CN114460287A

CN114460287A - Detection method and kit for neutralizing antibody

Info

Publication number: CN114460287A
Application number: CN202210116181.9A
Authority: CN
Inventors: 胡小龙; 屈武斐; 周丽
Original assignee: Venture Biotechnology Co ltd
Current assignee: Venture Biotechnology Co ltd
Priority date: 2022-01-28
Filing date: 2022-01-28
Publication date: 2022-05-10

Abstract

The invention relates to a detection method and a kit for a neutralizing antibody. The invention provides a method for processing carbon nano-materials, which comprises the following steps: (1) the carbon nano material in the solvent is subjected to ultrasonic treatment in the presence of a surfactant to obtain a nano carbon dispersion, and the nano carbon is optionally (2) separated and/or washed. The invention also relates to nanocarbon detection articles and kits made using the treated carbon nanomaterials. The invention has simple operation and intuitive result and is suitable for on-site rapid detection.

Description

Detection method and kit for neutralizing antibody

Technical Field

The invention relates to the field of biomedical detection, in particular to a detection method and a kit for a neutralizing antibody.

Background

The novel coronavirus (hereinafter referred to as "neo-corona") is a single-stranded positive-sense RNA virus, and the genome length is about 29.8 KB. The genome encodes mainly four structural proteins: spike protein S, envelope protein E, membrane protein M and nucleocapsid protein N. Wherein the S protein contains a receptor binding structural domain RBD, can recognize host cell receptor angiotensin converting enzyme 2(ACE2) and mediate membrane fusion, so that the virus adsorbs invaded cells. The specific antibody generated against S-RBD, namely the neutralizing antibody, can block the binding of the new coronavirus to ACE2, so that the virus cannot invade.

Vaccination is recognized as the most economical and effective method for controlling viral epidemics. After vaccination, the protective effect produced is not determined and one of the important indicators for evaluating the effectiveness of a vaccine is the level of neutralizing antibodies in the vaccinee.

The traditional method for evaluating vaccines is to verify the efficacy of neutralizing antibodies by using live viruses through a neutralization experiment virus infection verification method. The requirements on the safety of a laboratory and the skill of an operator are high, the operation is complex, and the time consumption is long. Therefore, it is very meaningful to establish a method for rapidly detecting the novel crown neutralizing antibody. Based on the combination characteristics of the neutralizing antibody and the new coronavirus and the virus infection mechanism, various products for rapidly detecting the new coronavirus exist in the market.

The detection main process of the common enzyme-linked immunosorbent assay (ELISA), such as a cPassTMsVNT new crown neutralizing antibody detection kit produced by King Rui, comprises the steps of coating S-RBD protein on an ELISA plate, incubating overnight, then washing, adding a sample to be detected and ACE2, incubating, then washing, adding HRP-labeled secondary antibody, incubating, washing, adding color-developing agent TMB, reacting, then adding stop solution, reading OD450 on an ELISA reader, and calculating the sample inhibition rate through the OD value to determine whether the neutralizing antibody is contained. The ELISA method needs plate washing and sample adding for many times in the detection process, has many steps and long time, needs instrument assistance, needs calculation of results, and is not very intuitive for quick detection results.

There are also conventional gold immunochromatographic assays, and competitive methods are often used in the market to detect neutralizing antibodies. The S-RBD protein is labeled by colloidal gold and then fixed on the combination pad, the ACE2 is coated on the nitrocellulose membrane to be used as a detection line, when a sample to be detected has a new crown neutralizing antibody, the neutralizing antibody can be specifically combined with the RBD antigen labeled by the colloidal gold, the combination efficiency of the RBD and the ACE2 is reduced, and the color development of the detection line can be reduced. After the reaction is finished, the level of the neutralizing antibody is judged by directly observing with naked eyes and comparing with a colorimetric card or by comparing the color development intensity of a detection line and a quality control line. When the neutralizing antibody is detected by a colloidal gold competition method, the resolution is reduced due to color development when the concentration of the neutralizing antibody is low, and the neutralizing antibody is difficult to interpret. In addition, because most of the detection of the neutralizing antibody is a whole blood sample, the colloidal gold red is easily influenced by blood.

The nano carbon material is used in the immunochromatography industry, can overcome the defects of colloidal gold but cannot be applied in a large scale, and mainly the processing method of nano carbon needs to be optimized. When the nano carbon is used in the conventional treatment method, the nano carbon is used as a marker, and the solubility, the storage stability and the marking stability are all required to be improved.

Disclosure of Invention

The invention aims to provide a simple and quick virus neutralizing antibody detection kit and a detection method.

In a first aspect, the present invention provides a method for processing a carbon nanomaterial, comprising the steps of:

(1) carrying out ultrasonic treatment on the carbon nano material in the solvent in the presence of a surfactant to obtain a nano carbon dispersion;

optionally (2) separating and/or washing the nanocarbon.

In one or more embodiments, the solvent is a polar solvent, such as water, preferably deionized water.

In one or more embodiments, the concentration of the carbon nanomaterial in the solvent is 1-30mg/mL, 2-25mg/mL, 5-20mg/mL, 7-15mg/mL, 8-12mg/mL, 10 mg/mL.

In one or more embodiments, the carbon nanomaterials include, but are not limited to, carbon nanotubes, carbon nanospheres, preferably multi-walled carbon nanotubes.

In one or more embodiments, the surfactant in step (1) is selected from the group consisting of Triton X-100, Tween-20, Tetronic 1307, S17, PEG and PVP; preferably PVP. The concentration of the surfactant is 0.1 to 3%, preferably 0.1 to 2%, more preferably 0.1 to 0.8%.

In one or more embodiments, the power of the ultrasound in step (1) may be 80-200W, preferably 100W. The ultrasonic time is 1-3 hours.

