WO2022151596A1 - Neutralizing antibody high-sensitivity detection method and product - Google Patents

Abstract

The present invention provides an antibody detection method and product. The detection method comprises the steps of: enabling a sample to be contact with a ligand-containing fragment, a reagent 1, and a reagent 2, wherein of the reagent 1 and the reagent 2, one is connected to a marker, and the other is fixed on a solid-phase carrier. The detection method and product of the present invention can be applied in neutralizing antibody detection and total antibody detection.

Description

Neutralizing antibody high-sensitivity detection method and product

CROSS-REFERENCE TO RELATED APPLICATIONS

The disclosure requires application numbers of 202110044189.4 (the application date is January 13, 2021, and the invention name is "neutralizing antibody high-sensitivity detection method and product") and 202110104780.4 (the application date is January 26, 2021, and the invention name is "China Antibody High Sensitivity Detection Method and Product"). and Antibody High Sensitivity Detection Methods and Products"), the entire contents of which are incorporated herein by reference.

technical field

The present disclosure relates to the field of antibody detection, in particular, to antibody detection methods and products.

Background technique

Receptor-ligand binding is one of the channels to achieve signal transduction, and receptors can recognize ligands and specifically bind to ligands. Taking the invasion of host cells by pathogens as an example, ligands on the surface of pathogens can bind to receptors on host cells, opening the door to infect host cells.

In 2019, the novel coronavirus raged around the world. As of January 9, 2021, Beijing time, the cumulative number of cases of infection with the new coronavirus (SARS-CoV-2, officially known as COVID-19) in the world has exceeded 80 million, and the number of deaths has exceeded 1.9 million. It is still in the pandemic stage. With the frequent occurrence of emergencies in China, the rapid approval and marketing of vaccines is particularly urgent. At present, some vaccines have obtained emergency use authorization or additional conditions for marketing around the world.

However, in the face of brand-new viruses, under the background of more relevant basic research but less thorough mechanism research, a large number of vaccine designs are rapidly entering the clinic, and there are still relatively high levels of vaccine effectiveness, protection period, quality control, and accessibility. Much content requires further research.

The first batch of new coronavirus vaccines in China are mainly inactivated vaccines. However, the BPL used in the inactivated vaccines is inactivated, and the Spike protein has a high proportion of post-fusion conformation and RBD "down" conformation. In the preliminary immune population analysis, Evidence of relatively low total antibody level responses and low neutralizing antibody titers were found.

In addition, judging from the existing basic research on the new coronavirus, not all individuals will produce sufficient titers of neutralizing antibodies after being infected with the new coronavirus, and there is a risk of secondary infection. Neutralizing antibody titers usually decline gradually after reaching a peak at 1 month, and a small number of low-titer recoveries will fall below the detection limit. Referring to other coronaviruses, antibodies against new coronaviruses may exist for about 1-2 years and cannot form long-term protection.

Neutralizing antibodies to SARS-CoV-2 can effectively control infection by blocking or inhibiting the interaction between SARS-CoV-2 and host cells. Among them, the most thoroughly studied mechanism is the interaction between the receptor binding domain (RBD) on the S1 subunit of the SARS-CoV-2 spike Spike protein and the host cell receptor ACE2, as well as the subsequent conformational transition and membrane fusion. ACE2, also known as ACEH, named angiotensin-converting enzyme 2, is a metalloproteinase with a full length of 805 amino acids and a type I transmembrane glycoprotein with a single extracellular catalytic domain.

At present, some new coronavirus vaccines show signs of low positive conversion rate and titer of neutralizing antibodies after immunization. In addition, as an RNA virus, SARS-CoV-2 has a very high mutation frequency, and the neutralizing effect of convalescent serum and vaccine shows lower neutralizing ability for some mutations, and the accumulation of these mutations may lead to the occurrence of immune escape. Therefore, higher requirements are placed on the sensitivity of neutralizing antibody detection.

SUMMARY OF THE INVENTION

The present disclosure provides at least one of the following embodiments:

In one or more embodiments, the present disclosure relates to a detection method comprising the steps of:

(1) The sample is contacted with the ligand-containing fragment, reagent 1, and reagent 2;

Reagent 1: Receptor including ligand;

Reagent 2: includes an antibody that binds a ligand, wherein the antibody competes with the receptor;

Wherein, one of reagent 1 or reagent 2 is connected with a label, and the other is immobilized on a solid-phase carrier;

(2) Detection signal.

In one or more embodiments, the contact means includes any of the following:

(a) The sample is contacted with the ligand-containing fragment, reagent 1, and reagent 2 at the same time;

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

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

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

(e) the sample is contacted first with the ligand-containing fragment, then with Reagent 1, and then with Reagent 2; and

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

In one or more embodiments, the ligand is a ligand for a pathogen to invade a host cell.

In one or more embodiments, the pathogen is a coronavirus. In one or more embodiments, the coronavirus includes HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, MERS-CoV or SARS-CoV-2.

In one or more embodiments, the coronavirus is SARS-CoV-2 or SARS-CoV. In one or more embodiments, SARS-CoV-2 includes wild strains of SARS-CoV-2 and variants thereof, and SARS-CoV includes wild strains of SARS-CoV and variants thereof.

In one or more embodiments, the ligand is RBD. In one or more embodiments, the RBD comprises an RBD or a core region thereof. In one or more embodiments, the RBD is the RBD of the SARS-CoV-2 S protein.

In one or more embodiments, the receptor is ACE2.

In one or more embodiments, the ligand-containing fragment comprises processing the fragment to obtain a structure that is more than dimeric. In one or more embodiments, the means of treatment include physical means or chemical means. In one or more embodiments, the manner of treatment includes the manner of photopolymerization, chemical cross-linking, or recombinant techniques.

