Rapid multi-strip immunochromatographic analysis method and apparatus capable of quantitative analysis and providing concentration value of disease marker for in vitro diagnosis

Abstract

The present invention relates to a rapid load strip immunochromatographic analysis method capable of quantitative analysis and providing a concentration value of a disease marker for in vitro diagnosis, and an apparatus therefor, the method comprising: i) preparing a separate strip to determine whether an immunochromatography method results in a false negative, mixing a standard material of a certain concentration with a sample and dropping the mixture, and if a detection signal of a test line becomes weaker (or continues to disappear), determining that the concentration of a target material in the sample is excessively high (higher than a concentration indicating a maximum signal value), and conversely, if the detection signal of the test line becomes stronger, determining that the original concentration of the target material in the sample is very low (lower than a concentration indicating a minimum signal value); ii) measuring signal intensity according to standard materials of various concentrations, obtaining a calibration curve thereof, determining the concentration of the target material, and repeatedly measuring the signal intensity of the target material in the sample to reduce analysis experiment errors; and iii) measuring the signal intensity of the standard material corresponding to the disease marker to obtain a calibration curve according to the concentration, and quantifying the target material, which is the disease marker, using the calibration curve so as to provide the concentration value of the disease marker for in vitro diagnosis of a specific disease of the human body.

Description

Rapid multi-strip immunochromatographic analysis method and device capable of quantitative analysis and providing concentration values ​​of disease markers for in vitro diagnosis

[0001] The present invention relates to an immunochromatographic analysis method and device utilizing a calibration curve of a standard material utilizing a multi-strip, which enables rapid on-site testing for quantifying target substances such as proteins in a living body. Furthermore, the present invention relates to a rapid quantitative analysis multi-strip immunochromatographic analysis method and analysis kit that can be used to provide concentration values ​​of disease markers for in vitro diagnosis of human diseases.

[0002]

[0003] The chromatographic immunoassay, also known as a rapid test or lateral flow immunochromatography assay (LFIA), is a method that can analyze small amounts of a target analyte in a short time by utilizing antigen-antibody reactions. It is used in various fields such as medicine, agriculture, livestock industry, food, military, and environment, as well as for the diagnosis or testing of various diseases. These immunochromatographic assays generally use an assay strip containing a reactant that can react with the target analyte to be detected and show a change, or an analysis device in the form of a device that mounts the assay strip in a plastic case.

[0004] A typical analysis strip is composed of a sample pad that receives a liquid sample, a conjugate pad that contains a conjugate that is a label that generates a signal that can be detected by the naked eye or a sensor and is conjugated to a ligand such as an antigen or antibody, a detection pad that has a test line that immobilizes a target substance in the sample or a ligand (antigen or antibody) that specifically binds to the conjugate and a control line that can confirm the development of the sample, and an absorbent pad that finally receives the liquid sample.

[0005] Immunochromatography assays measure target substances (e.g., proteins) in specimens such as blood, urine, saliva, spinal fluid, cell cultures, and microbial cultures. They are simple, non-invasive methods that do not require time-consuming or expensive equipment. Therefore, immunochromatography assays are widely used in settings where rapid testing is needed, such as at home or in healthcare settings. For example, pregnancy test kits commonly used at home are rapid testing devices that measure pregnancy-related hormones. It is well known that rapid diagnostic kits have recently been used to quickly detect coronavirus infection (COVID-19).

[0006] As a background technology of the present invention, a "multi-diagnostic kit" (hereinafter, "prior art 1") is disclosed in Korean Patent Registration No. 10-1941090. The multi-diagnostic kit of prior art 1 comprises multiple detection strips. Each of the multiple detection strips may be provided with a separate sample. For example, urine may be provided as a sample to the first sample pad, and blood may be provided as a sample to the second sample pad. Furthermore, antibodies for different diseases may be provided to each test line. Therefore, multiple diseases can be diagnosed on a single detection strip, depending on the number of test lines.

[0007] Prior art 1 utilized multiple strips for multiple samples and disease diagnoses, but suffered from the problem of inability to achieve quantitative measurements. The present invention differs in that it utilizes multiple detection strips to obtain a standard material calibration curve and quantify target substances based on the standard material curve.

[0008] As another background technology, Korean Patent No. 10-1540608 discloses “Analytical strip having a variable control line and a diagnostic kit having the same” (hereinafter, “Prior Art 2”). The analytical strip of Prior Art 2 overcomes the difficulty of measuring target substances (however, only qualitative measurement is possible) due to the Hook Effect (Wikipedia https: / en.wikipedia.org / wiki / Hook_effect) by having a variable control line. That is, in order to confirm whether or not the signal from the sandwich method of immunochromatographic analysis is false negative, a standard substance was fixed to the control line (variable control line).

[0009] As prior art 3, Korean Patent No. 10-1406923, “Quantitative immunochromatographic analysis assay strip having a captor compartment in the form of a rhomboid pattern and a quantitative immunochromatographic analysis method using the same” is known. This relates to a quantitative immunochromatographic analysis assay strip and a quantitative immunochromatographic analysis method using the same, in which spots composed of first captors are arranged in the form of a rhomboid pattern on a measurement pad in the assay strip, and first captors of various concentrations are fixed, thereby enabling clearer quantitative signaling than the linear captor alignment method of existing qualitative analysis assay strips, and in particular, by directly counting the number of spots by utilizing the fact that chromogenic particles develop color at each spot, and when the concentration of the analyte is high, many spots develop color, and when the concentration is low, few spots develop color, the concentration of the analyte can be quantified with the naked eye.