In one or more embodiments, the stability of the dispersion in step (1) can be expressed in terms of absorbance at 400 nm. The absorbance of the dispersion at 400nm can be maintained above 0.468 for 10 days, above 0.440 for 20 days, and above 0.455 for 30 days; preferably, it can be maintained at 0.431 or more for 60 days.

In one or more embodiments, the method may further comprise a step of determining the stability of the dispersion with an absorbance at 400nm after step (1).

In one or more embodiments, the separating the nanocarbon of step (2) comprises a centrifugation step. The rotational speed of the centrifugation may be 10000-. The time for centrifugation may be 10-40min, preferably 15-30 min. Washing the nanocarbon of step (2) includes a process of resuspending and separating the nanocarbon using a detergent.

The second aspect of the present invention provides a method for preparing a nanocarbon conjugate, comprising the steps of:

(2) separating and/or washing the nanocarbon;

(3) activating the nano carbon;

(4) and mixing the nano carbon and the antigen in the coupling solution and incubating to obtain the conjugate.

Steps (1), (2) are as described in steps (1), (2) of the first aspect herein.

In one or more embodiments, the nanocarbon is activated in step (3) using EDC and NHS. In one or more embodiments, the weight ratio of nanocarbon to EDC and NHS is in the range of 0.5-2:0.5-2:0.5-2, preferably 1:1: 1. The activation time may be 20-50min, preferably 30 min. The activation may be performed in a coupling solution. Ultrasonic dispersion is preferably performed before activation to accelerate activation.

In one or more embodiments, the weight ratio of nanocarbon to antigen in the coupling solution in step (4) is 30:1 to 10:1, preferably 20: 1. The nanocarbon and the antigen are preferably dissolved and diluted in the coupling solution before mixing. The incubation time may be 20-50min, preferably 30 min.

In one or more embodiments, the coupling solution contains 2-morpholinoethanesulfonic acid (MES). The concentration of MES in the coupling solution is 1-200mM, preferably 20-100mM, more preferably about 50 mM. The pH of the coupling solution is 5.0 to 7.0, preferably 5.5 to 6.5, more preferably about 6.0.

In one or more embodiments, the antigenic species coupled (labeled) to the nanocarbon can be any immunogenic macromolecule, including, but not limited to, proteins, polysaccharides, nucleic acids.

In one or more embodiments, the antigenic agent is an antibody, such as an IgG, IgM, or IgA.

In one or more embodiments, the antigenic material is a viral antigen, such as a protein or polysaccharide of a virus. Preferably, the antigen is the spike protein of a coronavirus or its receptor binding domain (e.g. S-RBD of a novel coronavirus) or murine IgG (e.g. for control).

In one or more embodiments, the method of preparing a nanocarbon conjugate may further comprise the steps of: and (4) blocking the uncoupled nanocarbon in the coupling solution.

In one or more embodiments, the method of preparing a nanocarbon conjugate may further comprise the steps of: and (3) suspending the nano-carbon conjugate in nano-carbon preservation solution.

In one or more embodiments, the nanocarbon preservation solution comprises Tris, preferably also containing Bovine Serum Albumin (BSA). The concentration of Tris is 1-200mM, preferably 20-100mM, more preferably about 50 mM. The concentration of bovine serum albumin is 0.1-2%, preferably 0.2-1%, more preferably about 0.5%. In some embodiments, the pH of the nanocarbon preservation solution is 7 to 9, preferably 7.5 to 8.5, more preferably about 8.

The present invention also provides a method of making a nanocarbon detection article, the method comprising: the nanocarbon conjugates prepared by the method according to any of the embodiments of the second aspect of the present disclosure are distributed on a solid support.

In one or more embodiments, the solid support is a glass fiber.

In one or more embodiments, the detection article is a detection card.

In one or more embodiments, the test card comprises: the device comprises a sample pad, a combination pad and a detection pad, wherein a solid phase carrier distributed with a nano carbon conjugate is positioned on the combination pad.

In one or more embodiments, the detection pad contains an antigen or antibody that binds to a neutralizing antibody to be detected.

In one or more embodiments, the binding pad further comprises a nanocarbon conjugate for use as a control, wherein the nanocarbon-conjugated antigen is a control antibody (e.g., IgG); the detection pad also contains an antigen or antibody that binds to the control antibody.

In one or more embodiments, the detection pad comprises a nitrocellulose membrane having immobilized thereon an antigen or antibody that binds to a neutralizing antibody to be detected and/or an antigen or antibody that binds to the control antibody. The method further comprises the step of immobilizing the antigen or antibody on a nitrocellulose membrane.

In one or more embodiments, the test card further contains a blood filtration membrane and/or an absorbent pad. The method further comprises the step of preparing a blood filtration membrane and/or the step of preparing an absorbent pad.

In one or more embodiments, the neutralizing antibody is a novel coronavirus neutralizing antibody.

In one or more embodiments, the method may further comprise the step of assembling a hemofiltration membrane, a sample pad, a conjugate pad, a detection pad, and an absorbent pad, wherein the sample pad, the conjugate pad, the detection pad, and the absorbent pad are sequentially adjacent, the hemofiltration membrane is positioned on the sample pad, and edges of adjacent regions overlap.

The present invention also provides a nanocarbon detection article for detecting neutralizing antibodies, comprising a nanocarbon conjugate prepared by a method according to any one of the embodiments of the second aspect herein.

In one or more embodiments, the nanocarbon conjugates are distributed on a solid support.

In one or more embodiments, the detection article is a detection card.

In one or more embodiments, the test card comprises: the kit comprises a sample pad, a combination pad and a detection pad, wherein a solid phase carrier distributed with a nano carbon conjugate is positioned on the combination pad.

In one or more embodiments, the detection pad comprises a nitrocellulose membrane having immobilized thereon an antigen or antibody that binds to a neutralizing antibody to be detected and/or an antigen or antibody that binds to the control antibody.

In one or more embodiments, the test card further contains a blood filtration membrane and/or an absorbent pad.