In one or more embodiments, ligand-containing fragments include fragments obtained by expressing two or more ligand sequences in tandem.

In one or more embodiments, the ligand-containing fragment is an RBD protein, an S1 protein, or an S protein.

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

In one or more embodiments, the sample is also contacted with a reagent 3 comprising a secondary antibody, the secondary antibody being an IgG against the species from which the sample is derived.

In one or more embodiments, the second antibody is an anti-human IgG.

In one or more embodiments, reagent 3 is attached to a label or solid support.

In one or more embodiments, if reagent 1 is attached to a label, then reagent 3 is attached to a label.

In one or more embodiments, if Reagent 1 is immobilized on the solid support, Reagent 3 is immobilized on the solid support. In one or more embodiments, the detection method can also be used to detect the presence or absence of total antibodies in a sample.

In one or more embodiments, the sample is selected from bodily fluids, excreta, or cells. In one or more embodiments, the sample is selected from serum, plasma, whole blood, lymph, cerebrospinal fluid, tissue fluid, saliva, urine, or lymphocytes.

In one or more embodiments, the present disclosure relates to detection assemblies, including:

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

(b) the reagent 1 of any one of the above embodiments; and

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

In one or more embodiments, the detection assembly further comprises (d) the reagent 3 described in any one of the above embodiments.

In one or more embodiments, the present disclosure relates to a chromatography assembly, including: a sample pad, a binding pad, a reaction membrane and an absorption pad, the reaction membrane is provided with a detection zone; the chromatography assembly further includes:

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

(b) the reagent 1 described in any one of the above embodiments;

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

In one or more embodiments, Reagent 1 is immobilized in the detection zone, Reagent 2 is attached with a label and placed on the binding pad; or Reagent 1 is attached with a label and placed on the binding pad, and Reagent 2 is immobilized in the detection zone.

In one or more embodiments, the chromatography assembly further includes (d) the reagent 3 described in any one of the above embodiments, and the reagent 3 is fixed in the detection area.

In one or more embodiments, the ligand-containing fragment is provided on a sample pad.

In one or more embodiments, the detection method described in any of the above embodiments, the detection assembly described in any of the above embodiments, or the chromatography assembly described in any of the above embodiments are used in antibody detection or antibody preparation. Application of detection reagents.

Detailed ways

Reference will now be made in detail to embodiments of the present disclosure, one or more examples of which are described below. Each example is provided by way of explanation and not limitation of the present disclosure. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the disclosure. For example, features illustrated or described as part of one embodiment can be used in another embodiment to yield a still further embodiment.

Herein, "ligand" can be understood as any protein or polypeptide capable of binding to a receptor. A "receptor" can be understood as a molecule capable of recognizing a ligand and specifically binding the ligand, the ligand-receptor binding can achieve 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 the ligand binds to the receptor and the strength of the binding are called affinity. The easier the two are combined, the stronger the binding after binding, the stronger the affinity, and vice versa.

Herein, "antibody" is different from "receptor", and antibody can be understood as an immune substance secreted by immune cells, which is used to identify and/or neutralize antigenic substances. A "neutralizing antibody" is an antibody produced by B lymphocytes to protect cells against an antigen or infectious agent.

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

Herein, "label" can be understood as being capable of directly generating a signal; or directly or indirectly triggering a specific substance to generate a signal, and the label can be directly or indirectly linked to the labeled object. For example, labels commonly used in immunodetection include metal ions, fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, electrochemiluminescent labels, radiolabels, nucleic acid labels, polypeptides Labels or enzymes, but not limited thereto. In one or more embodiments, the label can be colloidal gold, fluorescein, fluorescent microspheres, acridine esters, horseradish peroxidase, alkaline phosphatase, latex microspheres, triple ruthenium, luminol class, Eu chelate.

Herein, "solid support" can be understood as being able to be immobilized directly or indirectly with the immobilized substances (eg, proteins, polypeptides), such as solid supports commonly used in immunodetection, including plastics, microparticles or membrane supports. The plastic may be, for example, polystyrene; the microparticles may be, for example, magnetic microparticles; and the membrane carrier may be, for example, a nitrocellulose membrane, a glass cellulose membrane, or a nylon membrane.

Herein, the "detection signal" can be understood as obtaining or identifying the strength or level of the detection signal in a manner capable of identifying the marker.

Herein, a "ligand-containing fragment" can be understood as a protein or polypeptide comprising a ligand sequence.

Herein, "contacting" is understood to mean allowing it to bind. The contact time is not specifically limited, and the contact time varies with different implementations and detection platforms, but it belongs to the category that can be understood by those skilled in the art.

Herein, "reagent" can be understood as a substance or product, etc., which is not limited to its form or state, and can be either liquid or solid.

In this article, "colloidal gold" refers to gold sol, which is a stable, uniform and monodispersed gold particle suspension in liquid formed after gold salt is reduced to gold element. Gold sol particles consist of a gold atom surrounded by a double ion layer.

Herein, "magnetic particles" are magnetic particles, which refer to colloidal composite materials that can be uniformly dispersed in a certain base liquid. Because of its superparamagnetic properties, high specific surface area, and functional groups that can be modified. Therefore, antigens/antibodies, enzymes, nucleic acids/oligonucleotides, small molecule drugs, etc. are immobilized on its surface.

The present invention has higher sensitivity in neutralizing antibody detection. The present disclosure has generality and is not limited to a specific immunoassay platform and is not limited to a specific species.

The present disclosure relates to a neutralizing antibody detection method, comprising the following steps:

(1) The sample is contacted with the ligand-containing fragment, reagent 1, and reagent 2;

Reagent 1: a receptor including the ligand;

Reagent 2: comprising an antibody that binds the ligand, wherein the antibody competes with the receptor;

Wherein, one of reagent 1 or reagent 2 is connected with a label, and the other is immobilized on a solid-phase carrier;

(2) Detection signal.