[0010] As prior art 4, Korean Patent No. 10-2138192, “Immunochromatography strip for pregnancy diagnosis having multiple test lines and pregnancy diagnosis kit comprising same” is known. This relates to a chromatography strip for pregnancy diagnosis having multiple test lines, a pregnancy diagnosis kit having the chromatography strip, and a method for diagnosing pregnancy using the same, which overcomes the problem of false negatives caused by hCG variants, thereby enabling more accurate and convenient pregnancy diagnosis with the naked eye, and utilizes a chromatography strip for pregnancy diagnosis consisting of multiple test lines and control lines.

[0011] As prior art 5, Korean Patent Publication No. 2015-0030902, “Immunochromatography strip for pregnancy diagnosis having a heterologous test line and pregnancy diagnosis kit having the same” is known. This relates to an immunochromatography strip for pregnancy diagnosis having a heterologous test line, a pregnancy diagnosis kit having the immunochromatography strip, and a method for qualitatively or quantitatively analyzing human chorionic gonadotropin in a sample using the same. The immunochromatography strip for pregnancy diagnosis has a heterologous test line, thereby overcoming the problem of false negatives due to the hook phenomenon during quantitative measurement of high concentration human chorionic gonadotropin, and enabling not only qualitative analysis of pregnancy by the naked eye but also highly reliable quantitative analysis, and has the effect of improving the concentration measurement range of analyte through mutual complementation between the test line and the heterologous test line.

[0012] Unlike the above-mentioned conventional technologies, the present invention does not provide a variable control line but provides a separate independent strip. That is, in the separate independent strip, a certain concentration (S) is applied to the sample. Hook ) and mix the standard material and then drop it.

[0013] (i) If the target substance does not exist in the sample, the concentration (S) bound to the labeled detection ligand Hook ) is combined with a fixed capture ligand, and a detection signal appears.

[0014] (ii) In cases where a large amount of target material is present in the sample, the labeled detection ligand is saturated with the target material, i.e., the Hook phenomenon [at this time, the above concentration (S) added Hook ) accelerates the saturation of the detection ligand, thereby preventing the target substance bound to the fixed capture ligand from reacting.

[0015] In this way, it is possible to detect cases where the detection signal becomes weaker or disappears (false negative signal) because the target substance is excessively abundant and exceeds the maximum value of the range measured by the calibration curve (dynamic range).

[0016] [Prior Art Literature]

[0017] [Patent Document]

[0018] Prior art 1: Republic of Korea Patent No. 10-1941090.

[0019] Prior art 2: Republic of Korea Patent No. 10-1540608.

[0020] Prior art 3: Republic of Korea Patent No. 10-1406923.

[0021] Prior art 4: Republic of Korea Patent No. 10-2138192.

[0022] Prior Art 5: Republic of Korea Publication Patent No. 2015-0030902

[0023] [Non-patent literature]

[0024] 1) Arpasiri Srisrattakarn et al., Diagnostics, 2020, Vol. 10, pp 1-16.

[0025]

[0026] The purpose of the present invention is to provide a method for producing a multi-strip immunochromatography,

[0027] The present invention provides a method and device for distinguishing a case in which a detection signal disappears due to an “excessive” amount of a target substance in a sample (hook phenomenon) from a case in which a detection signal disappears due to a “weak or non-existent” target substance in a sample.

[0028] In addition, the present invention provides a rapid chromatograpic immunoassay method and device that are accurate and reproducible, i.e., capable of quantitative testing, by applying calibration of standard substances.

[0029] In addition, the method and device of the present invention as described above are used for the purpose of providing concentration values ​​of disease markers when applied to in vitro diagnostics (IVDs) for human diseases.

[0030]

[0031] In an immunochromatography analysis device comprising a plurality of unit strips connected in parallel, the unit strips comprising a sample pad for receiving a liquid sample of the present invention; a conjugate pad connected to one side of the sample pad and receiving a label and a detection ligand; a detection pad connected to one side of the conjugate pad and including a test line and a control line; and an absorption pad connected to one side of the detection pad and receiving a liquid remaining after a reaction,

[0032] The above conjugate pad is characterized by including a first conjugate (1st labeled-ligand conjugate) composed of a first detection ligand and a label that react with a target analyte or standard material of a sample, and a second conjugate (2nd labeled-ligand conjugate) composed of a second detection ligand and a label that do not react with the target analyte of the sample.

[0033] In addition, in the present invention, the detection pad includes a test line and a control line, and the test line has a capture ligand fixed thereon that reacts with a target analyte or a standard material in a sample, and the control line has a foreign material fixed thereon that does not react with the sample but reacts with a second detection ligand. In addition, the absorption pad is characterized by absorbing a developing solution of the sample to provide a driving force.

[0034] In addition, in the present invention, the conjugate of the first detection ligand and the label absorbed into the conjugate pad binds to a standard material or target material in the sample, and then proceeds to the detection pad and binds to the corresponding capture ligand at the test line to form a triple structure of the first conjugate-standard material (target material)-capture ligand; and the second conjugate absorbed into the conjugate pad, i.e., the conjugate of the second ligand and the label, proceeds to the detection pad and binds to the corresponding foreign material at the control line to form a double structure of the second conjugate-foreign material.