In one or more embodiments, the test card comprises a sample pad, a conjugate pad, a test pad, and an absorbent pad connected in series, and a blood filter membrane is positioned over the sample pad. Wherein the edges of adjacent regions overlap.

Methods of processing carbon nanomaterials, methods of making nanocarbon conjugates, and uses of the methods of making nanocarbon conjugates described herein in the preparation of nanocarbon detection articles. The test article is a test card.

Drawings

FIG. 1: the carbon nano material is schematically shown before and after activation, the left graph is before activation, and the right graph is after activation.

FIG. 2: schematic representation of negative samples.

FIG. 3: schematic representation of positive samples.

FIG. 4: and detecting the concentration gradient of the quality control product.

FIG. 5: and (5) carrying out national standard gradient detection.

FIG. 6: the effect before and after the nanocarbon treatment in example 1 was shown in the left figure and after the treatment in the right figure.

FIG. 7: the detection card is composed of an internal basic structure.

Detailed Description

To make the features and effects of the present invention comprehensible to those skilled in the art, general description and definitions are made below with reference to terms and expressions mentioned in the specification and claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The theory or mechanism described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, i.e., the present disclosure may be practiced without being limited by any particular theory or mechanism.

The terms "comprising," including, "" containing, "and the like, herein, encompass the meanings of" consisting essentially of … … "and" consisting of … …, "e.g., when" A comprises B and C, "A consists essentially of B and C" and "A consists of B and C" are disclosed herein, and are to be considered as having been disclosed herein.

All features defined herein as numerical ranges or percentage ranges, such as numbers, amounts, levels and concentrations, are for brevity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to cover and specifically disclose all possible subranges and individual numerical values (including integers and fractions) within the range.

Herein, unless otherwise specified, percentages refer to mass percentages and ratios to mass ratios.

Herein, when embodiments or examples are described, it is understood that they are not intended to limit the invention to these embodiments or examples. On the contrary, all alternatives, modifications, and equivalents of the methods and materials described herein are intended to be included within the scope of the invention as defined by the appended claims.

In this context, for the sake of brevity, not all possible combinations of features in the various embodiments or examples are described. Therefore, as long as there is no contradiction between combinations of these technical features, any combinations of the technical features in the respective embodiments or examples may be made, and all possible combinations should be considered as the scope of the present specification.

It is to be understood that within the scope of the present invention, the above-described technical features of the present invention and the technical features described in detail below (e.g., the embodiments) may be combined with each other to constitute a preferred embodiment.

The invention provides a method and a device for detecting an antibody by using a labeled antigen and a preparation method of the device. The invention is based on an immunochromatography technology, and adopts a nano carbon labeling sandwich method to detect the antibody. The inventors found that by treating the carbon nanomaterial to attach a surfactant to the surface thereof (as shown in fig. 1), the carbon nanomaterial can be uniformly and stably dispersed in an aqueous phase to facilitate labeling of an antigen or an antibody. The dispersed nanocarbon can be used to prepare a rapid detection device (e.g., a detection card) that can measure the level of neutralizing antibodies (e.g., neutralizing antibodies against the S protein or S-RBD of a novel coronavirus) within 15 min.

Accordingly, the present invention firstly provides a method for treating a carbon nanomaterial or for preparing nanocarbon, comprising the steps of:

optionally (2) separating and/or washing the nanocarbon.

Any form or shape of carbon nanomaterials are suitable in the art for use in the above methods, including but not limited to carbon nanotubes, carbon nanospheres, preferably multi-walled carbon nanotubes. The size of the carbon nanomaterial is not limited. The carbon nanomaterial treated by the method of the present invention is referred to herein as "nanocarbon".

Typically, the carbon nanomaterial forms a mixture with the solvent, with the majority of the carbon nanomaterial precipitating to the bottom of the solvent. The nanocarbon treated in step (1) is uniformly and dispersedly suspended in the solvent, as shown in fig. 6. The solvent is a polar solvent, such as water, preferably deionized water. The concentration of the carbon nano material in the solvent is 1-30mg/mL, 2-25mg/mL, 5-20mg/mL, 7-15mg/mL, 8-12mg/mL and 10 mg/mL.

The surfactant in the step (1) is selected from Triton X-100, Tween-20, Tetronic 1307, S17, PEG and PVP; preferably PVP. The concentration of the surfactant is 0.1-3%, such as 0.2-2.5%, 0.3-2.0, 0.4-1.5%, 0.5-1.0%, 0.6-0.8%, 2% -3% or 0.1-1%.

The power of the ultrasound in step (1) may be 80-200W, preferably 100W. The ultrasonic time is 1-3 hours. The ultrasound may be continuous ultrasound or cyclic ultrasound. For cyclic ultrasound, the ultrasound on time may be 1-3s, preferably 1 s; the ultrasound off time may be 1-3s, preferably 2 s; the total duration of the circulating ultrasound can be 1-10 h; preferably 2-8h, such as 2h, 4h, 5h, 6h, 8 h.

The stability of the dispersion in step (1) can be expressed in terms of absorbance at 400 nm. The absorbance of the dispersion at 400nm can be maintained above 0.468 for 10 days, above 0.440 for 20 days, and above 0.455 for 30 days; preferably, it can be maintained at 0.431 or more for 60 days. Therefore, in order to determine the quality of the dispersion, the nanocarbon treatment method may further comprise a step of measuring the stability of the dispersion with absorbance at 400nm after step (1).

The separation of the nanocarbon of step (2) includes any process of separating the nanocarbon from other components in the solution using chemical or physical properties of the nanocarbon. Exemplary methods are centrifugation. The rotational speed of centrifugation may be 10000-15000 rpm. Preferably 13000 revolutions per minute. The time for centrifugation may be 10-40min, preferably 15-30 min. Washing the nanocarbon of step (2) includes a process of resuspending and separating the nanocarbon using a detergent. Detergents include, but are not limited to: triton X-100, Tween-20, Tetronic 1307, S17, S9, PVA, PEG, PVP, preferably the solvent of step (1). The method for separating the nanocarbon is as described above, and centrifugation is preferable. The number of washing in step (2) may be plural, preferably at least two.