In one or more embodiments, the contacting manner includes: the sample is simultaneously contacted with the ligand-containing fragment, reagent 1, and reagent 2; in one or more embodiments, the contacting manner includes: the sample is first contacted with the ligand-containing fragment In one or more embodiments, the contact mode includes: the sample is first contacted with the ligand-containing fragment, reagent 2, and then contacted with reagent 1; in one or more In various embodiments, the contacting mode includes: the sample is first contacted with the ligand-containing fragment, and then contacted with reagent 1 and reagent 2; in one or more embodiments, the contacting mode includes: the sample is first contacted with the ligand-containing fragment. The fragments are contacted, then contacted with reagent 1, and then contacted with reagent 2; in one or more embodiments, the contact method includes: the sample is first contacted with the ligand-containing fragment, then contacted with reagent 2, and then contacted with reagent 1 .

In one or more embodiments, reagent 1 is attached with a label, and reagent 2 is immobilized on a solid support. In one or more embodiments, reagent 2 is attached with a label, and reagent 1 is immobilized on a solid support.

In one or more embodiments, the ligand is a ligand for a pathogen to invade a host cell. In one or more 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 one or more embodiments, the coronavirus is SARS-CoV-2 or SARS-CoV. Among them, SARS-CoV-2 includes SARS-CoV-2 wild strain and its variant strains. SARS-CoV includes SARS-CoV wild strains and their variants.

Herein, "pathogen" can be understood as a microorganism (including bacteria, viruses, rickettsia, fungi, etc.), parasites or other agents (such as recombinant microorganisms, including hybrids, which can cause human or animal and plant infection diseases) or mutant).

In one or more embodiments, the ligand is RBD, and the RBD includes RBD or its core region, as long as it can achieve the function of binding to the receptor, it should still be understood as the RBD described in the present disclosure, and RBD is the receptor binding Domain (Receptor Binding Domain). In one or more embodiments, the ligand is selected from the RBD of the S protein of SARS-CoV-2. In one or more embodiments, the sequence of the RBD of the S protein of SARS-CoV-2 can be referred to Wrapp Daniel, Wang Nianshuang, Corbett Kizzmekia S, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation, and its sequence identity is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequences that still function as ligand-binding receptors and/or antibodies. In one or more embodiments, ligand-containing fragments such as RBD protein, S1 protein, or S protein (see sequence Gene ID: 43740568).

In one or more embodiments, the receptor is selected from ACE2 (Angiotensin I Converting Enzyme 2), HDL (High Density Lipoprotein), HS (Heparan Sulphate) , SR-B1 (Scavenger Receptor Class B Member 1), APN (Aminopeptidase N), DPP4 (Dipeptidyl Peptidase 4), AGO4 (Argonaute 4), IFITM3 (Interferon Induced Transmembrane Protein 3), EGFR (Epidermal Growth Factor Receptor), ICAM1 (Intercellular Adhesion Molecule 1 (Intercellular Adhesion) Molecule 1)), HSPA1B (Heat Shock Protein A (Hsp70) 1B (Heat Shock Protein Family A (Hsp70) Member 1B)), ITGB6 (Integrin Subunit Beta 6)), WWTR1 (WW domain-containing transcriptional regulation Factor 1 (WW Domain Containing Transcription Regulator 1)), ALDH1A1 (Aldehyde Dehydrogenase 1 Family Member A1)), RUNX3 (RUNX Family Transcription Factor 3), NRP1 ( Neuropilin 1 (Neuropilin 1). In one or more embodiments, the receptor is ACE2. In one or more embodiments, the receptor is human ACE2. In one or more embodiments, the sequence of human ACE2 can be referenced to Gene ID: 59272 and is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% identical to its sequence , 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence, and these amino acid sequences still have the function of receptor binding ligand.

In one or more embodiments, the ligand-containing fragment includes processing the fragment to obtain a structure of more than dimerization; the processing method includes physical method or chemical method, etc., but is not limited thereto. In one or more embodiments, for example, photopolymerization is used to process the single-ligand-containing fragment to obtain a structure of more than dimerization. In one or more embodiments, for example, chemical cross-linking is used, for example, the amino group and carboxyl group on the fragment are used for cross-linking reaction, so as to obtain a structure of more than dimerization. In one or more embodiments, ligand-containing fragments include fragments obtained by expressing two or more ligand sequences in tandem. For example, using recombinant technology to achieve tandem expression of at least two ligand sequences, those skilled in the art should understand that the result of tandem expression is that the fragment obtains more than two ligand regions. In one or more embodiments, the ligand-containing fragment can be an RBD polymer, S1 protein polymer, or S protein polymer of SARS-CoV-2 S protein.

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

In one or more embodiments, the sample is also contacted with a reagent 3, and the reagent 3 includes a second antibody, and the second antibody is an IgG against the species from which the sample is derived; in one or more embodiments, when the present disclosure If the sample object detected by the method is human, the second antibody is anti-human IgG. In one or more embodiments, reagent 3 is attached to a label or solid support. In one or more embodiments, if reagent 1 is attached to a label, then reagent 3 is attached to a label. In one or more embodiments, if reagent 1 is immobilized on a solid support, then reagent 3 is immobilized on a solid support; in one or more embodiments, the detection method can also be used to detect the presence of total antibodies in the sample .

In one or more embodiments, the present disclosure relates to detection assemblies, including:

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

(b) the reagent 1 described in any one of the above embodiments;

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

In one or more embodiments, the detection assembly further comprises (d) the reagent 3 described in any one of the above embodiments.

