KR102350725B1 - Lateral flow assay strips for simultaneously testing antigens and antibodies and lateral flow assay cartridges using the same - Google Patents

Abstract

The present invention relates to a lateral flow assay strip for simultaneously testing antigen and antibody. The lateral flow analysis strip of the present invention includes a porous matrix in which a sample flows laterally, a first test line and a second test line formed apart from the porous matrix in a flow direction of the sample and fixed with different capture agents, and the porous matrix and a bonding pad formed upstream of the matrix and comprising an antigen-fluorescent conjugate and an antibody-fluorescent conjugate. In the first test line, an antibody detecting the antigen-fluorescent conjugate by an immune reaction is immobilized, and in the second test line, an immune antibody detecting the antibody-fluorescent conjugate by an immune reaction is immobilized. According to the present invention, there is an effect of simultaneously diagnosing various viral antigens-antibodies of companion animals regardless of viremia and simultaneously diagnosing whether or not an infectious disease is present.

Description

Lateral flow assay strips for simultaneously testing antigens and antibodies and lateral flow assay cartridges using the same

The present invention relates to a cartridge for testing a pathogen, and to a cartridge for simultaneously testing an antigen and an antibody of a pathogen using multiple fluorescence.

In the case of animals, it is difficult to directly appeal to the symptoms, so it is difficult to diagnose the disease and treatment accurately in the early stage of disease infection. In such complex screening, an infectious agent antigen or an infectious agent-specific antibody is used for diagnosis according to the characteristics of the causative infectious agent.

When diagnosing the antigen and specific antibody of a specific infectious agent for diagnosis of an infectious agent, there is a technical limitation in applying the combination to one diagnostic kit due to the opposite characteristics of the diagnostic kit that measures the antigen and the diagnostic kit that measures the antibody. .

In particular, in the case of cats, it is difficult to diagnose various diseases at the same time due to limited blood collection, and there are products in which antigen-antibody complex diagnosis is applied to a single cartridge, but there is a limitation in reliability because it has to be confirmed with the naked eye.

Republic of Korea Patent 10-0639776 "A method for quantitative assay of lateral flow and a strip and laser-induced surface fluorescence detection device and small scanner for the same" Republic of Korea Patent 10-1069391 “Antibody for feline retrovirus detection, and immunodetection kit comprising the same”

An object of the present invention is to simultaneously diagnose various viral antigens-antibodies of infectious diseases in companion animals.

In order to achieve the above object, the present invention provides a lateral flow assay strip for simultaneously testing antigen and antibody.

The present invention relates to a lateral flow analysis strip for simultaneously testing antigen and antibody, a porous matrix in which a sample flows laterally, and a first test line formed to be spaced apart from the porous matrix in the flow direction of the sample and to which different capture agents are fixed and a second test line and a bonding pad formed upstream of the porous matrix and comprising an antigen-fluorescent conjugate and an antibody-fluorescent conjugate, wherein the first test line detects the antigen-fluorescent conjugate by an immune reaction. the antibody is immobilized, and the second test line is characterized in that the immunoantibody for detecting the antibody-fluorescent conjugate by an immune reaction is immobilized.

Preferably, the antigen-fluorescence conjugate and the antibody-fluorescence conjugate include lanthanide-based atomic or organic phosphors. The antibody immobilized on the first test line is a Feline leukemia virus (FeLV) antibody, and the antibody immobilized on the second test line is an anti-feline antibody. The anti-feline antibody detects a Feline immunodeficiency virus (FIV) antibody or a Feline coronavirus (FCoV) antibody.

Another aspect of the present invention is a lateral flow analysis cartridge including the lateral flow analysis strip.

According to the present invention as described above, by providing a lateral flow analysis strip capable of diagnosing antigen and antibody at the same time, various viral antigens-antibodies of companion animals are simultaneously diagnosed regardless of viremia, and whether infectious diseases are infected effective in diagnosing

1 is a block diagram of a lateral flow analysis strip according to an embodiment of the present invention.
FIG. 2 shows an example of a reaction in a test line and a control line in the assay strip of FIG. 1 .
Figure 3 is a top view of the cartridge according to an embodiment of the present invention in which the side flow analysis strip and the housing protecting the side flow analysis strip are combined.
Figure 4 is a perspective view of the cartridge of Figure 3;

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1 is a block diagram of a lateral flow analysis strip 100 according to an embodiment of the present invention. As shown, the lateral flow type assay strip 100 is formed based on a porous matrix 110 that allows capillary flow. The porous matrix 110 may be one or more selected from nitrocellulose, cellulose, cellulose acetate, and polyethylene, preferably nitrocellulose.

