WO2025225941A1 - Quantitative multi-strip immunochromatographic assay kit using competitive immunoassay - Google Patents

Abstract

The present invention provides a quantitative measurement method using competitive immunochromatography, which has not been previously reported. The quantitative measurement method comprises: (1) creating a calibration curve based on the signal intensity of a test line according to the concentration of a standard material by using multiple strips, and calculating the concentration of a target analyte by using the calibration curve; (2) when the concentration of the target analyte increases to a significant level on the calibration curve of the standard material, a labeled signal of a detection ligand on a control line remains unchanged in a case where a detection ligand of a target analyte-detection ligand conjugate binds to and saturates a capture ligand on the control line, making it impossible to estimate the concentration beyond that level; however, by designing a binding site for the capture ligand on the control line to be the target analyte of the target analyte-detection ligand conjugate, a high concentration of the target analyte breaks down the triple conjugate of detection ligand-target analyte-capture ligand, leading to dissociation of the labeled detection ligand and thereby weakening the signal (hook effect); and (3) comparing the signal intensity patterns of the test line and the control line according to the calibration curve of the standard material with the signal intensity pattern of a sample target analyte, and determining how far the target analyte is from the saturation point of the detection ligand. This method is characterized in that, in cases where the target analyte is present in an excessively large amount and exceeds the maximum value of a measurement range (dynamic range) of the calibration curve, the calibration curve using multiple strips and the configurations (test line and control line) of an immunochromatographic device are designed differently to distinguish such cases.

Description

Quantitative analysis multi-strip immunochromatographic assay kit using competitive immunoassay

The present invention relates to an immunochromatographic analysis device, method and use thereof, which utilizes a calibration curve of a standard material utilizing a multiple strip and a competitive immunoassay for quantifying a target substance such as a protein in a living body.

Immunochromatographic assay (ICA), also known as rapid test or lateral flow immunochromatography assay (LFIA), is a type of immunoassay that utilizes antigen-antibody reactions. It is a method that can analyze small amounts of target analytes in a short period of time, and 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.

In this type of immunochromatographic analysis, an assay strip containing a reactant that can react with the target substance to be detected and exhibit a change, or an analysis device in the form of a device mounted on a plastic case is generally used. A typical assay strip is composed of a sample pad that accommodates a liquid sample, a conjugate pad containing a conjugate in which a label that generates a signal that can be detected by the naked eye or a sensor is conjugated to a ligand such as an antigen or antibody, a detection pad in which a test line is formed that has a ligand (antigen or antibody) that specifically binds to the target substance in the sample or the conjugate, and a control line that can confirm the development of the sample, and an absorbent pad that finally accommodates the liquid sample.

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 and do not require expensive or time-consuming equipment. Therefore, immunochromatography assays are widely used in settings where rapid testing is needed, such as at home or in medical settings. For example, it is well known that pregnancy test kits, commonly used at home, are rapid tests that measure pregnancy-related hormones, and the recent COVID-19 rapid test kits are rapid tests used to quickly detect coronavirus infection (COVID-19).

The difference between sandwich enzyme-linked immunosorbent assay (ELISA), a representative immunoassay method, and ICA in terms of the analysis process is that this assay is performed quickly in one step based on the principle of chromatographic capillary action; whereas, since there is no separate washing process in the middle, when a large amount of target substance is present, the target substance-detection ligand complex binds to the capture ligand immobilized on the test line, preventing the formation of a triple complex of capture ligand-target substance-detection ligand. In other words, the extra target substance breaks the triple complex into the form of capture ligand-target substance, target substance, and target substance-detection ligand. This causes the signal from the test line to decrease after a certain threshold when the actual target substance is increased and its concentration is measured, resulting in the so-called hook phenomenon, where the signal increases and then decreases again. This phenomenon is a common occurrence when applying the above-mentioned immunoassay method (sandwich immunoassay) to immunochromatographic analysis, and therefore, a separate device or method is required to overcome this difficulty in immunoassay.