In one or more embodiments, the present disclosure also relates to a chromatography assembly, including: a sample pad, a binding pad, a reaction membrane and an absorption pad, the reaction membrane is provided with a detection zone; the chromatography assembly further includes:

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

(b) the reagent 1 described in any one of the above embodiments;

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

In one or more embodiments, Reagent 1 is immobilized on the detection zone, and Reagent 2 has a label attached and is provided on the binding pad. In one or more embodiments, reagent 1 is conjugated with a label and disposed on the binding pad, and reagent 2 is immobilized on the detection zone.

In one or more embodiments, the chromatography assembly further comprises (d) the reagent 3 described in any of the above embodiments, and the reagent 3 is immobilized in the detection area; in one or more embodiments, the reagent 3 is Fragments of the ligand are placed on the sample pad.

In one or more embodiments, the chromatography component further includes a quality control reagent, wherein the quality control reagent is a pair of binding partners, such as biotin-avidin, antigen-antibody, receptor-ligand, digoxin Capryl-digoxigenin, carbohydrate-lectin, complementary two nucleic acids, aptamer-aptamer target; in one or more embodiments, the antigen described in the binding partner- Antibodies include antibodies and secondary antibodies, wherein the antibody is equivalent to the antigen of the secondary antibody, for example, the species of the antibody is derived from bovine, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat , rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, cock or human; the secondary antibody is an antibody against a specific species; the type of antibody is not limited, it can be IgG, IgM, IgY, IgE, IgD, etc.; in one or more embodiments, eg, chicken IgY and goat anti-chicken IgY; eg, murine IgG and goat anti-mouse IgG.

In one or more embodiments, a pair of binding partners are each independently attached to a label or immobilized on a solid support; eg, an antigen is attached to a label, and the antibody to the antigen is immobilized on a solid support; or, for example, The antigen is immobilized on the solid phase carrier, and the antibody of the antigen is connected with a label; in one or more embodiments, one of the quality control reagents connected with the label is arranged on the binding pad, and the quality control reagent immobilized on the solid phase carrier One of the reagents is immobilized in the detection area (eg, the quality control line, also known as the C line).

In one or more embodiments, the ligand-containing fragment may exist in liquid or solid form; in one or more embodiments, the ligand-containing fragment is dissolved in solution; in one or more embodiments In one or more embodiments, the solution may include a buffer (eg, PBS, MES, CB, or Tris, etc.) to allow the ligand-containing fragment to exist at a suitable pH; in one or more embodiments, the solution may Including protein protectants, such as BSA, or casein, etc.; in one or more embodiments, the solution may include surfactants, such as Tween, or triton, etc.; in one or more embodiments In one or more embodiments, the solution may include a salt ion, such as a sodium salt, or a magnesium salt, etc.; in one or more embodiments, the solution may include a sugar, such as sucrose, mannose, or glucose, and the like.

In one or more embodiments, the ligand-containing fragments are in solid state form, eg, immobilized on a sample pad; in one or more embodiments, the ligand-containing fragments are in the form of a dry powder (eg, lyophilized powder) Exist, dissolve in solution before use.

The examples of the present disclosure are only used to make the present disclosure clearer, and the use concentrations of the reagents involved (such as ligand-containing fragments, reagents 1-3, and quality control reagents) are not intended to limit the present disclosure. According to the principles of the scheme of the present disclosure, Practical concentrations for other specific embodiments can be obtained by those skilled in the art through a limited number of experiments.

The present disclosure also relates to the application of the detection method of any of the above embodiments, the detection assembly of any of the above embodiments, or the chromatography assembly of any of the above embodiments in antibody detection or preparation of antibody detection reagents.

Taking Embodiment 1 of the present disclosure as an example, the principle of the solution of the present disclosure is described. Those skilled in the art should understand that the extended solution thereof is still within the scope of the principle and belongs to the concept of the present disclosure. Contact the RBD protein polymer with the neutralizing antibody positive sample, the RBD protein polymer can react with the total RBD protein antibody (including the RBD neutralizing antibody) in the sample, and further neutralize with the RBD labeled colloidal gold through chromatography The antibody binds and is further chromatographed in contact with ACE2, resulting in a decolorizing reaction.

Herein, the numbers 1 and 2 in Reagent 1 and Reagent 2 have no meaning of order limitation, and are only set for the convenience of description.

Aspects and implementations 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 this application has been described in conjunction with exemplary embodiments, many equivalent modifications and variations will be apparent to those skilled in the art given this application. Thus, the exemplary embodiments of the present application are exemplary and non-limiting. Various changes may be made to the described embodiments without departing from the spirit and scope of the present application.

Example 1

1. Preparation of Colloidal Gold

The colloidal gold solution is prepared by the following method: chloroauric acid is prepared into a 1% solution, then the above solution with a final concentration of 4/10,000 is added to the boiled purified water, and the solution is continuously boiled for 3 min. Add 580 μL per 100 mL of chloroauric acid solution, continue to stir and heat for 10 min, and cool to room temperature to prepare a colloidal gold solution, which is stored at 2-8 °C for later use.

2. Colloidal Gold Labeling

Using SARS-CoV-2 S protein RBD antibody (No. G3, available from Guangdong Feipeng Bio Co., Ltd.), which can compete with human ACE2, the steps for connecting colloidal gold are as follows: add K ₂ CO to the colloidal gold solution _3. Adjust the pH to 8.0, add G3 to the pH-adjusted solution to make the protein content 10 μg/mL, couple for 5 min, and add 10% BSA to it to terminate the coupling reaction; Store at 2-8°C after dissolution.

3. Preparation of Binding Pads

The G3 solution connected with colloidal gold was diluted according to a 10% dilution ratio, spread evenly on the glass fiber, and placed in a drying room at 37° C. to dry overnight to obtain a binding pad.

4. Preparation of Reactive Membranes

T-line coating: Human ACE2 (from Guangdong Feipeng Biological Co., Ltd., Ag2) was diluted to 0.8 mg/mL, coated on nitrocellulose membrane using a gold spray synovial instrument, and placed in a 37 °C oven to dry for more than 2 hours. get the reaction membrane.

