratio, and reacting at 25 ℃ in a dark place for 3h to obtain a coated product; quenching buffer (10mM PBS, 1g/100ml BSA, 40mM glycine, pH 7.4) was added to stop the blocking reaction by keeping away from light at 25 ℃ for 1h, and S452 and 3F6 immobilized on magnetic microparticles were collected.

2. Linking acridine labels

3. Detection of

Taking 50 mu L of sample to react with 50 mu L of SARS-COV-2S1 protein (40ng/mL, Ag40) at 37 ℃ for 30min, then adding S452 and 3 F60.25mg/mL fixed on magnetic particles and ACE2 acridine marker at 1 mu g/mL to react at 37 ℃ for 15min, finally cleaning the reaction system and measuring luminescent signals. And (4) calculating a result: inhibition rate is 1-sample detection signal/negative quality control detection signal.

Example 4

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 ACE2 (purchased from Guangdong Fengcong biological Co., Ltd.), and reacting at room temperature in dark to obtain a coated product; the blocking reaction was terminated by adding a quenching buffer at room temperature away from light, and ACE2 immobilized on magnetic microparticles was collected.

2. Linking acridine labels

Taking an NTD neutralizing antibody 3F6 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; the unconjugated acridinium ester was removed by centrifugation and the 3F6 acridinium label was collected.

3. Detection of

Taking a sample to react with SARS-COV-2S1 protein Ag40 at 37 ℃, then reacting with 3F6 acridine marker at 37 ℃, then reacting with ACE2 fixed on magnetic particles 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.

Through detection evaluation, the inhibition rate of the example 4 is basically equivalent to that of the example 2, and the NTD neutralizing antibody and the RBD neutralizing antibody can be detected, and the effect of improving the sensitivity is achieved.

Example 5

The present example differs from example 1 in that detection: taking 50 mu L of sample, reacting with 50 mu L of SARS-COV-2S protein RBD (40ng/mL, purchased from Guangdong Fengpo biology Co., Ltd., number Ag27) and S4520.25mg/mL fixed on magnetic particles at 37 ℃ for 30min, adding 1 mu g/mL of ACE2 acridine marker, reacting at 37 ℃ for 15min, finally cleaning the reaction system, and measuring a luminescent signal. And (4) calculating a result: inhibition rate is 1-sample detection signal/negative quality control detection signal.

Through detection evaluation, the detection inhibition rate of the example 5 is basically equivalent to that of the example 1, RBD neutralizing antibodies of non-competitive ACE2 can be detected, and the effect of improving sensitivity is achieved.

Example 6

1. Preparation of colloidal gold

The colloidal gold solution was prepared by the following method: adding 1% chloroauric acid solution with final concentration of four ten-thousandths into boiled purified water, boiling for 3min, adding 0.1M sodium citrate (580 μ l per 100ml chloroauric acid solution), stirring and heating for 10min, cooling to room temperature to obtain colloidal gold solution, and storing at 2-8 deg.C.

2. Colloidal gold labeling

The method adopts human ACE2 protein Ag2, and comprises the following steps of: adding K to the colloidal gold solution₂CO₃Adding Ag2 into the solution with the adjusted pH value, coupling for 5min,to this was added 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: the NTD neutralizing antibody 3F6 is diluted to 1.0mg/ml, coated on a nitrocellulose membrane by using a gold spraying synoviograph, and placed in an oven for drying at 37 ℃ for more than 2h to obtain the reaction membrane.

5. Sample pad treatment

SARS-COV-2S protein (purchased from Guangdong Fengcong biological Co., Ltd.) was diluted to 1ug/ml with 1XPBST, spread on a sample pad, and dried at 37 ℃ for 1 h.

6. 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.

7. Detection of

The sample to be tested is loaded onto the sample pad.

Example 7

1. Preparation of colloidal gold

2. Colloidal gold labeling

The RBD neutralizing antibody S452 is adopted, and the steps of connecting colloidal gold are as follows: adding K2CO3 into the colloidal gold solution, adding S452 into the solution with the adjusted pH value, 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 S452 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, so as to obtain the combined 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 for drying at 37 ℃ for more than 2h to obtain the reaction membrane.