The sample pad 120 absorbs the liquid sample 104 in which the applied sample and the detection buffer are mixed to flow into the porous matrix 110 . A control line 140 and test lines 150 and 160 are positioned on the porous matrix 110 in a downstream direction from the sample pad 120 . A capture agent is fixed to the test line 150 , the test line 160 , and the control line 140 by a physical adsorption method. A capture agent can be a specific antibody that is responsive to an analyte, an antigen, or an oligonucleotide that hybridizes to a nucleic acid of constant sequence. And an absorbent pad 170 is disposed at the downstream end of the porous matrix 110 . Absorbent pad 170 absorbs excess liquid sample 104 and maintains lateral flow along porous matrix 110 .

The control line 140 may be located between the test lines 150 and 160 , upstream of the test line 150 , or on a downstream side of the test line 160 . A positive or negative control reagent or capture agent is immobilized on the control line 140 .

A conjugate pad 130 is further provided downstream of the sample pad 120 on the porous matrix 110 . The bonding pad 130 includes two or more different detection antibodies or detection antigens, wherein the detection antibodies or detection antigens are feline leukemia virus antibodies, feline immunodeficiency virus antigens or feline coronavirus antigens. Each detection antibody or detection antigen is specific for a different target antigen or target antibody, the feline leukemia virus antibody specifically reacts to the feline leukemia virus antigen and the feline immunodeficiency virus antigen is directed against the feline immunodeficiency virus antibody It reacts specifically, and the feline coronavirus antigen responds specifically to the feline coronavirus antibody. Each detection antibody or detection antigen is conjugated to one or more detectable labels such that it is present on the conjugation pad as an antigen-fluorescent conjugate or an antibody-fluorescent conjugate. Each label contains a different spectral emission, and the antibody-fluorescent conjugate to which the respective detection antibody and the fluorophore bind or the antigen-fluorescent conjugate to which the detection antigen binds can form a complex with its target analyte.

The detectable label is a group consisting of gold nanoparticles, colored latex beads, carbon nanoparticles, selenium nanoparticles, silver nanoparticles, quantum dots, up converting phosphors, and organic fluorophores. can be selected from The up-conversion phosphor is a lanthanum-based phosphor Lanthanum (La), Cerium (Cerium: Ce), Praseodymium (Pr), Neodymium (Neodymium: Nd), Promethium (Promethium: Pm), Samarium (Samarium: Sm) , Europium (EU), Gadolinium (Gd), Terbium (Tb), Dysprosium (Dy), Holmium (Holmium: Ho), Erbium (Erbium: Er), Ytterbium (Ytterbium: Yb) , one or more may be selected from the group consisting of Lutetium (Lu) atoms, and the organic phosphor may be a phosphor emitting light of 600-800 nm.

2 is a diagram illustrating the reaction of the liquid sample 104 in which the sample and the detection buffer are mixed in the test line 150 , the test line 160 , and the control line 140 in the lateral flow analysis strip 100 . The sample may be whole blood, serum or plasma of a cat.

The bonding pad 130 includes fluorescent conjugates 155, 167, and 169 in which the feline leukemia virus antibody 154, the feline immunodeficiency virus antigen 161, or the feline coronavirus antigen 163 is conjugated to a high-sensitivity atomic phosphor or an organic phosphor. to provide

A feline leukemia virus (FeLV) antibody 152 is fixed to the test line 150 as a capture agent, and a feline antibody is detected as a capture agent in the test line 160 . Anti-feline antibody 162 is immobilized, and chicken immunoglobulin Y (chicken IgY) 142 is immobilized on the control line 160 as a capture agent.

The test line 150 and the capture agents 152 and 162 fixed to the test line 160 are fixed so that the capture agents 152 and 162 are not the same between the test lines 150 and 160 .