5. Assembly

The above-mentioned sample pad, binding pad, reaction membrane, and water-absorbing pad were sequentially assembled on the bottom plate by overlapping, and cut into 2.7 mm to prepare corresponding chromatography components.

6. Detection

The sample was pre-mixed with RBD polyantigen (No. Ag29, from Guangdong Feipeng Biological Co., Ltd.) at a final concentration of 1 μg/mL for 30 min, and 60 μL of the sample was added.

7. Interpretation of results

Results are recorded as colloidal gold card readings:

When the reactivity of the T-line of the sample to be tested is more than 1C lower than that of the control sample, it means that the sample to be tested contains neutralizing antibodies. This indicates that the content of neutralizing antibodies against the new coronavirus in the sample to be tested is higher.

When the reactivity of the T-line of the sample to be tested is consistent with the color depth of the T-line of the control sample, it means that there is no neutralizing antibody against the new coronavirus in the sample to be tested.

Control samples were samples without neutralizing antibodies or quality controls.

Example 2

1. Preparation of Colloidal Gold

The colloidal gold solution is prepared by the following method: chloroauric acid is prepared into a 1% solution, then the above solution with a final concentration of 4/10,000 is added to the boiled purified water, and the solution is continuously boiled for 3 min. Add 580 μL per 100 mL of chloroauric acid solution, continue to stir and heat for 10 min, and cool to room temperature to prepare a colloidal gold solution, which is stored at 2-8 °C for later use.

2. Colloidal Gold Labeling

The SARS-CoV-2 S protein RBD antibody G3 used is used, and the steps for connecting colloidal gold are as follows: add K ₂ CO ₃ to the colloidal gold solution, adjust the pH to 8.0, add G3 to the adjusted pH solution to make its protein The content is 10 μg/mL, the coupling is carried out for 5 min, and 10% BSA is added to terminate the coupling reaction; after centrifugation, the supernatant is discarded, and the precipitate is reconstituted and stored at 2-8° C. for later use.

3. Preparation of Binding Pads

The G3 solution connected with colloidal gold was diluted according to a 10% dilution ratio, spread evenly on the glass fiber, and placed in a drying room at 37° C. to dry overnight to obtain a binding pad.

4. Preparation of Reactive Membranes

T-line coating: Human ACE2 protein Ag2 was diluted to 1.0 mg/mL, coated on nitrocellulose membrane using a gold spraying synovometer, and placed in a 37°C oven to dry for more than 2 hours to obtain a reaction membrane.

5. Sample Pad Handling

The RBD polyantigen Ag29 was diluted to 1 μg/mL with 1XPBST, spread on the sample pad, and dried at 37°C for 1 h before use.

6. Assembly

The above-mentioned sample pad, binding pad, reaction membrane, and water-absorbing pad were sequentially assembled on the bottom plate by overlapping, and cut into 2.7 mm to prepare corresponding chromatography components.

7. Detection

Take 60 μL of sample for loading.

8. Interpretation of results

Results are recorded as colloidal gold card readings:

When the reactivity of the T-line of the sample to be tested is more than 1C lower than that of the control sample, it means that the sample to be tested contains neutralizing antibodies. This indicates that the content of neutralizing antibodies against the new coronavirus in the sample to be tested is higher.

When the reactivity of the T-line of the sample to be tested is consistent with the color depth of the T-line of the control sample, it means that there is no neutralizing antibody against the new coronavirus in the sample to be tested.

Control samples were samples without neutralizing antibodies or quality controls.

Example 3

1. Preparation of Colloidal Gold

The colloidal gold solution is prepared by the following method: chloroauric acid is prepared into a 1% solution, then the above solution with a final concentration of 4/10,000 is added to the boiled purified water, and the solution is continuously boiled for 3 min. Add 580 μL per 100 mL of chloroauric acid solution, continue to stir and heat for 10 min, and cool to room temperature to prepare a colloidal gold solution, which is stored at 2-8 °C for later use.

2. Colloidal Gold Labeling

Using human ACE2 protein Ag2, the steps for connecting colloidal gold are as follows: add K ₂ CO ₃ to the colloidal gold solution, adjust the pH to 8.0, add Ag2 to the adjusted pH solution to make the protein content 10 μg/mL, and evenly After coupling for 5 min, 10% BSA was added to stop the coupling reaction; after centrifugation, the supernatant was discarded, and the precipitate was reconstituted and stored at 2-8°C for later use.

3. Preparation of Binding Pads

The Ag2 solution connected with colloidal gold was diluted according to a 10% dilution ratio, spread evenly on the glass fiber, and placed in a drying room at 37° C. to dry overnight to obtain a bonding pad.

4. Preparation of Reactive Membranes

T-line coating: SARS-CoV-2 S protein RBD antibody G3 diluted to 0.8mg/mL, coated on nitrocellulose membrane using a gold-spraying synovometer, and placed in a 37°C oven to dry for more than 2 hours to obtain a reaction membrane .

5. Assembly

The above-mentioned sample pad, binding pad, reaction membrane, and water-absorbing pad were sequentially assembled on the bottom plate by overlapping, and cut into 2.7 mm to prepare corresponding chromatography components.

6. Detection

The sample was pre-mixed with the RBD polyantigen Ag29 at a final concentration of 1 μg/mL for 30 min, and 60 μL of the sample was added.

7. Interpretation of results

Results are recorded as colloidal gold card readings:

When the reactivity of the T-line of the sample to be tested is more than 1C lower than that of the control sample, it means that the sample to be tested contains neutralizing antibodies. This indicates that the content of neutralizing antibodies against the new coronavirus in the sample to be tested is higher.

