WO2023080535A1

WO2023080535A1 - Lateral flow assay strip for detecting neutralizing antibodies against sars-cov-2, and kit comprising same

Info

Publication number: WO2023080535A1
Application number: PCT/KR2022/016513
Authority: WO
Inventors: 변희정; 박소영
Original assignee: 바디텍메드(주)
Priority date: 2021-11-05
Filing date: 2022-10-26
Publication date: 2023-05-11
Also published as: KR20230065789A

Abstract

The present invention relates to: a lateral flow assay strip for detecting neutralizing antibodies present in a sample obtained from a person infected with COVID-19 or vaccinated against COVID-19; and a kit comprising same. The lateral flow assay strip of the present invention comprises: a sample pad to which a sample is applied; a conjugation pad which is located downstream of the sample pad, and which comprises a complex of a label and a protein comprising the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, and a complex of a control antigen and a label; a porous matrix allowing capillary flow of the sample; a blocking line to which a receptor protein specifically reacting to the receptor-binding domain is fixed; a test line to which a protein comprising the receptor-binding domain is fixed; a control line to which an antibody specifically reacting to the control antigen is fixed; and an absorption pad for absorbing excess sample from the sample applied to the sample pad.

Description

A lateral flow assay strip for detecting neutralizing antibodies against SARS-COV-2 and a kit including the same

The present invention relates to a lateral flow assay strip for detecting neutralizing antibodies to SARS-CoV-2 and a kit including the same, and more particularly, to SARS-CoV-2 using a double antigen sandwich assay. It relates to a lateral flow assay strip capable of detecting a neutralizing antibody against -2 and a kit including the same.

Around December 2019, the third human coronavirus (human CoV), which causes a group infection in Wuhan, Hubei Province, China, and causes fatal pneumonia, is spreading globally through Asia starting in China, and by mid-2022 Until now, there are very high levels of new infections in each country. When the coronavirus enters through the respiratory system of our body, it settles on the surface of respiratory epithelial cells, binds to its receptor protein, Angiotensin converting enzyme -2 (ACE-2) on the cell surface, and penetrates into the body. do.

Currently, the fatality rate due to COVID-19 virus infection is about 2-3%, but the US Centers for Disease Control and Prevention predicts the actual fatality rate due to COVID-19 virus infection to be about 10%, which is about 10% of SARS in the past. Similar, and lower than about 34% of MERS. As of August 17, 2022, the number of confirmed COVID-19 infections worldwide is about 590 million, of which the number of deaths exceeds about 6.46 million, with a fatality rate of about 1.08%, and this figure continues. is increasing

Neutralizing antibodies are antibodies that neutralize the effects of pathogens on cellular lesions by invading cells when infected with pathogens. It is produced by an immune response when infected with a pathogen one or more times, and if it has never been infected with a pathogen, the titer of neutralizing antibodies against the pathogen is very low or non-detectable. When SARS-CoV-2 enters the human respiratory tract or body mucous membrane, it recognizes ACE-2 on the cell surface, binds to the cell, and penetrates into the cell in order to penetrate the host cell (human respiratory epithelial cell). At this time, the viral protein that binds to ACE-2 and helps to penetrate into epithelial cells is Spike protein (S protein). The S protein of SARS-CoV-2 is on the surface of the virus and consists of two subunits, S1 and S2. ACE-2 on the cell surface serves as a receptor for the Receptor Binding Domain (RBD) of the S1 subunit, and the S2 subunit is responsible for passing through the host cell. In order to obtain immunity against the virus by interfering with this intracellular penetration, the human body makes antibodies to the S protein, which are known as SARS-CoV-2 neutralizing antibodies.