5. Assembly

6. Detection of

The sample to be tested was premixed with SARS-COV-2S protein (purchased from Guangdong Fengcong Bio Inc.) and loaded onto a sample pad.

Through detection evaluation, both example 6 and example 7 are superior to comparative example 2, a more comprehensive detection of neutralizing antibodies can be detected, while comparative example 2 cannot detect RBD neutralizing antibodies and NTD neutralizing antibodies of non-competitive ACE2, and the embodiment scheme has higher sensitivity.

Example 8

The present example differs from example 1 in that detection: taking 50 mu L of sample, simultaneously reacting with RBD protein Ag27, S452 fixed on magnetic particles and ACE2 acridine marker at 37 ℃, finally cleaning the reaction system, and measuring luminescent signals. And (4) calculating a result: inhibition rate is 1-sample detection signal/negative quality control detection signal.

Through detection evaluation, the detection inhibition rate of the example 8 is basically equivalent to that of the example 1, and the RBD neutralizing antibody of the non-competitive ACE2 can be detected, and the effect of improving the sensitivity is achieved.

Comparative example 1

1. Magnetic particle immobilization

Mixing 1 μ M magnetic particles with 0.1M MES buffer solution with pH of 5.0, mixing, placing on a magnetic separator until the supernatant is not turbid, removing the supernatant, and collecting magnetic particles; adding MES buffer solution into the magnetic particles to fully suspend the magnetic beads, separating and re-suspending according to the method; adding 10mg/ml carbodiimide (EDAC) into the washed magnetic particles, reacting at 25 deg.C for 30min, placing on a blood mixing machine, and mixing at medium speed; adding human ACE2 (number Ag2), and reacting at 25 deg.C in dark for 3h to obtain coated product; the blocking reaction was stopped by adding a quenching buffer (10mM PBS, 1g/100ml BSA, 40mM glycine, pH 7.4) and protecting from light at 25 ℃ for 1h, and the ACE2 immobilized on magnetic microparticles was collected.

2. Linking acridine labels

Taking SARS-COV-2S protein RBD (number Ag27), ultrafiltering, centrifuging, replacing with 0.1M MES buffer solution with pH of 5.0, centrifuging, adding acridine ester, mixing, labeling, and labeling at 37 deg.C for 4 hr; after the labeling reaction is finished, adding a blocking buffer (10% BSA buffer with the pH of 8.0 is used as a blocking buffer), and blocking for 1 h; and (4) performing ultrafiltration and centrifugation after blocking to remove unconjugated acridinium ester, and collecting the RBD acridinium marker.

3. Detection of

Taking 50 mu L of sample and 50 mu L of RBD acridine marker (40ng/mL) to react for 15min at 37 ℃, adding ACE20.25mg/mL fixed on magnetic particles to react for 15min at 37 ℃, finally cleaning the 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. Colloidal gold labeling

Colloidal gold was prepared as in example 1. Labeling RBD antigen (accession number Ag26, from Guangdong Fengcong biological Co., Ltd.) with gold, and adding K to the colloidal gold solution₂CO₃Adjusting the pH value to 7.5, adding Ag26 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.

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

The sample to be tested is loaded onto the sample pad.

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

experimental example 1

The quality control product (as a negative sample), the neutralizing antibody of non-competitive ACE2 in the S452 simulated sample, and the neutralizing antibody of competitive ACE2 in the RBD neutralizing antibody (Guangdong Fengcong biological limited, No. G3) simulated sample were used, and the quality control product and the simulating antibody were calibrated by a pseudovirus neutralization test. Methods for the Neutralization of pseudoviruses reference the Protocol and Reagents for pseudoviral viral Particles with SARS-CoV-2 Spike Protein for neural lysis Assays.