The prepared nano carbon can be used for coupling an antigen. The invention also provides a method for preparing the nanocarbon conjugate, which comprises

(2) separating and/or washing the nanocarbon;

(3) activating the nano carbon;

Steps (1), (2) are as described herein for the method of treating carbon nanomaterials.

Typically, the nanocarbon is activated to improve the antigen coupling efficiency. The activation refers to activating groups on the nanocarbon, such as carboxyl groups. Illustratively, EDC and NHS are common reagents used to couple the amino group of the antigen substance and the carboxyl group of the nanocarbon. The weight ratio of the nano carbon to EDC and NHS is 0.5-2:0.5-2:0.5-2, preferably 1:1: 1. The activation time may be 20-50min, preferably 30 min. The activation may be performed in a coupling solution. Ultrasonic dispersion is preferably performed before activation to accelerate activation.

The weight ratio of the nanocarbon to the antigen in the coupling solution in the step (4) is 30:1 to 10:1, preferably 20: 1. The nano carbon and the macromolecules are preferably dissolved and diluted in the coupling solution and then mixed. The incubation time may be 20-50min, preferably 30 min. The ratio of the amino group to the nano carbon and the incubation time can be determined according to the content of the amino group on the antigen. The carbon nano conjugate obtained in the step (4) can be separated from liquid by centrifugation.

The coupling solution may be 2-morpholinoethanesulfonic acid (MES), the concentration of MES in the coupling solution may be about 50mM, and the pH of the coupling solution may be about 6.0.

In the nanocarbon conjugates herein, the antigenic material conjugated (labeled) to the nanocarbon can be any macromolecule having immunogenicity, including but not limited to proteins, polysaccharides, nucleic acids. The antigen itself may be an antibody, for example IgG, IgM or IgA. The antigenic substance is preferably a viral antigen, such as a viral protein or polysaccharide, in particular a viral surface protein or its Receptor Binding Domain (RBD), preferably the S protein of a coronavirus or its RBD. In a preferred embodiment, the antigenic material is a neocoronavirus S-RBD (neocoronavirus S-protein receptor binding domain) or murine IgG (for control).

In order not to affect the detection, the method for preparing the nanocarbon conjugate may further comprise the steps of: and (4) blocking the uncoupled nanocarbon in the coupling solution. The blocking agent may be any agent suitable for blocking nanocarbons, such as BSA, Tween20 or a combination of blocking agents. The amount of BSA blocking agent may be 2% of the total blocking solution, and the concentration of Tween20 blocking solution may be 1% of the total blocking solution. For example 2% BSA in 50mM Tris, or 10% Tween 20. The blocking time may be 20-40min, preferably 30 min. The nanocarbon conjugates obtained by blocking can be centrifuged and separated from the liquid.

The method for preparing a nanocarbon conjugate may further comprise the steps of: the nanocarbon conjugate is suspended in a nanocarbon preservation solution, which can be any reagent suitable for preserving nanocarbon conjugates, such as Tris (hydroxymethyl) aminomethane (Tris). The nanocarbon preservation solution may contain Bovine Serum Albumin (BSA). An exemplary nanocarbon preservation solution is 0.5% BSA in 50mM Tris. The resulting liquid may be shaken, sonicated, and agitated for uniform mixing. The step can be carried out for a plurality of times, the preserving fluid is added again after each centrifugation, and the using amount of the preserving fluid is decreased gradually. The nano carbon conjugate in the preservation solution can be directly used for detection or distributed on a dry solid phase carrier in the form of solution.

In a specific embodiment, the method of preparing a nanocarbon conjugate comprises the steps of:

(1) ultrasonically dispersing the prepared nano carbon in the coupling solution, and placing the nano carbon in a centrifugal tube;

(2) EDC and NHS are prepared in situ by coupling liquid, and are respectively added into a centrifuge tube containing nano-carbon standby liquid, mixed evenly and stood for room temperature activation;

(3) diluting the antibody to be marked in the coupling solution, adding the activated nano carbon solution, uniformly mixing, and coupling at room temperature;

(4) adding the sealing liquid, uniformly mixing, and sealing at room temperature;

(5) centrifuging and discarding the supernatant;

(6) adding nano-carbon preservation solution, performing ultrasonic homogenization, centrifuging, and discarding the supernatant;

(7) adding nano carbon preservation solution, and performing ultrasonic homogenization;

(8) diluting the marked nano carbon (1:12-1:20, preferably 1:15), spreading on a bonding pad, drying at 37 ℃ overnight, cutting, drying and sealing. The bonding pad material can be glass fiber.

The present invention also provides a method of making a nanocarbon detection article, based on the method of making a nanocarbon conjugate described herein, the method comprising: the nanocarbon conjugates prepared by the methods of preparing nanocarbon conjugates described herein are distributed on a solid support. The preferred solid support is glass fiber. A preferred example of the detection article is a detection card.

In some embodiments, a test card comprises: a sample pad (or sample area), a conjugate pad (or junction area), and a detection pad (or detection area), wherein the solid phase carrier distributed with the nanocarbon conjugate is located on the conjugate pad.

The sample pad is for receiving a sample. The step of preparing the sample pad comprises: the sample pad substrate was soaked with 40ml of sample pad treatment solution until the surface was bubble free and dried overnight at 37 ℃. And cutting the sample pad, drying and sealing. The composition of the sample pad treatment solution is well known in the art and may be, for example, sodium tetraborate buffer. The amount of the sample pad treatment solution used may be 30 to 50ml, preferably 40 ml. Any sample pad substrate known in the art may be used, for example: polyester fiber films, glass fibers 8955, 8964, Z90B, Z60B, KB50, etc., preferably glass fiber 8964.