[0035] In addition, in the present invention, when quantifying (or measuring) a target substance, first, several strips are used to measure the signal intensity of a standard substance at various concentration levels (S0, S1, S2, S3, S4, S5, S6, S7) to obtain a calibration curve; and this is used to quantify the target substance of the sample. That is, the signal intensity according to the concentration of the standard substance is applied to a model such as a linear function, a quadratic function, a 4-parameter logistic function, or a 5-parameter logistic function, and a model (function) with the smallest error between the predicted value of the signal intensity according to the concentration and the actual value is selected as the calibration curve; and the signal intensity of the target substance of the sample is applied to the calibration curve to determine its concentration.

[0036] In addition, in the concentration measurement based on a general 'sandwich' immunoassay such as the present invention, when the concentration of the target substance exceeds the maximum value of the dynamic range (the width between the maximum and minimum values ​​of the signal measurement amount), the Hook effect (https: / en.wikipedia.org / wiki /

[0037] Hook_effect) may occur.

[0038] Meanwhile, in the enzyme-linked immunosorbent assay (ELISA), which is a commonly used immunoassay method in places equipped with a central instrument analysis laboratory, there is a washing step in the analysis experiment process. For example, in the case of the sandwich enzyme-linked immunosorbent assay, which is the most common method, the target substance is reacted with the capture ligand, and the unbound target substance is washed away so that the hook phenomenon can be prevented by reacting with the labeled detection ligand later.

[0039] On the other hand, in immunochromatographic analysis without a washing step during the analysis experiment process, it is necessary to distinguish between cases where the signal disappears due to the hook phenomenon and cases where the signal is weak due to the absence of the target substance in the sample from the beginning.

[0040] To address this, an additional concentration (S) of a substance was added to the sample suspected of having the hook effect. Hook ) is mixed and dropped onto the sample pad; if the detection signal of the test line becomes stronger, it is determined that the target substance is absent or exists in a trace amount in the original sample; conversely, if the detection signal of the test line becomes weak or remains completely absent, it is determined that the concentration of the target substance is too high and the Hook effect has occurred.

[0041] That is, the multi-strip immunochromatography analysis method of the present invention is a method for analyzing a sample and an added constant concentration (S Hook ) is dropped onto a sample pad (1), and if the detection signal of the label (8) appearing on the test line (3) becomes stronger than that of the test line (3) where only the sample is dropped, it is determined that the concentration of the target analyte is lower than the concentration that saturates the detection and capture ligand; conversely, if the detection signal of the label (8) appearing on the test line (3) becomes weaker or continues to disappear compared to the test line where only the sample is dropped, it is determined that the concentration of the target analyte is excessively high, resulting in a false negative. The mixture is characterized in that it is dropped onto a sample pad (1), which is a separate independent strip among the multiple strips.

[0042] In addition, the multi-strip immunochromatography analysis method of the present invention is characterized by measuring the signal intensity of the standard material at various concentrations on the multi-strip, obtaining a calibration curve according to the concentration using the signal intensity, and using the calibration curve to quantify the target analyte.

[0043] In addition, the multi-strip immunochromatography analysis method of the present invention is characterized in that the signal intensity of the target analyte is repeatedly measured and the average value is used to determine the concentration based on the calibration curve.

[0044] In addition, in the present invention, the label is characterized by being any one of a fluorescent dye, gold colloid, latex particles, colored polystyrene microparticles, or enzyme.

[0045] In addition, in the present invention, the signal measuring device of the marker is characterized by being either a CCD or a CMOS.

[0046] In addition, in the present invention, the target material is characterized by being any one of a protein, an antigen, an antibody, DNA, RNA, PNA, or an aptamer.

[0047] In addition, in the present invention, the sample is characterized in that it is any one of blood, urine, saliva, spinal fluid, cell culture fluid, or microbial culture fluid.

[0048] In addition, the present invention is characterized in that, for in vitro diagnosis of infectious diseases (using human-derived substances such as blood for disease diagnosis), a blood marker that is already widely used for diagnosis, differentiation, and prognosis of diseases is rapidly quantitatively measured at the point-of-care and its value (e.g., blood concentration) is provided. For example, in cases where a conventional single-strip immunochromatography method that helps in the diagnosis of infectious viral diseases such as coronavirus and influenza virus does not provide information on whether it is false negative, a separate strip can be provided to determine this.

[0049] Additionally, in patients presenting with systemic inflammatory response syndrome (SIRS)—that is, suspected sepsis—blood concentrations of procalcitonin (PCT), a marker of bacterial infection, can be rapidly provided on-site. While less specific, blood concentrations of CRP (C-reactive protein), a widely used marker, can also be provided.

[0050] In addition, the present invention is characterized by providing a rapid quantitative measure of blood markers already widely used for diagnosis, differentiation, and prognosis estimation at the point-of-care for in vitro diagnosis of cardiovascular diseases (cardio-cerebrovascular diseases) and providing the numerical value (e.g., blood concentration). For example, it provides blood concentration values ​​of troponin I, troponin T, and creatin kinase-MB (CK-MB), which are widely used as markers for diagnosing diseases in myocardial infarction, and also provides blood concentration values ​​of brain natriuretic peptide (BNP) and N-terminal prohormone of brain natriuretic peptide (NT-proBNP) as markers for differentiating whether acute dyspnea is due to heart failure from respiratory disease in patients complaining of acute dyspnea, and can provide blood concentration values ​​of S100B and neuron-specific enolase (NSE) as markers for predicting neurological prognosis in patients whose circulation has recovered after cardiac arrest.