The results of the tests carried out using the method and reagents of example 1 and comparative example 1, respectively, are shown in the following table:

the experimental data show that comparative example 1 can only detect the neutralizing antibody G3 blocking ACE2 binding, while the inventive protocol can simultaneously detect the neutralizing antibody S452 on RBD that does not compete for ACE2 binding, similar to a pseudovirus, suggesting that more neutralizing antibodies can be detected. In addition, compared with comparative example 1, the detection inhibition rate of the scheme of the invention on the neutralizing antibody G3 of the blocking type is improved from 25.9% to 47.6%, and the detection inhibition rate on the mixed sample of G3 and S452 is improved from 26.8% to 68.4%, which indicates higher detection sensitivity.

Experimental example 2

The quality control product (as a negative sample), 3F6 simulation sample NTD neutralizing antibody, G3 simulation sample ACE2 competing neutralizing antibody, the quality control and simulation antibodies through the false virus neutralization test for calibration.

The results of the tests carried out using the method and reagents of example 2 and comparative example 1, respectively, are shown in the following table:

the experimental data show that comparative example 1 can only detect the neutralizing antibody G3 blocking the binding of ACE2, while the experimental protocol can detect the neutralizing antibody 3F6 in the NTD region simultaneously, similar to the pseudovirus, suggesting that more neutralizing antibodies can be detected. In addition, compared with the prior art, the test scheme has the advantages that the detection inhibition rate of the test scheme on the blocking type neutralizing antibody G3 is improved from 25.9% to 46.9%, and the detection inhibition rate of the test scheme on the mixed sample of G3 and 3F6 is improved from 27% to 69.9%, so that higher detection sensitivity is prompted.

Experimental example 3

The quality control sample (as a negative sample), 3F6 and S452 are adopted to simulate NTD neutralizing antibodies in the sample, neutralizing antibodies of non-competitive ACE2 in the sample, G3 (Guangdong Fengcong biological limited, the product number ncov-PS-GonABG3) and neutralizing antibodies of competitive ACE2 in the sample are simulated, and the quality control and the simulating antibodies are calibrated through a pseudovirus neutralizing test.

The results of the tests carried out using the method and reagents of example 3 and comparative example 1, respectively, are shown in the following table:

the experimental data show that comparative example 1 can only detect the neutralizing antibody G3 blocking ACE2 binding, while the scheme of the present invention can simultaneously detect the neutralizing antibody S452 binding to non-competitive ACE2 on RBD and the neutralizing antibody 3F6 in NTD region, similar to pseudoviruses, suggesting that more neutralizing antibodies can be detected. In addition, compared with comparative example 1, the detection inhibition rate of the scheme of the invention on the neutralizing antibody G3 of the blocking type is improved from 25.9% to 47.4%, and the detection inhibition rate on the mixed sample of G3, S452 and 3F6 is improved from 26.9% to 86.2%, which indicates higher detection sensitivity.

The other contact modes are adopted to process the sample, the other steps are respectively the same as the embodiment 1, the embodiment 2 or the embodiment 3, and the obtained sensitivity and the detection rate are basically equivalent to the corresponding embodiment.

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:

(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 comprises the following steps:

(2) and detecting the signal.

4. The detection method according to claim 3, wherein the contact manner includes any one of:

5. The detection method according to any one of claims 1 to 4, wherein the antibody is a neutralizing antibody.

6. The detection method according to any one of claims 1 to 4, wherein the ligand is a ligand for invasion of a host cell by a pathogen;

optionally, the pathogen is a coronavirus;

optionally, the coronavirus is SARS-CoV-2;

optionally, the ligand is selected from RBD, NTD;

optionally, the ligand is selected from the group consisting of RBD of SARS-CoV-2S protein, NTD of SARS-CoV-2S protein;

optionally, the receptor is ACE 2.

7. The assay of any one of claims 1 to 4 wherein the assay is for detecting the presence or absence of neutralising antibodies in a sample.

8. A detection assembly, comprising:

(a) a ligand-containing fragment according to any one of claims 1-2, 5-7;

(b) the reagent 1 of any one of claims 1-2, 5-7;

(c) the reagent 2 according to any one of claims 1 to 2 and 5 to 7.

9. A detection assembly, comprising:

(a) a fragment containing a first ligand and a second ligand according to any one of claims 3 to 7;

(b) an agent A according to any one of claims 3 to 7;

(c) agent B according to any one of claims 3 to 7.

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