The sample pad may be covered or partially covered with a blood filtration membrane. The blood filter membrane is used for retaining interfering components such as red blood cells in blood when a sample moves through. The method further comprises the step of preparing a blood filtration membrane. The step of preparing the blood filtration membrane may comprise: soaking the filter membrane substrate with the filter membrane treatment solution until no bubbles exist on the surface, and drying at 37 ℃ overnight. Cutting the blood filtering membrane, drying and sealing. The composition of the blood filter membrane treatment solution is well known in the art, and for example, a 5-10mg/ml trisodium citrate solution containing 0.1-1.0mg/ml RBC antibody can be used. The amount of the blood filtration membrane treatment solution used is determined according to the size of the blood filtration membrane, and may be 30 to 50ml, preferably 40 ml. Any filter membrane substrate known in the art may be used, for example LF1, VF1, VF2, MF1, Cobert RB0.45, RB1.1, PSM0180-B, etc., of Whatman, preferably Cobert RB 0.45.

The binding pad comprises a solid support having nanocarbon conjugates distributed thereon, wherein the nanocarbon conjugates are antigens capable of specifically binding to neutralizing antibodies (e.g., S-RBD of neocoronavirus). The solid support may be any material to which the nanocarbon conjugate can be attached, such as glass fiber. The attached nanocarbon conjugates detach from the appendage supports upon contact with the sample and move forward along with the capillary action of the sample. For example, the nanocarbon may be attached to glass fibers and dissolve upon contact with the sample and move with the sample. The conjugate pad (e.g., on a solid support therein) may further comprise a nanocarbon conjugate for use as a control, wherein the nanocarbon-conjugated antigen is a control antibody (e.g., IgG). The nanocarbon conjugates for detecting neutralizing antibodies in the sample and the nanocarbon conjugates used as a control may be mixedly distributed on the binding pad, or may be separately located on different binding pads. The conjugate pads containing the nanocarbon conjugates are preferably contiguous. The preparation steps of the bonding pad comprise: and (3) paving the nanocarbon conjugate for detecting the neutralizing antibody and the nanocarbon conjugate used as a control on the base material of the bonding pad according to a proper proportion (preferably 1:15), and drying at 37 ℃ to prepare the bonding pad. Specifically, the preparation step of the conjugate pad includes: the nanocarbon conjugates for detecting the neutralizing antibodies of the sample and the nanocarbon conjugates used as the control are diluted by the treatment solution of the binding pad according to the proper proportion (preferably 1:15), the base material of the binding pad is treated by 14ml of the dilution until the surface has no bubbles, the base material is dried at 37 ℃ overnight, and the binding pad is dried and sealed after being cut. The composition of the conjugate pad treatment solution is well known in the art and may be, for example, TRIS buffer. The amount of the treatment solution for the conjugate pad may be 12 to 15ml, preferably 14 ml. Any bond pad substrate known in the art may be used, for example: glass fibers 8955, 8964, Z90B, Z60B, KB50 and the like, preferably glass fiber KB 50.

The detection pad contains an antigen or antibody (also referred to herein as a T-wire) that binds to a neutralizing antibody to be detected. The antigen is preferably S-RBD of the novel coronavirus. The detection pad is a solid support, such as a nitrocellulose membrane, for immobilizing the antigen or antibody. Thus, the method further comprises the step of immobilizing the antigen or antibody on a nitrocellulose membrane. In addition, the detection pad also contains (e.g., immobilized on a nitrocellulose membrane) an antigen or antibody (also referred to herein as C-line) that binds to the control antibody. The antigen or antibody immobilized on the detection pad does not move with the movement of the sample.

The preparation steps of the detection pad containing the nitrocellulose membrane comprise: (1) preparing coating liquid of a quality control line (C line) and a detection line (T line); (2) and respectively coating the C-line antibody and the T-line antigen on a nitrocellulose membrane fixed on a PVC (polyvinyl chloride) bottom plate by adopting a gold spraying and membrane scratching instrument. The detection line (T line) coating solution is prepared by diluting S-RBD to about 1.5mg/ml with buffer solution; the quality control line (line C) coating was formulated by diluting goat anti-mouse IgG to about 2.0mg/ml with buffer. The dilution used is well known in the art, for example, a buffer comprising about 10mM PBS, about 1% sucrose, and about 0.1% proclin300 (pH 7.4).

The test card may also contain an absorbent pad for providing suction for liquid flowing in the test card through the conjugate pad and the test pad. The material of the absorbent pad is a thick cellulose material with a fast climbing speed and a very high water adsorption capacity. The method further comprises the step of preparing an absorbent pad.

The method of making the detection article may further comprise the step of assembling the blood filtration membrane, the sample zone, the conjugate zone, the detection zone, and/or the absorbent pad such that edges of adjacent zones or components overlap. In particular, the assembly may be: the prepared detection pad is pasted with absorbent paper (namely an absorption pad), is pasted on a base plate (such as a PVC base plate), and is sequentially pasted according to the sequence: binding pad (IgG), binding pad (S-RBD), sample pad, hemofilter, resulting in the test card (shown in FIG. 6). The method also comprises the step of cutting the detection card into reagent strips with the width of 3-4 mm.

The invention also provides a kit for detecting neutralizing antibodies (e.g., neutralizing antibodies of a novel coronavirus) by nanocarbon-labeled immunochromatography, which kit comprises a detection article, e.g., a detection card, as described herein. The kit is simple to operate, has an intuitive result, is suitable for on-site rapid detection, and can be used for detecting the novel coronavirus neutralizing antibody in serum, plasma or whole blood of a suspected group infected by the novel coronavirus and a novel coronavirus vaccine immunized person. The kit also includes instructions for using the test article to detect neutralizing antibodies, a reagent strip housing, optional blood pretreatment reagents (e.g., 10mM PBS), and the like. In the invention, the detection sensitivity of the kit is high, and the detection limit is as low as 500 ng/ml.