When the liquid sample 104 is applied to the sample pad 120 and flows laterally in the porous matrix 110, the fluorescent conjugate 155 of the bonding pad 130 is Feline leukemia virus (FeLV) (153). After conjugation with the antigen, the Feline leukemia virus (FeLV) antibody 152 is captured in the immobilized test line 150 . After the fluorescent conjugate 167 is conjugated with a Feline immunodeficiency virus (FIV) antibody 164 , the anti-feline antibody 162 is captured in the immobilized test line 160 . The fluorescent conjugate 169 is conjugated with the Feline coronavirus (FCov) antibody 165 and then captured in the test line 160 to which the anti-feline antibody 162 is immobilized. And up converting phosphors ), a fluorescent conjugate (147) in which a lanthanum-based europium (EU) atomic phosphor and anti-chicken immunoglobulin Y (chicken IgY) are conjugated, and an organic phosphor emitting light at a wavelength of 600-800 nm and anti-chicken immunoglobulin Y ( The fluorescent conjugate 149 to which chicken IgY is conjugated is captured by chicken immunoglobulin Y (chicken IgY), which is the capture agent 142 of the control line 140 .

The fluorescence results of the atomic phosphor and the organic fluorescent agent of the fluorescent conjugates 147 and 149 captured by the control line 140 have different wavelength bands and different light emission times, respectively, and result from different wavelength bands and different light emission times, respectively. of fluorescence signal data is formed.

In order to reduce the variation that occurs when the same phosphor is used, fluorescence of different wavelength bands and different emission times is used as described above. Data is input using two optical systems in the same measuring device. The calculation is performed using each of the measured phosphor data values as a reference.

The light emitted from the phosphor 146 or the phosphor 148 captured by the control line 140 is analyzed in an optical system (not shown) so that the liquid sample 104 is normally sideways in the lateral flow analysis strip 100 . It is determined whether the flow is flowing or not, and is used as a reference value of the degree of lateral flow.

Light generated from the phosphor 156 captured by the test line 150 indicates the amount of Feline leukemia virus (FeLV) antigen 153, and from the phosphor 156 captured by the test line 150 By calculating the amount of light generated, the amount of Feline leukemia virus (FeLV) antigen (153) is measured.

The light generated by the phosphor 168 captured by the test line 160 indicates the amount of the Feline immunodeficiency virus (FIV) antibody 164, and the phosphor 166 captured by the test line 160. The light generated by the Feline coronavirus (FCoV) indicates the amount of antibody 165. Calculate the amount of light generated by the phosphor 168 or the phosphor 166 captured in the test line 160 to measure the amount of Feline immunodeficiency virus (FIV) antibody (164) or Feline coronavirus (FCoV) antibody (165).

According to this embodiment, a feline immunodeficiency virus (FIV) antibody 164, a feline leukemia virus (FeLV) antigen 153, and a feline coronavirus ((Feline coronavirus: FCoV) antibody 165 can be detected at once. Viruses have antigens in the blood depending on the immunity of the subject to be infected or the situation of the individual, or the antigen is present in the blood after being infected. There may be differences in non-viremia, however, by testing antigen and antibody at the same time as described above, virus infection can be detected regardless of differences caused by viremia.

When detecting three viruses at the same time, it is very important to eliminate the interference reaction that may be caused by each other on the sample and to maintain the fluorescence signal at the same time. To this end, the movement of nanoparticles on the porous matrix 110 and the antigen-antibody reaction should be smoothly progressed, and non-specific reactions that may appear in cat blood samples should be suppressed.

To implement this, in this embodiment, fluorescence having different wavelength and emission time regions is applied as multi-fluorescence in order to minimize the cross-reaction of the fluorescence afterimage, which is the most important part of the multi-fluorescence analysis, and the fluorescence between the test line intervals is An interval of 2 to 3 mm, which is a standard between the test lines 150 and 160, which minimizes the effect on the flow of the fluid without affecting the signal saturation in the situation was applied. In addition, a glass fiber pad was used as a bonding pad so that an excess of fluorescent conjugate was easily re-dissolved in a liquid sample and flow was excellent. ) was applied to the appropriate amount of bonding pad.

3 is a top view of the lateral flow analysis cartridge 30 in which the lateral flow analysis strip 100 and the housings 10 and 20 are combined.

Referring to FIG. 3 , the lateral flow analysis cartridge 30 includes a handle derivative 13 so as to know the difference between the portion injected into the fluorescent LFI and the handle portion. It is possible to reduce the probability of making a mistake when performing an experiment using the lateral flow analysis cartridge 30 by enabling tactile recognition as well as an arrow display that can visually recognize the injection direction.