When the reactivity of the T-line of the sample to be tested is consistent with the color depth of the T-line of the control sample, it means that there is no neutralizing antibody against the new coronavirus in the sample to be tested.

Control samples were samples without neutralizing antibodies or quality controls.

Comparative Example 1

1. Colloidal Gold Labeling

The preparation of colloidal gold is the same as that in Example 1. For RBD antigen (No. Ag26, from Guangdong Feipeng Biological Co., Ltd.), add K ₂ CO ₃ to the colloidal gold solution, adjust the pH to 7.5, and add Ag26 to the adjusted pH solution to make the protein content 10μg /mL, couple for 5 min, add 10% BSA to it to terminate the coupling reaction; discard the supernatant after centrifugation, and store the pellet at 2-8° C. for later use after reconstitution.

2. Preparation of Binding Pads

The Ag26 solution connected with colloidal gold was diluted according to a 10% dilution ratio, spread evenly on the glass fiber, and placed in a drying room at 37° C. to dry overnight to obtain a bonding pad.

3. Preparation of Reaction Membrane

T-line coating: Human ACE2 protein Ag2 was diluted to 0.8 mg/mL, coated on nitrocellulose membrane using a gold spray synovial instrument, and placed in a 37°C oven to dry for more than 2 hours to obtain a reaction membrane.

4. Assembly

The above-mentioned sample pad, binding pad, reaction membrane, and water-absorbing pad were sequentially assembled on the bottom plate by overlapping, and cut to 2.7 mm to prepare a corresponding chromatography component.

5. Detection

Take 60 μL of sample for loading.

6. Interpretation of results

Results are recorded as colloidal gold card readings:

When the reactivity of the T-line of the sample to be tested is more than 1C lower than that of the control sample, it means that the sample to be tested contains neutralizing antibodies. This indicates that the content of neutralizing antibodies against the new coronavirus in the sample to be tested is higher.

When the reactivity of the T-line of the sample to be tested is consistent with the color depth of the T-line of the control sample, it means that there is no neutralizing antibody against the new coronavirus in the sample to be tested.

Control samples were samples without neutralizing antibodies or quality controls.

Example 4

1. Preparation of Colloidal Gold

The colloidal gold solution is prepared by the following method: chloroauric acid is prepared into a 1% solution, then the above solution with a final concentration of 4/10,000 is added to the boiled purified water, and the solution is continuously boiled for 3 min. Add 580 μL per 100 mL of chloroauric acid solution, continue to stir and heat for 10 min, and cool to room temperature to prepare a colloidal gold solution, which is stored at 2-8 °C for later use.

2. Colloidal Gold Labeling

Using the SARS-CoV-2 S protein RBD antibody (No. Ab7, available from Guangdong Feipeng Biological Co., Ltd.), which can compete with human ACE2, the steps for connecting colloidal gold are as follows: add K ₂ CO to the colloidal gold solution _3. Adjust the pH to 8.0, add Ab7 to the pH-adjusted solution to make the protein content 10 μg/mL, couple for 5 min, and add 10% BSA to it to terminate the coupling reaction; after centrifugation, discard the supernatant and reprecipitate. Store at 2-8°C after dissolution.

3. Preparation of Binding Pads

The Ab7 solution connected with colloidal gold was diluted according to a 10% dilution ratio, spread evenly on the glass fiber, and placed in a drying room at 37° C. to dry overnight to obtain a binding pad.

4. Preparation of Reactive Membranes

T1 line coating: dilute anti-human IgG antibody to 1.0mg/mL, and coat on nitrocellulose membrane using a gold-spraying synovometer; T2 line coating: human ACE2 (No. ACE2-Ag13, from Guangdong Feipeng) Biotechnology Co., Ltd.) was diluted to 1.0 mg/mL, coated on a nitrocellulose membrane using a gold-spraying synovial membrane, and placed in a 37 °C oven to dry for more than 2 h to obtain a reaction membrane. In this reaction membrane, the T1 line is arranged below the T2 line, and the T1 line is close to the bonding pad after assembly.

5. Assembly

The above-mentioned sample pad, binding pad, reaction membrane, and water-absorbing pad were sequentially assembled on the bottom plate by overlapping, and cut into 2.7 mm to prepare corresponding chromatography components.

6. Detection

The sample was pre-mixed with 1 μg/mL final concentration of S protein polyantigen (number S-Ag21, from Guangdong Feipeng Biological Co., Ltd.) for 30 min, and 60 μL of the sample was added.

7. Interpretation of results

Results are recorded as colloidal gold card readings:

When the reactivity of the T2 line of the sample to be tested is more than 1C lower than that of the control sample, it means that the sample to be tested contains neutralizing antibodies. This indicates that the content of neutralizing antibodies against the new coronavirus in the sample to be tested is higher.

When the reactivity of the T2 line of the sample to be tested is consistent with the color depth of the T2 line of the control sample, it means that there is no neutralizing antibody against the new coronavirus in the sample to be tested.

When the T1 line of the sample to be tested develops color, it means that there are total antibodies against the new coronavirus in the sample to be tested.

Control samples were samples or controls without neutralizing antibodies and total antibodies.

In this example, total antibody and neutralizing antibody can be detected at the same time, and the detection sensitivity and detection rate of neutralizing antibody are comparable to those in Example 1.

Example 5

1. Magnetic particle immobilization

Wash the magnetic particles, resuspend in MES buffer, add carbodiimide (EDAC) for reaction, place on a blood mixer and mix at medium speed; add SARS-CoV-2 S protein RBD antibody G3, and protect from light at room temperature The reaction was carried out to obtain a coated product; the blocking reaction was terminated by adding a quenching buffer to avoid light at room temperature, and the G3 immobilized on the magnetic particles was collected.