Meanwhile, a competitive immunoassay is commonly used along with the sandwich immunoassay in immunochromatographic analysis. This is an immunoassay in which, instead of directly measuring the target substance of the target substance-detector ligand complex with the capture ligand immobilized on the test line, the detector ligand that has not formed the target substance-detector ligand complex binds to a standard substance (a synthetic substance with the same properties as the target substance in the sample) immobilized on the test line, so that the signal intensity of the detector ligand is inversely proportional to the amount of the target substance. This immunoassay has the characteristic that the signal intensity of the test line weakens as the amount of target substance increases, which is contrary to instinctive visual recognition, and the signal intensity weakens and eventually disappears as the target substance increases.

As a background technology of the present invention, a ‘competitive immunoassay device (hereinafter referred to as ‘prior art 1’)’ is disclosed in U.S. Patent No. 5,648,274 (publication date July 15, 1997). The competitive immunoassay device of prior art 1 uses an immunochromatographic analysis method based on the principle of chromatographic capillary phenomenon. Specifically, an empty (labeled ligand that does not bind to a target substance) labeled ligand (also biotinylated) is designed to bind to a standard substance fixed to a test line, and the target substance-detection ligand complex formed when the labeled detection ligand (first) binds to a target substance (analyte) is captured by a capture ligand (avidin) in a control line (first) using an avidin-biotin bond. And there is a detection ligand (second) designed to bind to a foreign substance attached to another control line (second) so as to determine whether the solution flows normally in the device. In addition, in the prior art 1, a standard substance of a certain concentration is fixed to the bottom or to the detection ligand (second), and then immunochromatographic analysis is performed together with the target substance, and then the intensity of the signal appearing in the control line (first) is compared to determine whether the concentration of the target substance is high or low compared to the concentration of the standard substance.

As another background technology, a commonly used ‘typical competitive immunochromatographic assay for the detection of the organophosphorus pesticide EPN’ (Eun-Hye Lee, et al, Food and Agricultural Immunology, 2013; vol 24: 129-138) discloses a ‘typical competitive immunochromatographic assay (hereinafter referred to as ‘prior art 2’)’. The competitive immunochromatographic apparatus of prior art 2 is designed such that a labeled detection ligand (antibody-gold conjugate) binds to a standard substance (pesticide-ova) immobilized on a test line in an empty (non-bound) state, and the target substance-detection ligand conjugate formed by binding of the labeled detection ligand to the target substance (pesticide) is captured by binding of the detection ligand portion of the conjugate to a capture ligand (anti-mouse antibody) on a control line. Prior art 2 lacks a control line immobilized with another detection ligand or external substance, which is used to confirm the device's operation. These techniques are often used to confirm only the presence of a target substance, making them "qualitative measurement" rather than "quantitative measurement."

[Prior Art Literature]

[Patent Document]

(Patent Document 1) Prior Art 1: U.S. Patent No. 5,648,274 (Published on July 15, 1997)

[Non-patent literature]

(Non-patent literature 1) Prior art 2: Eun-Hye Lee, et al. Food and Agricultural Immunology 2013; 24:129-138

First, let's examine what quantitative analysis is. Unlike qualitative analysis, which deals with the presence (1) or absence (0) of a substance, quantitative analysis measures the continuous amount of a substance. It involves measuring and estimating the signal value that corresponds to the amount of that substance. It's a well-known fact that the signal value cannot be measured in perfect proportion to the substance, and a certain degree of error is inherent in this measurement. Now, let's examine the errors that arise during the quantitative measurement of an analyte. These errors can be divided into systematic errors and random errors.

(see https:/en.wikipedia.org/wiki/Accuracy_and_precision ).

Systematic errors arise from misalignment (or tuning) of the analytical instrument system, and are related to so-called accuracy issues. These can vary depending on factors such as ambient temperature and humidity, the experimenter, and the date of manufacture of the instrument during quantitative measurements. Therefore, any instrument that requires quantitative measurement must undergo a tuning process, similar to "zero adjustment," before starting analysis. Next, there are random errors caused by random errors occurring in nature, and these are related to so-called precision issues. These cannot be overcome through instrument adjustment, but rather through statistical processing (regression analysis, central limit theorem) that utilizes the average value of repeated measurements of the analyte.