As a standard method for testing neutralizing antibodies, the plaque reduction neutralization test (PRNT) method is mainly used. The PRNT method is known as the best immunoassay method for determining whether a person has defense against various viruses. The PRNT method is a method of measuring the neutralizing antibody ability of a virus through a cytopathic effect while infecting cells and culturing for a certain period of time after reacting stepwisely diluted serum with a virus for a certain period of time. In general, the dilution ratio of a sample at which plaque formation is reduced by 50% by a neutralizing antibody is expressed as a titer (eg, PRNT ₅₀ =160). Although a clear titer standard for positive/negative results has not been defined through PRNT measurement results, in the case of the COVID-19 virus, the US FDA recommends a titer of 1:160 as a value that can produce an immune effect as a neutralizing antibody. A lower concentration of 1:80 is suggested as an acceptable (immunizing) neutralizing antibody level in some cases. Since the PRNT method requires the use of live viruses, it requires a facility of Bio-safety level 3, takes about 5 days to confirm the result through cell culture, and has a limit on the number of experiments that can be processed by one person. Because of this, there are many disadvantages in large-scale experiments. As a neutralizing antibody test reagent that has improved the disadvantages of the PRNT test, there are SVNT (Surrogate Virus Neutralization Test) using the principle of PRNT but using the ELISA method, and rapid test products that simplify the test method. . In general, in order to test the amount of only a specific antibody among all antibodies present in the body, a competitive reaction test method should be used. However, when the competitive reaction method is used, it is not easy to distinguish weakly positive samples, so sensitivity and specificity are low.

In view of the above circumstances, the present invention is to provide a lateral flow assay strip for detecting neutralizing antibodies present in a sample obtained from a person infected with COVID-19 or vaccinated with COVID-19 and a kit containing the same. for work purposes

Therefore, the present invention improves the competitive reaction method, filters out a complex of an antibody and a detector that is not a neutralizing antibody or a detector that does not form a complex at the blocking line, which is in front of the test line reaction line, and the neutralizing antibody and the detector Another object is to develop a new system capable of detecting only the complex by a normal sandwich method, thereby confirming the result of a reaction line proportional to the concentration of neutralizing antibody in a sample.

In addition, another object of the present invention is to suppress unnecessary waste of a vaccine or treatment by helping to select the need for vaccination and the timing of vaccination through a neutralizing antibody test for COVID-19.

One embodiment of the present invention, the sample pad to which the sample is applied; A conjugation pad located downstream of the sample pad and having a complex of a label and a protein having a receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, and a complex of a control antibody and a label ; a porous matrix allowing capillary flow of the sample; A blocking line fixed with ACE-2 (Angiotensin-converting Enzyme 2) protein, which is a receptor that specifically reacts to the receptor binding region; a test line immobilized with the protein having the receptor-binding region; a control line immobilized with an antigen that specifically reacts with the control antibody; and an absorbent pad for absorbing an excessive amount of the sample among the samples applied to the sample pad.

The lateral flow assay strip may further include a support plate, and the sample pad, the conjugation pad, the porous matrix, and the absorbent pad may be positioned on the support plate.

The protein immobilized on the blocking line reacts specifically to the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, and the excess complex 210 that is not bound to the antibody and the neutralizing antibody It is possible to capture a receptor binding region bound to an antibody other than that.

The label may be at least one selected from the group consisting of gold nanoparticles, colored latex beads, carbon nanoparticles, selenium nanoparticles, silver nanoparticles, quantum dots, and organic fluorophores.

The antibody immobilized on the blocking line may be ACE-2 (Angiotensin Converting Enzyme 2).

One embodiment of the present invention may also provide a kit comprising the lateral flow assay strip and a housing, wherein the housing covers the sample pad, conjugate pad, blocking line, and absorbent pad.

The present invention can provide a lateral flow assay strip for detecting neutralizing antibodies to COVID-19 present in a sample. In addition, the present invention can provide a lateral flow assay strip that rapidly detects whether or not neutralizing antibodies are formed in a person infected with SARS-CoV-2 or vaccinated against SARS-CoV-2.