Principle of detection

The kit adopts a nano-carbon immunochromatography technology and a double-antigen sandwich method to detect the content of the new crown neutralizing antibody. The reagent strip adopts a Nitrocellulose (NC) membrane as a matrix, and fixed S-RBD (detection line T line) and goat anti-mouse IgG antibody (quality control line C line) as detection lines. The combination pad is marked by S-RBD and mouse IgG nanocarbon, when serum or whole blood inoculated with the vaccine is dripped into a sample adding area (sample pad) of a test strip during detection, the sample can be chromatographed forwards under the capillary action, and the nanocarbon on the combination pad can be dissolved and then moves forwards together with the sample. After the sample reaches the detection area, the S-RBD marked by the nano carbon, the neutralizing antibody in the sample and the S-RBD coated on the detection line form a compound, the detection line is shown to be gray or black, and the content of the antibody in the sample is in positive correlation with the color development intensity of the detection line. After the reaction is finished, the color development condition is directly observed by naked eyes for testing, and the content of the RBD antibody can be judged through the color development intensity of the colloidal carbon on the detection line. The quality control line should appear black regardless of the detection line color intensity. The absence of a colored signal on the control line means that the test is invalid and the sample must be retested.

The invention has the following beneficial effects:

according to the invention, a new crown neutralizing antibody kit is prepared by adopting a colloidal carbon labeling method, the nano carbon raw material is cheap and environment-friendly, and is easier to distinguish compared with the black and white color of a nitrocellulose membrane, so that the problems of low resolution and difficulty in interpretation when the neutralizing antibody concentration is low in a colloidal gold competition method are solved; treating a whole blood sample through a blood filtering membrane, dropwise adding a sample buffer solution by using a trace sample, and quickly detecting the content of a novel coronavirus neutralizing antibody in a human serum sample, a plasma sample and the whole blood sample within 15 min; meanwhile, the defects of complex technology, high cost and the like in the detection by adopting a fluorescence immunochromatographic competitive method are overcome. The method has the advantages of simple operation, high specificity, high sensitivity, visual result, safety and low price, and is a simple, accurate and economic field detection method.

The invention adopts a sandwich method to detect the total neutralizing antibody of the new corona, selects the S-RBD of the S protein of the new corona virus with high specificity, and can effectively detect the total neutralizing antibody generated by a human body, including IgA, IgM and IgG.

Examples

Example 1 nanocarbon treatment

And (3) nano-carbon treatment process:

1. adding 10mg of multi-walled carbon nanotubes into 1mL of deionized water;

2. polyvinylpyrrolidone (PVP) was added to the liquid in step 1 and sonication was performed, with a PVP concentration of 0.6%. Ultrasonic parameters: ultrasonic power: 100W; ultrasonic on-time: 1 s; ultrasonic off time: 2 s; total time of ultrasound: 2-8 h.

Content of PVP	0	0.1	0.3	0.6	0.8	1.0	2.0
								Absorbance at 400nm	/	0.476	0.482	0.492	0.432	0.401	0.368

3. The stability of the dispersions was investigated by measuring the absorbance on days 10, 20, 30 and 60 after the initial dispersion. Data show that the nanocarbon system remains basically stable after being processed;

days of dispersion (sky)	0	10	20	30	60
						Absorbance at 400nm	0.489	0.468	0.440	0.455	0.431

4. Centrifuging the nano carbon subjected to ultrasonic treatment, washing with pure water for 2 times, and washing off redundant surfactant;

5. and removing the supernatant. Performing third centrifugation by using the coupling solution (centrifugation parameters: 13000 r/min, 30min of centrifugation);

6. removing supernatant, and resuspending and mixing with 5ml of coupling solution for later use.

Example 2 preparation of test cards

Preparation of blood filtration membranes

The blood filtering membrane treatment liquid is trisodium citrate (8mg/ml), RBC antibody (0.5mg/ml, Guangzhou ghrelin, product number G0093R), each blood filtering membrane (Corbert RB0.45, 250mm × 300mm) is soaked by 40ml of blood filtering membrane treatment liquid until the surface is free of bubbles, and the blood filtering membrane treatment liquid is dried at 37 ℃ overnight; and drying and sealing after cutting.

Preparation of sample pad

The sample pad treatment solution is sodium tetraborate buffer solution, each sample pad (glass fiber 8964, 254mm × 300mm) is soaked by 40ml of sample pad treatment solution until the surface is free of bubbles, and the sample pad treatment solution is dried at 37 ℃ overnight; and drying and sealing after cutting.

Preparation of carbon pad (conjugate pad)

Firstly, marking S-RBD nano carbon, and specifically comprising the following steps:

(1) dispersing the nano-carbon prepared in example 1 by an ultrasonic instrument, and putting 400ul of nano-carbon in a 1.5ml centrifuge tube;

(2) using coupling solution to prepare 0.8mg/ml EDC and 0.8mg/ml NHS in situ;

(3) adding 100ul of 0.8mg/ml EDC and 100ul of 0.8mg/ml NHS into 400ul of nanocarbon, mixing uniformly, standing and activating at room temperature for 30 min;

(4) diluting 40ug of new coronavirus S-RBD (or control mouse IgG) to 100ul of coupling solution, adding into activated nanocarbon solution, mixing, and coupling at room temperature for 30 min;

(5) adding blocking solution No. 1 (50mM Tris (Tris hydroxymethyl aminomethane), 2% BSA (bovine serum albumin), pH 8.0)300ul, adding blocking solution No. 2 (10% Tween20)100ul, mixing, and blocking at room temperature for 30 min;

(6) centrifuging at 13000rpm for 15min, and then discarding the supernatant;

(7) adding 400ul of carbon preservation solution (50mM Tris, 0.5% Bovine Serum Albumin (BSA), pH 8.0), homogenizing with ultrasound, centrifuging at 13000rpm for 15min, and discarding the supernatant;

(8) adding 200ul of carbon preservation solution, and performing ultrasonic treatment;

(9) and (6) paving. The marked nano carbon conjugate for detecting the neutralizing antibody of the sample and the nano carbon conjugate used as the control are mixed according to the proportion of 1:15 diluted with conjugate pad treatment and then 14ml of the dilution was spread onto glass fiber KB50 (200mm x 280mm, sandiskei (fossa) paper limited) until no bubbles were present on the surface;

(10) oven dried at 37 degrees overnight to prepare conjugate pads (RBD carbon pads).