[0051] In addition, the present invention is characterized by providing a rapid quantitative measure (e.g., blood concentration) of blood markers already widely used for diagnosis, differentiation, and prognosis estimation at the point-of-care for in vitro diagnosis of neoplastic diseases. For example, blood concentration values ​​of AFP (marker for liver cancer, etc.), CEA (marker for colon cancer, etc.), PSA (marker for prostate cancer), ferritin (marker for leukemia, etc.), TG (marker for thyroid cancer), SCC (marker for cervical cancer, etc.), Free light chain (marker for multiple myeloma), CA19-9 (marker for colon cancer, etc.), CA125 (marker for ovarian cancer), CA15-3 (marker for breast cancer), beta-HCG (marker for placental tumor, etc.), NSE (marker for small cell lung cancer, etc.), cyfra21-1 (marker for lung cancer, etc.), Pepsinogen I / II (marker for stomach cancer, etc.), HE4 (marker for ovarian cancer, etc.) can be quickly provided on-site.

[0052]

[0053] Although the conventional single-strip rapid test method has improvements such as using each control line to check whether the device is functioning, including the expansion of the sample solution, and to check whether the test (target substance measurement) is false negative, it was impossible to obtain a calibration curve necessary for accurate quantitation because a single strip could not obtain signal values ​​for standard substances of various concentrations.

[0054] However, according to the present invention, a quantitative measurement device for biological substances can be provided that can quickly perform reproducible quantitative testing on site by applying a calibration curve of a standard substance through a multi-strip in a single-strip immunochromatography analysis method that was difficult to perform in the past, which was difficult to perform in a conventional “quantitative” test.

[0055] In addition, the immunochromatography analysis device according to the present invention, when it is suspected that a large amount of a sample target substance is present and the hook effect occurs, resulting in a weak signal, is used to confirm this by placing a sample and a certain concentration (S) on a separate strip provided. Hook ) can be additionally injected to determine whether the sample signal is false negative by observing the increase or decrease (or continued disappearance) of the signal intensity.

[0056] In addition, the present invention has the advantage of being able to confirm whether quantitative results are reproducible by repeatedly measuring a sample, and reducing error (variance) due to analysis by using an average value instead of a single value as the test value used in post-analysis.

[0057] In addition, the multi-strip immunochromatography device according to the present invention has the effect of drastically reducing the testing time, testing cost, and device cost compared to devices that require a central laboratory, such as enzyme-linked immunosorbent assay (ELISA) commonly used for quantitative measurement, and has the effect of providing a solution for point-of-care testing (POCT) with limitations in testing location, testing equipment, testing time, etc., especially in cases where quantitative testing related to specific diseases of the human body is required.

[0058] In addition, the analysis method and analysis device of the present invention can provide an in vitro analysis method and an in vitro analysis kit using quantitative analysis multi-strip immunochromatography, which are characterized by providing a disease marker concentration value for in vitro diagnostics (IVDs) of a specific disease of the human body.

[0059]

[0060] Figure 1 is a perspective view of a unit strip structure of the present invention for measuring a target substance of a sample.

[0061] Figure 2 is an explanatory drawing of a combination that is coupled to a test line on a detection pad of a multi-strip of the present invention.

[0062] Figure 3 is a conceptual diagram for quantitative measurement of a sample target substance through calibration of a standard substance using a multi-strip of the present invention.

[0063] Figure 4 is a representative calibration curve shown in the immunoassay of the target substance platelet factor 4 (PF-4) present in human serum in the present invention.

[0064]

[0065] To avoid confusion and clarify the terms used in the present invention, the symbols are written in English.

[0066] Generally, immunochromatographic assays can be categorized into qualitative and quantitative tests in terms of the precision with which they measure target substances. Qualitative tests, such as those found in pregnancy test kits or rapid COVID-19 diagnostic kits, have a control line, indicating normal functioning, and a test line, which may or may not appear depending on the pregnancy or COVID-19 response. This means that a signal does not appear below a certain threshold concentration of the target substance being tested, and only after this threshold is exceeded does a signal appear. In fact, the signal can be visually confirmed through color development when colloidal gold is used as a marker.

[0067] Meanwhile, quantitative testing, unlike qualitative measurements, does not judge every signal as "positive" or "negative." Instead, it measures and treats the signal as a continuous quantity, requiring accuracy and reproducibility of the measured quantity. For example, situations requiring actual concentrations, such as cancer markers in blood, differ from situations like pregnancy tests or COVID-19 infections, where the amount present varies significantly and qualitative analysis alone is sufficient.

[0068] To date, research efforts to develop effective quantitative assays for immunochromatographic analysis have included the advancement of optical devices (e.g., complementary metal-oxide-semiconductor; CMOS, charge-coupled device; CCD) to improve the correlation between target substance concentration on the test line and label signal measurements, and non-optical measurement methods (e.g., magnetic immunoassay; MIA). Furthermore, devices to maintain a constant flow rate to reduce the variability of capillary pumping of the sample solution have been developed. Such technical research can be said to have improved the instrumental aspects of quantitative measurement in immunochromatographic analysis.