When the substance to be identified is mixed in the total analyte, it is common to use the antigen-antibody reaction of the competition method, but in this case, the negative sample has the highest reactivity (line thickness is On the other hand, as the concentration of the positive substance (neutralizing antibody) in the sample increases, the reactivity decreases (the line decreases in thickness), so it is distinguished between negative and weakly positive. This is not easy, and as a result, there is a disadvantage that the sensitivity and specificity of the product are lowered. The present invention modifies the existing competitive reaction method, which is a method for selectively examining only a specific antibody among all antibodies, to preemptively remove non-target antibodies (non-neutralizing antibodies) to compete with. An in vitro diagnostic cartridge of the system is provided in which a blocking line is added to detect the reaction with the neutralizing antibody of interest by a general sandwich reaction. Through these changes in the test system, the advantage of increasing the sensitivity and specificity, which are the disadvantages of competitive reactions, is created, and it has the advantage of making it easier to distinguish changes in antibody concentration, making it possible to perform quantitative as well as qualitative tests. expandability is also increased.

1 is a view showing a lateral flow analysis strip according to an embodiment of the present invention.

Figure 2 is a diagram illustrating the reaction in the conjugation pad shown in Figure 1.

FIG. 3 is a diagram illustrating a reaction at the blocking line shown in FIG. 1 .

FIG. 4 is a diagram illustrating reactions in the test line and control line shown in FIG. 1 .

As used herein, the term "porous material" refers to a material capable of providing capillary motion or lateral flow. Porous materials include nitrocellulose, polyesters or nitrocellulose blends such as cellulose, porous paper, rayon, etc. ), glass fiber, acrylonitrile copolymer, nylon, etc. In the present specification, the porous material constitutes a porous matrix and allows the sample to pass through.

As used herein, the term “sample” refers to any sample presumed to contain neutralizing antibodies, for example infected with SARS-CoV-2 or vaccinated against SARS-CoV-2. It may be obtained from a person with immunity. The sample is preferably liquid, preferably whole blood (plasma, blood, serum), saliva, urine or dilutions thereof.

As used herein, the term "neutralizing antibody" refers to an antibody that defends cells by neutralizing the biological effects of pathogens or infectious particles when they penetrate the body. For example, people infected with SARS-CoV-2 or vaccinated against SARS-CoV-2 develop neutralizing antibodies, which are produced in the receptor-binding region of the SARS-CoV-2 spike protein (RBD). ), inhibiting the binding of SARS-CoV-2 to receptor proteins on the cell surface.

As used herein, the term "receptor binding domain" is a part required to interact with an endogenous receptor to promote membrane fusion and delivery to cells.

Hereinafter, embodiments of the present invention will be described in detail using the accompanying drawings. However, the following examples are merely illustrative of one embodiment of the present invention, and the content of the present invention is not limited to the following examples.

1 is a view showing a lateral flow analysis strip 100 according to an embodiment of the present invention. As shown in FIG. 1, the lateral flow assay strip 100 includes a sample pad 102, a conjugation pad 103, a porous matrix 104, a blocking line 105, and a test line 106 on a support plate 101. ), a control line 107 and an absorbent pad 108. The sample applied to the sample pad 102 flows along the conjugation pad 103 and the porous matrix 104 to reach the absorbent pad 108 .

The support plate 101 includes the sample pad 102 of the lateral flow assay strip 100, the conjugation pad 103, the porous matrix 104, the blocking line 105, the test line 106, the control line 107 and As the support plate 101 supporting the absorbent pad 108, its material is not particularly limited.