The same method used nanocarbon labeling of murine IgG.

The dried mouse IgG and RBD carbon pads were cut to 5mm width, dried and sealed.

Preparation of detection pad (containing nitrocellulose membrane)

Firstly, C, T preparation of a coating liquid:

(1) preparing a T-line coating liquid: S-RBD was diluted to 1.5mg/ml with buffer (buffer pH 7.4, including 10mM PBS, 1% sucrose, and 0.1% proclin 300);

(2) c, preparing a coating solution for the wire: the goat anti-mouse IgG was diluted to 2.0mg/ml with buffer (same as T line);

(3) sticking a nitrocellulose membrane: tearing off the protective paper in the middle by using a PVC (polyvinyl chloride) bottom plate, and sticking the nitrocellulose membrane along the lower edge of the protective paper on the upper surface;

(4) coating the C-line antibody and the T-line antibody on a nitrocellulose membrane fixed on a bottom plate respectively by adopting a gold spraying and membrane scratching instrument;

(5) and drying the scratched PVC plate at 37 ℃ overnight, adding a drying agent, and sealing for later use.

Assembling a large card: firstly, sticking 17mm of absorbent paper (an absorption pad) on a PVC (polyvinyl chloride) bottom plate, and sequentially sticking a carbon pad mouse IgG, a carbon pad RBD (binding domain protein), a sample pad and a blood filtering membrane;

and after forming a large plate, cutting the large plate into test strips with the width of 4mm, installing the test strips on a shell, adding a drying agent, and sealing and storing at normal temperature to obtain the detection card.

Embodiment 3 detection method of detection card

Negative samples: selecting human venous blood whole blood, plasma, serum and fingertip blood which are not inoculated with the vaccine as negative samples;

according to the kit instruction: adding 25 mul of negative sample, dripping 3 drops of sample diluent, and judging the result after 15 min. The test result is shown in fig. 1, the negative sample test quality control line is normal, the negative whole blood, the negative fingertip blood, the negative serum and the negative plasma have no obvious difference and are all negative, and the test backgrounds of the whole blood and the fingertip blood are clean;

positive samples: the non-vaccinated human venous blood whole blood, plasma, serum negative samples and sample diluent (10 mM PBS, pH 7.4) are used, and the national Xinguan neutralizing antibody standard substance is diluted by 100 times respectively to be used as a positive sample test.

According to the kit instruction: adding 25 mul of positive sample, dripping 3 drops of sample diluent, and judging and reading the result after 15 min. The test result is shown in fig. 2, C, T lines exist in the test of the positive samples, the color development is uniform, and the positive quality control products diluted by the negative whole blood, the negative serum, the negative plasma and the sample diluent have no obvious difference and do not influence the experimental result.

Gradient detection

Diluting the neutralizing antibody quality control product with sample diluent at different concentrations by 20ug/ml, 10ug/ml, 5ug/ml, 2.5ug/ml, 1ug/ml, 0.5ug/ml, 0.25ug/ml, 0.1ug/ml, and performing gradient dilution on the national Xinguan neutralizing antibody standard product: 1:10, 1:20, 1:50, 1:100, 1:200 and 1:400, and the results are shown in figure 3, the sensitivity of the detection quality control product of the kit disclosed by the invention reaches 500ng/ml, and the sensitivity of the national neutralizing antibody standard product reaches 1:100, namely 10U/ml; for repeated detection of national standard products 1:50 and 1:100 (as shown in fig. 4), the results are consistent in color development and stable in experimental results, and the detection data are as follows:

table 1: gradient test results

Concentration of quality control product

0.1ug/ml

0.25ug/ml

0.5ug/ml

1ug/ml

2.5ug/ml

5ug/ml

Colorimetric results

C1

C3.5

C4

C5

C7

Dilution ratio of standard

1:400

1:200

1:100

1:50

1:20

1:10

Colorimetric results

C1

C3

C3.5

C4

C5+

C6+

Example 4 real sample detection

According to the kit instruction: 25ul of fingertip blood is taken, 3 drops of sample diluent are added dropwise, and the result is read after 15 min.

By testing 125 clinical specimens: 64 negative samples were not vaccinated with new corona vaccine and uninfected, 7 unvaccinated infected, 31 vaccinated with pfeiri vaccine, 18 vaccinated with danio-na vaccine, 5 vaccinated with booster vaccine. The detection results are as follows:

all testers detected a clean background and could detect different levels of neutralizing antibody.

Table 2: real sample test results

From the preparation of the detection card of the kit in the embodiment 1 and the verification of the detection method in the embodiment 2, the nanocarbon labeling immunochromatography method and the kit for detecting the novel coronavirus neutralizing antibody provided by the patent have the advantages of simple operation, high sensitivity and visual result, and are a simple, accurate and economic field rapid detection method.

Claims

1. A method of processing carbon nanomaterials comprising the steps of:

(1) performing ultrasonic treatment on the carbon nano material in the solvent in the presence of a surfactant to obtain a nano carbon dispersion,

optionally (2) separating and/or washing the nanocarbon;

preferably, the solvent is a polar solvent, such as water,

preferably, the surfactant is selected from one or more of the following: triton X-100, Tween-20, Tetronic 1307, S17, PEG and PVP,

preferably, the carbon nanomaterial includes carbon nanotubes and carbon nanospheres.