2. Attach the acridine label

The human ACE2 protein Ag2 was resuspended in MES buffer, then acridine ester was added, and the labeling was performed after mixing. After the labeling reaction, 10% BSA was added to block; centrifugation to remove unconjugated acridine ester and collect ACE2 acridine pyridine label.

3. Detection

The samples were reacted with the RBD polyantigen Ag29 at 37°C, and then the G3 and ACE2 acridine markers immobilized on the magnetic particles were added to react at 37°C. Finally, the reaction system was washed and the luminescence signal was measured. Calculation of results: Inhibition rate=1-sample detection signal/negative control substance detection signal.

Example 6

1. Magnetic particle immobilization

The magnetic particles were washed, resuspended in MES buffer, added with carbodiimide (EDAC) for reaction, placed on a blood mixer and mixed at medium speed; added human ACE2 protein Ag2, reacted at room temperature in the dark to obtain a coated product ; Add quenching buffer at room temperature in the dark to stop the blocking reaction, and collect the Ag2 immobilized on the magnetic particles.

2. Attach the acridine label

Take SARS-CoV-2 S protein RBD antibody (No. G3, which can be purchased from Fipeng Bio, which competes with ACE2) and resuspend it in MES buffer, then add acridine ester, mix well and label it, and the labeling reaction ends After that, 10% BSA was added to block; the unconjugated acridine ester was removed by centrifugation, and the G3 acridine label was collected.

3. Detection

The samples were reacted with the RBD polyantigen Ag29, the Ag2 and G3 acridine labels immobilized on the magnetic particles at 37°C, and finally the reaction system was washed and the luminescence signal was measured. Calculation of results: Inhibition rate=1-sample detection signal/negative control substance detection signal.

Comparative Example 2

1. Magnetic particle immobilization

The magnetic particles were washed, resuspended in MES buffer, added with carbodiimide (EDAC) for reaction, placed on a blood mixer and mixed at medium speed; added human ACE2 protein Ag2, reacted at room temperature in the dark to obtain a coated product ; Add quenching buffer at room temperature in the dark to stop the blocking reaction, and collect the Ag2 immobilized on the magnetic particles.

2. Attach the acridine label

The RBD antigen Ag26 was resuspended in MES buffer, then acridine ester was added, and the labeling was performed after mixing. After the labeling reaction, 10% BSA was added to block; centrifugation to remove unconjugated acridine ester and collect Ag26 acridine Mark.

3. Detection

Take the sample and react with Ag26 acridine label at 37 °C, then add Ag2 immobilized on the magnetic particles to react at 37 °C, and finally wash the reaction system and measure the luminescence signal. Calculation of results: Inhibition rate=1-sample detection signal/negative control substance detection signal.

Through experimental evaluation, both Example 5 and Example 6 can effectively detect clinical vaccine samples, and the sensitivity of inhibition efficiency of Example 5 and Example 6 is higher than that of Comparative Example 2.

Example 7

1. Latex Cross-Linking RBD Neutralizing Antibodies or ACE2

Take polystyrene latex microspheres and resuspend in MES buffer, add EDC and NHS to activate at 37 °C, add CB buffer and RBD neutralizing antibody (G3) or human ACE2 (Ag2), mix well, and react at 37 °C For 2-3 hours, block with 10% BSA, centrifuge, remove supernatant, wash 1-2 times, and resuspend to obtain G3 or Ag2 of cross-linked latex.

2. Detection

Take the sample and mix it with RBD polyantigen Ag29 for 7.5min, add cross-linked latex G3 to react for 6min, read A1, add cross-linked latex Ag2 to react for 10min to read A2, calculate ΔA=A2-A1.

Using the method of this embodiment, negative and positive samples can be effectively distinguished. When testing 50ng/mL G3 sample (simulated positive sample), the inhibition rate can reach 15%-20%, and when testing 8000ng/mL G3 sample (simulated positive sample), the inhibition rate can reach more than 80%.

In order to facilitate the understanding of the present disclosure, some experimental results are shown as follows:

Experimental result 1

Using the protocols of Examples 1-3 and Comparative Example 1, RBD neutralizing antibodies were used to simulate the content of neutralizing antibodies in vaccine samples, and the test results were shown in the following table. In the table, the smaller the reading value of the color card, the deeper the color is, "+" means the color is deeper under the same reading value, and "B" means no color.

It can be seen from the table that Examples 1-3 are all better than Comparative Example 1, indicating that the solution of the present disclosure has higher detection sensitivity.

Experimental result 2

Using the scheme of Example 1, RBD neutralizing antibodies were used to simulate the content of neutralizing antibodies in vaccine samples, and the detection results of different temperatures and times were shown in the following table. In the table, the smaller the reading value of the color card, the darker the color rendering, "+" means that the color rendering is deeper under the same reading value, and "B" means no color rendering.

As can be seen from the table, the disclosed scheme has high stability and wide detection time.

Experimental result 3

Using the protocol of Example 1, neutralizing antibody detection was performed on 5 vaccinated specimens, and the results are shown in the following table. In the table, the smaller the reading value of the color card, the deeper the color development, "+" means the color development is deeper under the same reading value, "B" means no color development, "P" means positive, and "N" means negative. In addition, it was compared with a neutralizing antibody detection kit of EUA.

It can be seen from the table that the scheme of the present disclosure has a high detection rate for clinical vaccine specimens and has a more sensitive inhibitory effect.