[0069] However, in immunoassays based on antigen-antibody reactions, such as immunochromatography, it is essential to first perform calibration using standard substances, in addition to improvements in equipment such as automated devices. This is a natural process, similar to a kind of "zero adjustment." This is because antigen-antibody reactions generally vary slightly depending on the temperature and humidity at the time of the analysis experiment, as well as the person or machine performing the experiment. In other words, standard substances of various concentrations must be prepared at the time of the experiment, the detection signal must be measured, a calibration curve must be obtained, and the concentration of the target substance in the sample must be accurately measured based on this.

[0070]

[0071] FIG. 1 of the present invention is a perspective view of the structure of a unit strip for measuring a target substance present in a body fluid such as blood, and is configured to include a sample pad (1), a conjugate pad (2), a test line (3), a control line (4), a detection pad (5), and an absorbent pad (7) on an adhesive plastic support (6). The sample pad (1) absorbs a sample (liquid sample or analysis sample or analyte) and ensures uniform flow of the sample. Samples such as whole blood, plasma, serum, tears, saliva, urine, nasal mucus, and body fluids can be used. The sample pad (1) may additionally include a filtering function to further improve selectivity for the sample or to minimize the influence of interfering substances that may be included in the sample.

[0072] A sample dropped onto a sample pad (1) passes through a conjugate pad (2) including a first conjugate composed of a first detection ligand (9)-label (8) that reacts with a sample target substance and a second conjugate composed of a second detection ligand (12)-label (8) that does not react with a sample target substance, and the labeled conjugates move to a detection pad (5) that indicates a detection result.

[0073] The above detection pad (5) includes a test line (3) and a control line (4) that are positioned at a distance from each other, and the test line (3) has a capture ligand (11) fixed thereto that reacts with a target substance in a sample. The control line (4) has a foreign substance (13) fixed thereto that does not react with the sample but reacts with a second detection ligand (12).

[0074]

[0075] Figure 2 is an explanatory diagram of a combination that binds to a test line (3) on a detection pad (5) of the present invention. In Figure 2, (a) is a case where standard substances of various concentrations (S0-S7) are dropped onto a sample pad to obtain a calibration curve, (b) is a case where a sample is dropped onto a sample pad to measure a target substance, and (c1) is a case where a sample without a target substance and an additional sample with a certain concentration (S Hook) are injected together with the standard material, and (c2) is a sample with an excess of target material beyond the maximum signal value and an additional concentration (S Hook ) is injected together with the standard substance.

[0076] In Fig. 2(a), the test line (3) includes a capture ligand (11) that binds to a site (epitope) different from the site to which the first detection ligand (9) of the first conjugate to which the label (8) is conjugated binds among the sites of the standard material (14) injected from S0 to S7.

[0077] In Fig. 2(b), the test line (3) includes a capture ligand (11) that binds to a site (epitope) different from the site where the first detection ligand (9) to which the label (8) is conjugated binds among the sites of the target material (10) of the specimen.

[0078] In Fig. 2(c1), the inspection line (3) is an added constant concentration (S Hook ) and a sample that does not contain a target substance are injected together, and a capture ligand (11) is included that binds to a site different from the site of the standard substance (14) that binds to the labeled first detection ligand (9).

[0079] In Fig. 2(c2), the inspection line (3) is an added constant concentration (S Hook ) and a sample containing an excess (exceeding the maximum measurable amount) of the target substance are introduced together, and the labeled first detection ligand (9) includes a capture ligand (11) that binds to a site different from the site of the target substance (10) to which it binds.

[0080]

[0081] Figure 3 is a conceptual diagram for quantitative measurement of a sample target substance through calibration of a standard substance using a multi-strip of the present invention, including a section for injecting standard substances of various concentrations (S0-S7) to obtain a calibration curve, and a section for additionally mixing a standard substance of a certain concentration with the sample (S) to distinguish a false negative signal appearing from an excessive amount of target substance. Hook+ It consists of a part that drops the sample, and a part that drops the sample repeatedly (sample).

[0082] To obtain a calibration curve, standard substances of various concentrations (S0-S7) are prepared in a state (S0) where no standard substance is added to account for the background noise signal of the buffer, and the concentrations of standard substances corresponding to the dynamic range (the range of minimum and maximum values ​​of signal values) are injected starting from the minimum concentration (S1) to the maximum concentration (S7).

[0083] Added constant concentration (S Hook ) is injected together with the standard material and the sample to distinguish between the case where the detection signal becomes weak or not measured due to the presence of an excess target material and saturating the labeled detection ligand (hook effect) and the case where the target material is in trace amounts or does not exist in the first place. If the detection signal becomes weak or disappears but continues, it is determined that there is an excess target material, and if the detection signal that was weak or absent becomes strong, it is determined that there was no target material in the first place.

[0084] Multiple input ports for sample drop are used to repeat the measurement of the sample target substance, and the detection signal of the test line is observed to be reproducible and the average value is used to reduce the error caused by the analysis experiment.

[0085] In the present invention, the models or functions that can be applied when obtaining a calibration curve using standard substances of various concentrations are as follows.