4 is an exploded perspective view of the lateral flow analysis cartridge 30 in which the lateral flow analysis strip 100 of FIG. 3 and the housings 10 and 20 are combined. Referring to FIG. 4 , the lateral flow analysis cartridge 30 includes a lateral flow analysis strip 100 that absorbs a sample and displays a detection result, a lower housing 20 and an upper coupled to each other to contain the lateral flow analysis strip 100 . It includes a housing (10).

The lower housing 20 has a single side fixing part 21 to which the lateral flow analysis strip 100 can be fixed is located on both sides, and includes a support part 24 coupled to the upper housing 10 correspondingly.

The upper housing 10 has a sample inlet 11 formed to correspond to the sample pad 120 , a porous membrane 110 , a control line 140 , and a result display window 12 formed to correspond to the test lines 150 and 160 . ), a handle derivative 13 for guiding the handle, and a support 14 corresponding to the lower housing 20 and coupled thereto.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1.

Anti-feline leukemia virus antibody and anti-feline antibody as a capture agent on the nitrocellulose (NC) membrane were instilled into the first and second test lines, respectively, and chicken immunoglobulin Y (chicken IgY) was instilled at the control line position. After drying, it is prepared by drying at 37°C for 24 hours.

Feline immunodeficiency virus (FIV) antigen is reacted with organic fluorescent fluorescent beads to prepare a fluorescent conjugated antibody conjugated with fluorescent material and antigen, and Feline coronavirus (FCoV) antigen is reacted with high-sensitivity fluorescent fluorescent beads A fluorescent conjugate is prepared in which a fluorescent substance and an antigen are conjugated, and an anti-feline leukemia virus (FeLV) antibody is reacted with a high-sensitivity fluorescent substance to prepare a fluorescent conjugated antibody in which the fluorescent substance and the antibody are conjugated. At this time, the high-sensitivity phosphor is prepared by using a fluorescent bead that is a ceramic particle into which a lanthanum-based phosphor Europium (EU) atomic phosphor is introduced, and a fluorescent substance that is a ceramic particle into which an organic phosphor that emits light at a wavelength of 600-800 nm is introduced. Prepare fluorescent conjugates using beads. In addition, Europium-anti chicken immunoglobulin Y conjugate (Eu-Anti chicken IgY conjugate), which is a fluorescent conjugate (147) conjugated with anti-chicken IgY, which can be used as a reference value for fluorescence emission, and 600-800 nm A fluorescent conjugate (149) conjugated with an organic fluorescent substance emitting light at a wavelength of 149 and anti-chicken immunoglobulin Y (chicken IgY) is prepared. The five types of fluorescent conjugated antibodies prepared in this way are sprayed onto the bonding pad 130 made of glass fiber to be absorbed, and then dried to prepare the bonding pad.

The dried nitrocellulose (NC) film is placed on the support, and the sample pad, bonding pad, and absorbent pad to which the sample is applied are placed in the direction of lateral flow without overlapping the first test line, the second test line and the control line. Assemble and prepare the lateral flow analysis strips so that they can be placed in sequence.

Comparative Example 1.

A first sample is prepared by collecting blood from a cat infected with feline immunodeficiency virus (FIV).

To confirm the clinical results of the first sample, it was measured using “ViraCHEK® / FIV”, an enzyme-linked immunosorbent assay (ELISA) device for the detection of feline immunodeficiency virus (FIV) antibody in whole cat blood, serum or plasma. , the order and samples related to the measurement follow the VirachekFIV measurement method provided previously.

Comparative Example 2.

An enzyme-linked immunosorbent assay (ELISA) device for detecting FeLV antigen to prepare a second sample by removing blood from a cat infected with feline leukemia virus (FeLV) and confirm the clinical result of the second sample It was measured through “ViraCHEK® / FeLV”, and the procedure and samples related to the measurement follow the ViraCHEK® / FeLV measurement method provided previously.

Comparative Example 3.

A third sample is prepared by collecting blood from a cat infected with Feline coronavirus (FCoV).

Immunofluorescence assay (IFA) was performed to confirm the clinical results of the third sample, and the results were measured.

Measurement example 1.

The same sample as in Comparative Example 1 was measured, but 50 μl of the first sample was added to 1000 μl of the extraction solution, and the sample was prepared by shaking and mixing 10 times or more.