Experimental result 4

Negative blood, 2 mixed vaccine sera, 2 single vaccine sera were respectively used, and the scheme of Example 5 and Comparative Example 2 were used to test, and the results were as shown in the following table:

Experimental result 5

Negative diluents and neutralizing antibody G3 of different concentrations (0.0625 μg/mL, 0.3125 μg/mL) were respectively used to test using the schemes of Example 5 and Comparative Example 2. The results are shown in the following table:

Experimental result 6

Negative blood, 2 mixed vaccine sera, and 1 single vaccine serum with different concentrations (0.0625 μg/mL, 0.3125 μg/mL) of neutralizing antibody G3 were used to test using the schemes of Example 6 and Comparative Example 2. The results As shown in the table below:

The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity, all possible combinations of the technical features in the above-described embodiments are not described. However, as long as there is no contradiction between the combinations of these technical features, All should be regarded as the scope described in this specification.

The above-mentioned embodiments only represent several embodiments of the present disclosure, and the descriptions thereof are relatively specific and detailed, but should not be construed as a limitation on the scope of the disclosed patent. It should be noted that, for those skilled in the art, without departing from the concept of the present disclosure, several modifications and improvements can be made, which all belong to the protection scope of the present disclosure. Accordingly, the scope of protection of the present disclosure should be determined by the appended claims.

Industrial Applicability

The detection method provided by the present disclosure can be implemented in industry, and the detection components and chromatography components provided by the present disclosure can also be mass-produced in industry, and their application in antibody detection or preparation of antibody detection reagents has higher sensitivity , is also versatile, not limited to a specific immunoassay platform, nor to a specific substance. Therefore, the antibody detection method, detection component and chromatography component provided by the present disclosure have broad market application value in the detection of neutralizing antibodies and total antibodies against novel coronavirus.

Claims (19)

1. The detection method includes the steps:

   (1) The sample is contacted with the ligand-containing fragment, reagent 1, and reagent 2;

   Reagent 1: a receptor comprising the ligand;

   Reagent 2: comprising an antibody that binds the ligand, wherein the antibody competes with the receptor;

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

   (2) Detection signal.
2. The detection method according to claim 1, wherein the contact mode comprises any one of the following:

   (a) The sample is contacted with the ligand-containing fragment, reagent 1, and reagent 2 at the same time;

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

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

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

   (e) the sample is contacted first with the ligand-containing fragment, then with Reagent 1, and then with Reagent 2; and

   (f) The sample is first contacted with the ligand-containing fragment, then with Reagent 2, and then with Reagent 1.
3. The detection method according to claim 1 or 2, wherein the ligand is a ligand for a pathogen to invade a host cell;

   Optionally, the pathogen is a coronavirus;

   Optionally, the ligand is RBD;

   Optionally, the receptor is ACE2.
4. The detection method according to any one of claims 1-3, wherein the coronavirus comprises HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, MERS-CoV or SARS-CoV -2.
5. The detection method according to claim 4, wherein the SARS-CoV-2 includes wild strains of SARS-CoV-2 and variants thereof, and the SARS-CoV includes wild strains of SARS-CoV and variants thereof.
6. The detection method according to claim 3, wherein the RBD comprises an RBD or a core region thereof.
7. The detection method according to claim 3, wherein the RBD is the RBD of the SARS-CoV-2 S protein.
8. The detection method according to any one of claims 1-7, wherein the ligand-containing fragment comprises processing the fragment to obtain a structure of more than dimerization;

   Optionally, ligand-containing fragments include fragments obtained by expressing two or more ligand sequences in tandem.
9. The detection method according to any one of claims 1-8, wherein the ligand-containing fragment is RBD protein, S1 protein or S protein.
10. The detection method according to claim 8, wherein the treatment method includes physical method or chemical method.
11. The detection method according to claim 8 or 10, wherein the processing method includes photopolymerization, chemical cross-linking or recombinant technology.
12. The detection method according to any one of claims 1-11, wherein the detection method is used to detect the presence or absence of neutralizing antibodies in a sample.
13. The detection method according to any one of claims 1-12, wherein the sample is further contacted with a reagent 3, and the reagent 3 includes a second antibody, and the second antibody is an IgG against the species from which the sample is derived;

    Optionally, the second antibody is anti-human IgG;

    Optionally, the reagent 3 is connected with a label or a solid phase carrier;

    Optionally, if reagent 1 is connected to the label, then reagent 3 is connected to the label;

    Optionally, if reagent 1 is immobilized on the solid phase carrier, then reagent 3 is immobilized on the solid phase carrier;

    Optionally, the detection method can also be used to detect the presence or absence of total antibodies in the sample.
14. The detection method according to any one of claims 1-13, wherein the sample is selected from bodily fluids, excreta or cells.
15. The detection method according to any one of claims 1-13, wherein the sample is selected from serum, plasma, whole blood, lymph, cerebrospinal fluid, tissue fluid, saliva, urine or lymphocytes.
16. Inspection components, including:

    (a) the ligand-containing fragment of any one of claims 1-15;

    (b) the reagent 1 of any one of claims 1-15; and

    (c) The reagent 2 of any one of claims 1-15.
17. The detection assembly of claim 15 , wherein the detection assembly further comprises: (d) the reagent 3 of claim 13 .
18. The chromatography assembly includes: a sample pad, a binding pad, a reaction membrane and an absorption pad, the reaction membrane is provided with a detection area; the chromatography assembly further includes:

    (a) the ligand-containing fragment of any one of claims 1-15;

    (b) the reagent 1 of any one of claims 1-15;

    (c) the reagent 2 of any one of claims 1-15;

    Optionally, the reagent 1 is fixed on the detection area, and the reagent 2 is connected with a label and arranged on the binding pad; or the reagent 1 is connected with a label and arranged on the binding pad, and the reagent 2 is fixed on the detection area;

    Optionally, the chromatography assembly further comprises: (d) the reagent 3 according to claim 13 , the reagent 3 is fixed in the detection area;

    Optionally, the ligand-containing fragments are provided on a sample pad.
19. Application of the detection method of any one of claims 1 to 15, the detection assembly of claim 16 or 17, or the chromatography assembly of claim 18 in antibody detection or preparation of antibody detection reagents.