[0086] (1) Linear model: (a, b: coefficients, error: error term, concentration: concentration)

[0087]

[0088] (2) Quadratic model: (a,b,c: coefficients, error: error term, concentration: concentration)

[0089]

[0090] (3) Four parameter logistic model:

[0091] (Top: upper asymptotes, Bottom: lower asymptotes, EC50: effective concentration representing 50% of the maximum response, Slope: slope, concentration: concentration)

[0092]

[0093] (4) Five parameter logistic model:

[0094] (Top: upper asymptotes, Bottom: lower asymptotes, EC 50 : Effective concentration that produces 50% of the maximum response, Slope: slope, concentration: concentration, Asymmetry: EC of a sigmoid curve 50 Asymmetry for)

[0095]

[0096] The signal intensity according to the concentration of the standard substance is applied to a model such as a linear function, a quadratic function, a 4-parameter logistic function, or a 5-parameter logistic function, and the model (function) with the smallest error between the predicted value of the signal intensity according to the concentration and the actual value is selected as the calibration curve, and the signal intensity of the target substance of the sample is applied to the above calibration curve to determine its concentration.

[0097]

[0098] Next, as an embodiment of the present invention, a rapid multi-strip immunochromatography analysis method and device for quantitatively measuring human serum PF4 are described.

[0099] The unit strip is manufactured by fixing mouse anti-PF4 antibody (monoclonal antibody; R&D Systems, USA) as a capture ligand (11) to the test line (3) of the nitrocellulose membrane, which is the detection pad (5), and rabbit IgG (polyclonal antibody; R&D Systems, USA) as a foreign substance (13) to the control line (4).

[0100] The first conjugate uses a rabbit anti-PF4 antibody (polyclonal antibody; R&D Systems, USA) (first detection ligand (9)) that recognizes an epitope at a different site from the antibody (capture ligand (11)) used in the test line (3) and a fluorescent label (8) conjugated thereto.

[0101] The second conjugate uses a goat anti-rabbit IgG antibody (polyclonal antibody; R&D Systems, USA) (second detection ligand (12)) conjugated with a fluorescent label (8).

[0102] And when the first and second conjugates are dried on a conjugate pad (Millipore, USA) (2), they are diluted with phosphate buffer and used.

[0103] A nitrocellulose membrane is attached as a detection pad (5) to a plastic support (6) containing an adhesive material, and a conjugate pad (2) and a sample pad (1) are sequentially attached by overlapping them at the bottom, and an absorbent pad (7) is overlapped and attached at the top, and then cut with a cutter to manufacture an immunochromatography strip, thereby completing the process.

[0104] A total of 12 of these unit strips are connected in parallel as shown in Figure 3 to ultimately provide a quantitative multi-strip immunochromatography analysis device.

[0105] Dilute the standard substance PF4 in buffer solution to prepare 0 μg / mL (S0), 0.8 μg / mL (S1), 1.6 μg / mL (S2), 3.2 μg / mL (S3), 6.4 μg / mL (S4), 12.8 μg / mL (S5), 25.6 μg / mL (S6), and 51.2 μg / mL (S7), and sequentially drop them onto the sample pads marked 'S0' to 'S7', respectively.

[0106] Additionally, a constant concentration (S) diluted in a buffer solution Hook ; For example, EC is the concentration that produces half the response of the maximum signal value. 50 A standard substance (using [Half maximal effective concentration]) was prepared by mixing human serum samples together, and 'S' in Figure 3 Hook Drop onto the specimen pad marked ‘+ specimen’.

[0107] Finally, prepare the same amount of serum (diluted in buffer) for repeated injections and inject it into each corresponding sample pad marked as 'sample' in Figure 3.

[0108] The detection signal, which is the result of the test line and control line reaction, can be measured by a device that detects the fluorescence of the label (8) generated using a fluorescent light source.

[0109] Figure 4 is an example of a calibration curve (log-log linear function applied) for a standard substance (PF4 protein) for measuring the target substance platelet factor 4 (PF-4) present in human serum in the present invention. The X-axis of the graph is log concentration (ng / mL), and the Y-axis is log signal intensity (unit). In other words, the standard substance calibration curve is a linear model in which the X-coordinate is the concentration on a log scale, and the Y-coordinate is the signal intensity on a log scale.

[0110] The concentration of protein PF-4, which represents the fluorescence signal intensity measured in a blood sample, is determined by reading the X-coordinate corresponding to the fluorescence signal intensity of PF-4 in the calibration curve (Fig. 4).

[0111] The fluorescence signal intensity of the target substance is repeatedly measured through three sample input ports separate from the input port for standard material calibration, and the average value is used as the final signal intensity for calibration.

[0112]

[0113] Next, the analysis method and device of the present invention can be used to provide a concentration value of a target substance, which is a disease marker, for in vitro diagnostics (IVDs) of a specific disease of the human body.

[0114] The above independent strip can be used to determine whether the single-strip immunochromatography method for analyzing infectious viral disease infection, including coronavirus and influenza virus, is false negative. That is, if no signal is observed in the single strip, an additional concentration (S) is required. Hook ) is added together with the sample to see whether the signal increases or the signal disappears continuously. If the signal increases, it means that the target substance does not exist in the sample (negative); if it disappears continuously, it means that the target substance existing in excess in the sample has saturated the detection ligand (primary) and capture ligand of the label, so that binding between the detection ligand (label) - target substance - capture ligand does not occur (false negative).