2. The method of claim 1, further comprising one or more features selected from the group consisting of,

the concentration of the surfactant is 0.1-3%,

the concentration of the carbon nano material in the solvent is 1-30mg/mL,

the power of the ultrasound in the step (1) is 80-200W,

the ultrasonic treatment in the step (1) is carried out for 1 to 3 hours,

the method further comprises a step of determining the stability of the dispersion with an absorbance at 400nm after step (1),

the separation of the nanocarbon in step (2) comprises a centrifugation step,

washing the nanocarbon of step (2) includes a process of resuspending and separating the nanocarbon using a detergent.

3. A method of preparing a nanocarbon conjugate, comprising the steps of:

(2) separating and/or washing the nanocarbon;

(3) activating the nano carbon;

(4) mixing nano carbon and an antigen in a coupling solution and incubating to obtain a conjugate,

the method further comprises one or more features selected from,

the solvent is a polar solvent, such as water,

the surfactant is selected from one or more of the following: triton X-100, Tween-20, Tetronic 1307, S17, PEG and PVP,

the concentration of the surfactant is 0.1-3%,

the carbon nano material comprises carbon nano tubes and carbon nano spheres,

the concentration of the carbon nano material in the solvent is 1-30mg/mL,

the power of the ultrasound in the step (1) is 80-200W,

the ultrasonic treatment in the step (1) is carried out for 1 to 3 hours,

the separation of the nanocarbon in step (2) comprises centrifugation,

4. The method of claim 3,

EDC and NHS are used for activating the nano-carbon in the step (3),

the weight ratio of the nano carbon to the EDC and the NHS is 0.5-2:0.5-2:0.5-2,

the activation time is 20-50min,

the activation is carried out in a coupling solution.

5. The method of claim 3,

the weight ratio of the nano carbon to the antigen in the coupling solution in the step (4) is 30:1 to 10:1,

the incubation time in step (4) is 20-50min,

the coupling solution contains 2-morpholine ethanesulfonic acid,

the concentration of the 2-morpholine ethanesulfonic acid in the coupling solution is 1-200mM,

the pH value of the coupling solution is 5.0-7.0,

the antigen substance coupled with the nano carbon is protein, polysaccharide or nucleic acid with immunogenicity; preferably, the antigen is an antibody or a viral antigen; more preferably, the antigen is S-RBD or murine IgG of a new coronavirus.

6. The method of claim 3,

the method further comprises the steps of: sealing the uncoupled nano carbon in the coupling solution,

the method further comprises the steps of: suspending the nano carbon conjugate in nano carbon preservative solution,

the nanocarbon preservation solution contains Tris, preferably bovine serum albumin.

7. A method of making a nanocarbon detection article, the method comprising: distributing the nanocarbon conjugates prepared by the method of any one of claims 3 to 5 on a solid support,

the detection article is for detecting neutralizing antibodies, preferably neutralizing antibodies to neocoronaviruses,

the solid-phase carrier is a glass fiber,

the test article is a test card and,

the detection card includes: the device comprises a sample pad, a combination pad and a detection pad, wherein a solid phase carrier distributed with a nano carbon conjugate is positioned on the combination pad.

8. The method of claim 7,

the detection pad contains an antigen or antibody that binds to a neutralizing antibody to be detected, and the method further comprises the step of immobilizing the antigen or antibody on the detection pad; preferably, the antigen is a new coronavirus S-RBD,

the nano carbon conjugate is characterized in that an antigen coupled with nano carbon is a new coronavirus S-RBD; the binding pad further comprising a nanocarbon conjugate serving as a control, wherein the antigen substance conjugated to the nanocarbon is a control antibody, and the detection pad further contains an antigen or an antibody bound to the control antibody, the method further comprising the step of immobilizing the antigen or the antibody bound to the control antibody on the detection pad,

the test card also contains a blood filtration membrane and/or an absorption pad, the method also comprises a step of preparing the blood filtration membrane and/or a step of preparing the absorption pad,

the method may further comprise the step of assembling a blood filtration membrane, a sample pad, a conjugate pad, a detection pad and an absorbent pad, wherein the sample pad, the conjugate pad, the detection pad and the absorbent pad are connected in series, the blood filtration membrane is positioned on the sample pad, and edges of adjacent regions overlap.

9. A nanocarbon detection article for detecting neutralizing antibodies, comprising the nanocarbon conjugate prepared by the method of any one of claims 3-5,

in a preferred embodiment of the method of the invention,

the neutralizing antibody is a neutralizing antibody of a novel coronavirus,

the nano-carbon conjugate is distributed on a solid phase carrier,

the test article is a test card and,

the detection card includes: a sample pad, a combination pad and a detection pad, wherein the solid phase carrier distributed with the nano carbon conjugate is positioned on the combination pad,

the detection pad contains an antigen or antibody that binds to the neutralizing antibody to be detected, preferably, the antigen is neocoronavirus S-RBD,

the nano carbon conjugate is characterized in that an antigen coupled with nano carbon is a new coronavirus S protein or a new coronavirus S-RBD; the binding pad further comprises a nanocarbon conjugate serving as a control, wherein the nanocarbon-conjugated antigen is a control antibody, and the detection pad further contains an antigen or an antibody that binds to the control antibody,

the detection pad comprises a nitrocellulose membrane on which an antigen or antibody that binds to a neutralizing antibody to be detected and/or an antigen or antibody that binds to the control antibody is immobilized,

the test card also contains a blood filtration membrane and/or an absorbent pad,

the test card comprises a sample pad, a conjugate pad, a test pad, and an absorbent pad connected in series, and a hemofilter membrane positioned on the sample pad, wherein edges of adjacent regions overlap.

10. Use of the method of processing carbon nanomaterial of claim 1 or 2, the method of producing a nanocarbon conjugate of claim 3 in the production of a nanocarbon detection article, preferably the detection article is a detection card.