A sample to be analyzed is applied to the sample pad 102 and the sample is provided to the conjugation pad 103 . A sample pad 102 is disposed at an upstream end on the support plate 101 . The material constituting the sample pad 102 is not limited as long as it can absorb the sample. In addition, a blocker for controlling a non-specific reaction by a sample material may be applied to the sample pad 102 and used after drying. The blocking agent may include bovine serum albumin (BSA), skim milk, casein, and polymer materials such as poly ethylene glycol (PEG) and poly vinyl alcohol (PVA). Whole blood (plasma, blood, serum) or secretion (saliva, brain) of a person may be used as the sample, and neutralizing antibodies against SARS-CoV-2 may be included in the sample.

The conjugation pad 103 is a complex 210 of a protein 211 having a receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 and a label 212, and a control antibody 221 ) and a label 222. A conjugation pad 103 is disposed downstream of the sample pad 102 on the support plate 101 .

The label 212 or 222 means a detectable label, for example, gold nanoparticles, colored latex beads, carbon nanoparticles, selenium nanoparticles, silver nanoparticles, quantum dots, organic fluorophores ), preferably gold nanoparticles. The label 212 attached to the protein 211 having the receptor binding region and the label 222 attached to the control antibody 221 may be the same or different.

The control antibody 221 is an antibody that can confirm whether the lateral flow assay has been properly operated, and is, for example, chiken IgY, anti-chiken IgY, KLH, anti-KLH, rabbit IgG, anti-rabbit IgG, goat IgG, It may be anti-goat IgG, but is not limited thereto.

FIG. 2 is a diagram illustrating the reaction at the conjugation pad 103 shown in FIG. 1 . In the conjugation pad 103, the neutralizing antibody 230 present in the sample binds to the complex 210 of a protein having a receptor-binding region and a label. More specifically, the neutralizing antibody 230 binds to the receptor binding region present in the protein 211 having the receptor binding region. Here, since various regions other than the receptor binding region exist in the protein 211 having the receptor binding region, only the neutralizing antibody 230 does not bind to the complex 210 . That is, an antibody 240 that specifically reacts with a protein 211 having a receptor binding region, other than a neutralizing antibody, may bind to the complex 210 .

Regardless of whether the neutralizing antibody 230 is present in the sample, the

complexes

210 and 220 present in the conjugation pad 103 move in a downstream direction according to the flow of the sample.

The porous matrix 104 is composed of a porous material, allowing for capillary flow of the sample, such that the sample flows along the matrix. A blocking line 105 , a test line 106 , and a control line 107 are positioned in the porous matrix 104 .

A receptor protein 250 that specifically reacts to the receptor binding region is fixed to the blocking line 105 . Blocking lines 105 are disposed spaced apart from conjugation pads 103 in matrix 104 . The position of the blocking line 105 is located on the porous matrix 104 23.1 mm from the end of the sample pad 102, and the position can be changed between the conjugation pad 103 and the test line 106.

FIG. 3 is a diagram illustrating the reaction at the blocking line 105 shown in FIG. 1 . The blocking line 105 filters out excessive complexes 210 that do not bind to the antibody and receptor binding regions that bind to the antibody 240 that is not the neutralizing antibody, thereby increasing the accuracy of detecting the neutralizing antibody. More specifically, the protein 250 fixed to the blocking line 105 is a receptor protein that specifically reacts to the receptor binding region of the spike protein of SARS-CoV-2. Therefore, since the receptor binding region of the complex 210 of the protein having the receptor binding region to which the antibody 240, other than the neutralizing antibody, is bound is in an open state, the receptor protein 250 immobilized on the blocking line 105 are captured On the other hand, since the complex 210 of the protein having the receptor-binding region to which the neutralizing antibody 230 is bound is in a state in which the receptor-binding region can no longer bind to other antibodies, the receptor protein fixed to the blocking line 105 It is not captured by (250). In addition, the excess complex 210 that is not bound to the antibody in the conjugation pad 103 is also filtered out in the blocking line 105, so that the detection accuracy of the neutralizing antibody can be further improved.