[0115] Until now, one of the most helpful methods for in vitro diagnosis of human diseases has been measuring the concentration of disease markers using human-derived substances such as blood. By constructing a device based on this multi-strip immunochromatography analysis method, it is possible to measure the concentration of target substances such as blood proteins, which are disease markers, and provide the results. Examples of disease markers frequently used in in vitro diagnosis are as follows: {'Specific disease:Disease marker [target substance]'}.

[0116] Sepsis: PCT (procalcitonin), CRP (C-reactive protein);

[0117] Myocardial infarction: troponin I, troponin T, creatin kinase-MB (CK-MB);

[0118] Differential diagnosis of heart failure in dyspnea: Brain natriuretic peptide (BNP), N-terminal prohormone of brain natriuretic peptide (NT-proBNP);

[0119] Neurological prognosis after cardiac arrest: S100B, neuron-specific enolase (NSE);

[0120] Oncological diseases: AFP (liver cancer, etc.), CEA (colon cancer, etc.), PSA (prostate cancer), ferritin (leukemia, etc.), TG (thyroid cancer), SCC (cervical cancer, etc.), Free light chain (multiple myeloma), CA19-9 (colon cancer, etc.), CA125 (ovarian cancer), CA15-3 (breast cancer), beta-HCG (placental tumor, etc.), NSE (small cell lung cancer, etc.), cyfra21-1 (lung cancer, etc.), Pepsinogen I / II (stomach cancer, etc.), HE4 (ovarian cancer, etc.).

[0121]

[0122] <Explanation of symbols>

[0123] 1: Sample pad

[0124] 2: Conjugate pad

[0125] 3: Test line

[0126] 4: Control line

[0127] 5: Detection pad

[0128] 6: Support

[0129] 7: Absorption pad

[0130] 8: Label

[0131] 9: 1st detection ligand

[0132] 10: Target analyte

[0133] 11: Capture ligand

[0134] 12: 2nd detection ligand

[0135] 13: Foreign material

[0136] 14: Standard material

[0137]

[0138] The multi-strip immunochromatographic analysis method and device using the calibration curve of the standard material of the present invention enable rapid quantitative measurement of biological substances, which was previously difficult, thereby providing a solution for point-of-care testing (POCT) with limitations in testing location, testing equipment, and testing time, especially in cases where quantitative testing is required, thereby opening up a new medical market.

Claims

1. A mixture of a sample and a standard substance of a certain concentration is added to the sample pad (1), and when the detection signal of the label (8) appearing on the test line (3) becomes stronger than that of the test line (3) where only the sample is added, it is determined that the concentration of the target analyte is lower than the concentration that saturates the detection and capture ligand; On the other hand, if the detection signal of the marker (8) appearing on the test line (3) becomes weaker or continues to disappear compared to the test line where only the sample is dropped, it is judged that the concentration of the target analyte is excessively high, resulting in a false negative result. A multi-strip immunochromatography analysis method characterized in that the above mixture is applied to a sample pad (1) of a separate independent strip among the multi-strips.

2. In claim 1, the signal intensity of the standard material at various concentrations is measured on the multi-strip, and a calibration curve according to the concentration is obtained using the signal intensity; A multi-strip immunochromatography analysis method characterized in that the calibration curve is used to quantify the target analyte.

3. A multi-strip immunochromatography analysis method according to claim 1, characterized in that the signal intensity of the target analyte is repeatedly measured and the average value is used to determine the concentration based on the calibration curve.

4. A multi-strip immunochromatography analysis method according to claim 1, wherein the material of the label is any one of a fluorescent dye, gold colloid, latex particles, colored polystyrene microparticles, and an enzyme; and the device for measuring the signal of the label is a CCD or CMOS.

5. A multi-strip immunochromatography analysis method according to claim 1, wherein the target material is any one of a protein, an antigen, an antibody, DNA, RNA, PNA, or an aptamer; and the sample is any one of blood, urine, saliva, spinal fluid, cell culture fluid, or microbial culture fluid.

6. A mixture of a sample and a standard substance of a certain concentration is added to the sample pad (1), and if the detection signal of the label (8) appearing on the test line (3) becomes stronger than that of the test line (3) where only the sample is added, it is determined that the concentration of the target analyte is lower than the concentration that saturates the detection and capture ligand; On the other hand, if the detection signal of the marker (8) appearing on the test line (3) becomes weaker or continues to disappear compared to the test line where only the sample is dropped, it is judged that the concentration of the target analyte is excessively high, resulting in a false negative. In a multi-strip immunochromatography analysis device, characterized in that the above mixture is dropped onto a sample pad of a separate independent strip among the multi-strips, Each single strip constituting the above multi-strip immunochromatography is composed of a sample pad (1), a conjugate pad (2), a detection pad (5), and an absorbent pad (7). A sample is administered to the above sample pad (1); The above conjugate pad (2) includes a first conjugate composed of a first detection ligand (9)-label (8) that reacts with the target material, and a second conjugate composed of a second detection ligand (12)-label (8) that does not react with the target material; The above detection pad (5) has a test line (3) on which a capture ligand (11) that reacts with a target substance in the sample is fixed; It includes a control line (4) on which a foreign substance (13) that does not react with the target substance in the sample but reacts with the second detection ligand (12) is fixed; A multi-strip immunochromatography analysis device characterized in that the above absorbent pad (7) absorbs a solution and provides power to the immunochromatography device.