In the extraction solution, a reaction inhibitor and a surfactant are added to prevent an interference reaction or a non-specific reaction. In addition, the composition of the detection buffer used for the purpose of diluting the sample and allowing it to be developed in the kit consists of 3% BSA, 1% Sucrose, 100 mM Tris-HCl, 0.5% detergent (surfactant) and distilled water. The amount is 1 ml.

The sample prepared by shaking is put into the sample pad of the lateral flow analysis strip prepared in Example 1 and reacted for 12 minutes. The lateral flow assay strip reacted for 12 minutes is mounted on a fluorescence lateral flow immunoassay (LFI) “Vetchroma” (manufacturer: Anybet) to measure the result.

Measurement example 2.

It was carried out in the same manner as in Measurement Example 1, but using the second sample prepared in Comparative Example 2.

Measurement example 3.

It was carried out in the same manner as in Measurement Example 1, except that the third sample prepared in Comparative Example 3 was used.

Tables comparing the results of Comparative Example 1 and Measurement Example 1, Comparative Example 2 and Measurement Example 2, Comparative Example 3 and Measurement Example 3 are shown in Tables 1 to 3 below.

predicate'(VIRACHEK) Positive negative Total Sensitivity Specificity FIV Ab Positive 68 One 69 97.1% 99.0% negative 2 103 105 Total 70 104 174

predicate0VIRACHEK) Positive negative Total Sensitivity Specificity FeLV Ag Positive 62 One 63 95.4% 98.9% negative 3 89 92 Total 65 90 155

predicate (IFA) Positive negative Total Sensitivity Specificity FCoV Ab Positive 39 5 44 90.7% 95.4% negative 4 103 107 Total 43 108 151

In Tables 1 to 3, Sensitivity is the rate at which a sample and subject with disease receive a positive result when tested. is the divided value.

The specificity of Tables 1 to 3 is the ratio of samples and subjects without disease receiving a negative result when tested, and the number of actually detected samples in each (-) group is calculated as the total number of samples (-) group. divided by the number of samples.

Above, a specific part of the present invention has been described in detail, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

10: upper housing 11: sample inlet
12: result display window 13: handle derivative
14: support 20: lower housing
21: one side fixed part 24: support part
100: analysis strip 104: liquid sample
110: porous matrix 120: sample pad
130: bonding pad 140: control line
142: Chicken Immunoglobulin Y
146: phosphor 148: phosphor
156: phosphor 166: phosphor
168: phosphor 147: fluorescent conjugate
149 Fluorescent Conjugate 155 Fluorescent Conjugate
167 Fluorescent Conjugate 169 Fluorescent Conjugate
150: test line
152: Feline Leukemia Virus Antibody
153: Feline Leukemia Virus Antigen
161: Feline Leukemia Virus Antibody
160: test line 161: feline immunodeficiency virus antigen
162: anti-feline antibody 163: feline coronavirus antigen
164: Feline Immunodeficiency Virus Antibody
165: Feline Coronavirus Antibody
170: absorbent pad

Claims (5)

In the lateral flow analysis strip for simultaneously testing antigen and antibody,
A porous matrix in which a sample containing an infectious agent antigen and an infectious agent antibody flows laterally;
A first test line and a second test line formed to be spaced apart in the flow direction of the sample in the porous matrix and to which different capture agents are fixed;
a bonding pad formed upstream of the porous matrix and comprising an antigen-fluorescent conjugate and an antibody-fluorescent conjugate;
In the first test line, an antibody that captures the reaction product of the antigen-fluorescent conjugate and the infectious agent antibody by reaction with the infectious agent antibody is fixed as a capture agent, and in the second test line, the antibody-fluorescent conjugate and the A lateral flow analysis strip, characterized in that an antibody that captures the reaction product of the infectious agent antigen by reaction with the infectious agent antigen is fixed as a capture agent.
According to claim 1,
The antigen-fluorescent conjugate and the antibody-fluorescent conjugate are lateral flow analysis strips, characterized in that they include lanthanum-based atomic phosphors and organic phosphors.
According to claim 1,
The capture agent immobilized on the first test line is an anti-feline antibody,
The lateral flow assay strip, characterized in that the capture agent immobilized on the second test line is a feline leukemia virus antibody.
According to claim 1,
The lateral flow assay strip according to claim 1, wherein the bonding pad further comprises a blocking agent for controlling a non-specific reaction between the antigen-fluorescent conjugate and the antibody-fluorescent conjugate.
A lateral flow analysis cartridge comprising the lateral flow analysis strip of any one of claims 1 to 4.

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