The protein fixed to the blocking line 105 is not limited as long as it is a protein that specifically reacts to the receptor binding region, but is preferably specific to the receptor binding region of the spike protein of SARS-CoV-2 present on the epithelial cell surface. The receptor protein that responds is ACE-2 (angiotensin-converting enzyme 2).

In the test line 106, a protein 211 having a receptor binding region is immobilized. Test lines 106 are spaced apart from blocking lines 105 in the matrix. The position of the test line 106 is located on top of the porous matrix 104, 28.6 mm from the end of the sample pad 102, and its position can be changed between the blocking line 105 and the control line 107. .

FIG. 4 is a diagram illustrating reactions in the test line 106 and control line 107 shown in FIG. 1 . Since the protein 211 having a receptor binding region present in the test line 106 specifically reacts with the neutralizing antibody 230, the neutralizing antibody 230 to which the complex 210 of the protein having a receptor binding region is bound to capture By detecting a signal emitted from the complex 230 captured in the test line 106, the amount of neutralizing antibody in the sample can be measured.

An antigen 260 that specifically reacts with the control antibody 221 is immobilized on the control line 107 . The control lines 107 are spaced apart from the test lines 106 in the matrix. The position of the control line 107 is located on top of the porous matrix 104, 33.6 mm from the end of the sample pad 102, and its position can be changed between the test line 106 and the absorbent pad 108. .

The antibody 260 immobilized on the control line 107 is a substance that specifically reacts with the control antigen 221, for example, chiken IgY, anti-chiken IgY, KLH, anti-KLH, rabbit IgG, anti-rabbit It may be IgG, goat IgG, anti-goat IgG, but is not limited thereto.

An antibody 260 immobilized on the control line 107 captures the control antigen 221 . By detecting a signal emitted from the complex 220 captured in the control line 107, it can be confirmed whether the lateral flow assay has been normally operated.

The absorbent pad 108 absorbs an excess sample from among the samples applied to the sample pad 102 . An absorbent pad 108 is disposed at the downstream end on the support plate 101 . Cellulose, glass fiber, polypropylene, polyethylene, etc. constituting the absorbent pad 108 are used, but any material capable of absorbing the sample is not limited.

Although this invention has been specifically shown and described with reference to its preferred embodiments, it should be appreciated by those skilled in the art that various changes in form and detail may be made without departing from the scope of the invention as covered by the appended claims. It will be understood. It should also be understood that the embodiments described herein are not mutually exclusive, and that features of the various embodiments may be combined in whole or in part in accordance with the present invention.

Claims

a sample pad to which a sample is applied;

A conjugation pad located downstream of the sample pad and having a complex of a label and a protein having a receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, and a complex of a control antibody and a label ;

a porous matrix allowing capillary flow of the sample;

A blocking line immobilized with an antibody specifically reactive to the receptor binding region;

a test line immobilized with the protein having the receptor-binding region;

a control line immobilized with an antibody specifically reactive to the control antigen; and

A lateral flow assay strip comprising an absorbent pad that absorbs an excess of a sample among samples applied to the sample pad.
According to claim 1,

further comprising a support plate;

The lateral flow assay strip, wherein the sample pad, the conjugation pad, the porous matrix, and the absorbent pad are positioned on the support plate.
According to claim 1 or 2,

The protein immobilized on the blocking line reacts specifically to the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, and forms a complex with an antibody other than the neutralizing antibody present in the sample and a neutralizing antibody. A lateral flow assay strip that captures conjugates that fail to form.
According to claim 1 or 2,

The label is at least one selected from the group consisting of gold nanoparticles, colored latex beads, carbon nanoparticles, selenium nanoparticles, silver nanoparticles, quantum dots, and organic fluorophores. assay strip.
According to claim 1 or 2,

The antibody immobilized on the blocking line is ACE-2, a lateral flow assay strip.
Comprising the lateral flow assay strip of claim 1 or 2 and a housing;

The kit, wherein the housing covers the sample pad, the conjugation pad, the blocking line, and the absorbent pad.