7. A mixture of the sample and a standard substance of a certain concentration is added to the sample pad (1), and when the detection signal of the label (8) appearing on the test line (3) becomes stronger, it is judged that the concentration of the target analyte is lower than the concentration that saturates the detection and capture ligand, and thus signal measurement is possible; On the other hand, if the detection signal of the marker (8) appearing on the test line (3) becomes weaker or disappears continuously, it is judged that the concentration of the target analyte is excessively high, resulting in a false negative result; The above mixture is applied to the sample pad of a separate independent strip among the multiple strips; In an analysis method of measuring the signal intensity of the standard material at various concentrations on the multi-strip, obtaining a calibration curve according to the concentration using the signal intensity, and quantitatively measuring the target analyte using the calibration curve, The above analysis method is a quantitative analysis multi-strip immunochromatography analysis method characterized in that it is a method for providing a concentration value of a target analyte, which is a disease marker (biomarker), for in vitro diagnostics (IVDs) of a specific disease of the human body.

8. A quantitative analysis multi-strip immunochromatography analysis method according to claim 7, characterized in that the independent strip is used to determine whether a single-strip immunochromatography method for analyzing infectious viral disease infection, including coronavirus and influenza virus, is false negative.

9. In claim 7, the specific disease and the corresponding target substance {disease: target substance} are, respectively, sepsis: PCT (procalcitonin), CRP (C-reactive protein); myocardial infarction: troponin I, troponin T, creatin kinase-MB (CK-MB); Differentiation of heart failure in dyspnea: Brain natriuretic peptide (BNP), N-terminal prohormone of brain natriuretic peptide (NT-proBNP); Post-cardiac arrest neurological prognosis: S100B, neuron-specific enolase (NSE); Tumor disease: A quantitative analysis multi-strip immunochromatographic analysis method characterized by any one of AFP (liver cancer, etc.), CEA (colon cancer, etc.), PSA (prostate cancer), ferritin (leukemia, etc.), TG (thyroid cancer), SCC (cervical cancer, etc.), Free light chain (multiple myeloma), CA19-9 (colon cancer, etc.), CA125 (ovarian cancer), CA15-3 (breast cancer), beta-HCG (placental tumor, etc.), NSE (small cell lung cancer, etc.), cyfra21-1 (lung cancer, etc.), Pepsinogen I / II (gastric cancer, etc.), HE4 (ovarian cancer, etc.).

10. The in vitro diagnostic multi-strip immunochromatography in vitro analysis kit includes a sample pad (1), a conjugate pad (2), a detection pad (5), and an absorbent pad (7). Specimens and additional concentrations (S Hook ) The sample pad (1) onto which the mixture of standard substances is injected is formed as a separate independent strip among the multiple strips; The above-mentioned conjugate pad (2) includes a marker (8), and the marker (8) is used to confirm that when the detection signal appearing on the test line (3) in the independent strip becomes stronger, the concentration of the target analyte is lower than the concentration that saturates the detection and capture ligand, so that signal measurement is possible. On the other hand, if the detection signal appearing on the test line (3) becomes weaker or disappears continuously, it is to confirm that the concentration of the target analyte is excessively high, indicating a false negative; The above detection pad (5) includes a test line (3) and a control line (4); The above absorbent pad (7) absorbs the mixture; In an in vitro analysis kit that measures the signal intensity of the standard material at various concentrations on the multi-strip, obtains a calibration curve according to the concentration using the signal intensity, and quantitatively measures the target analyte using the calibration curve, The above in vitro analysis kit is an in vitro analysis kit using quantitative analysis multi-strip immunochromatography for in vitro diagnosis, characterized in that it is an in vitro analysis kit that provides a concentration value of a target substance, which is a disease marker (biomarker), for in vitro diagnosis (in vitro diagnostics, IVDs) of a specific disease of the human body.

11. In claim 10, the independent strip is characterized in that it is a strip for confirming whether or not a false negative result is obtained in a single-strip immunochromatography method for analyzing infectious viral disease infection, including a coronavirus or influenza virus. An in vitro diagnostic kit using a quantitative analysis multi-strip immunochromatography.

12. In claim 10, the specific disease and the corresponding target substance {disease: target substance} are, respectively, sepsis: PCT (procalcitonin), CRP (C-reactive protein); myocardial infarction: troponin I, troponin T, creatin kinase-MB (CK-MB); Differentiation of heart failure in dyspnea: Brain natriuretic peptide (BNP), N-terminal prohormone of brain natriuretic peptide (NT-proBNP); Post-cardiac arrest neurological prognosis: S100B, neuron-specific enolase (NSE); Oncological diseases: An in vitro diagnostic kit using a quantitative analysis multi-strip immunochromatography characterized by any one of AFP (liver cancer, etc.), CEA (colon cancer, etc.), PSA (prostate cancer), ferritin (leukemia, etc.), TG (thyroid cancer), SCC (cervical cancer, etc.), Free light chain (multiple myeloma), CA19-9 (colon cancer, etc.), CA125 (ovarian cancer), CA15-3 (breast cancer), beta-HCG (placental tumor, etc.), NSE (small cell lung cancer, etc.), cyfra21-1 (lung cancer, etc.), Pepsinogen I / II (stomach cancer, etc.), HE4 (ovarian cancer, etc.).

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