Now, let's explore regression analysis, the most commonly used method in quantitative analysis. The regression function (curve) derived from standard substances with known concentrations can vary slightly depending on the conditions of the instrument and surrounding environment at the time of analysis (accuracy issue). Therefore, each time quantitative analysis is performed, a regression function using standard substances is derived and used as a calibration curve. Furthermore, by repeatedly measuring the analyte (often two or three times) and using the average value (to overcome chance error), the regression function derived through regression analysis can be used.

This provides a brief explanation of what quantitative analysis is. In immunochromatography using a single strip, signal values are obtained at several concentrations, a functional equation representing their correlation is derived, and the amount of analyte is later estimated from the signal values in the sample. This process is sometimes claimed as quantitative measurement. However, in this case, the correlation function cannot reflect the ambient environment, such as temperature, humidity, or the experimenter, at the time of actual sample analysis.

Therefore, in order to secure the aforementioned accuracy, a kind of ‘zero point adjustment’ or ‘tuning process’ must be performed each time the analyte in the sample is measured, and a calibration function (curve) for the standard substance must be obtained by considering the surrounding environment at the time. This can be easily understood by looking at the usage of ELISA (Enzyme-Linked Immunosorbent Assay), which is currently widely used in laboratories. In ELISA, a calibration curve, which is a regression function, is obtained each time the amount of analyte is measured using a standard substance.

As mentioned above, quantitative analysis requires an analytical device equipped with solutions that can define and improve accuracy and precision. Even for single-strip immunochromatography analyzers, there is no device that generates a calibration curve every time analyte is quantified, and there is no process of utilizing the average value of repeated measurements (regression analysis), so it is not considered quantitative measurement. Therefore, careful literature (papers, inventions) makes concessions in such cases and uses the term "semi-quantitative."

As mentioned above, the purpose of the present invention is to provide a “quantitative” immunochromatographic analysis method that goes beyond the existing qualitative or semi-quantitative immunochromatographic analysis.

To this end, first, a calibration curve is obtained according to various concentrations of standard substances, and then a method and device capable of measuring the concentration of the target substance are provided.

A method and device for detecting a case where the amount of a target substance in a sample is so high that it falls outside the measurement range of the calibration curve are included.

Also included in the present invention is the measurement of target substances in human-derived materials (e.g., blood) for in vitro diagnosis of human diseases.

The quantitative analysis multi-strip immunochromatography assay kit utilizing the competitive immunoassay of the present invention includes the method, device and use thereof.

First, the basic configuration of the multi-strip immunochromatography analysis kit of the present invention is characterized by configuring a multi-strip by connecting multiple unit strips in parallel, including a sample pad for receiving a liquid sample; a conjugate pad connected to one side of the sample pad and receiving first and second detection ligands conjugated to a label; a detection pad connected to one side of the conjugate pad and including a test line, first and second control lines; and an absorption pad connected to one side of the detection pad for receiving a liquid remaining after the reaction.

In addition, the conjugate pad includes a first conjugate in which a first detection ligand that reacts with a target analyte or standard material of a sample is conjugated to a label, and a second conjugate in which a second detection ligand that does not react with a target analyte of a sample is conjugated to a label.

In addition, the detection pad includes a test line and a first reference line and a second reference line, and the test line has a standard substance fixed thereon, and reacts with a first detection ligand that binds to the standard substance (or a target substance in the sample) to capture it in the form of a two-layer structure of a first conjugate-standard substance; the first reference line has a capture ligand fixed thereon, and reacts with a standard substance (target substance) portion of a standard substance (target substance)-first detection ligand conjugate formed by binding the standard substance (or a target substance in the sample) and the first detection ligand to capture it in the form of a three-layer structure of a first conjugate-standard substance (target substance)-capture ligand; and the second reference line has a foreign body fixed thereon, and reacts with a second detection ligand to capture it in the form of a two-layer structure of a second conjugate-foreign body. The role of the second ship here is to determine the normal operation of the deployed liquid in the device and to establish a standard state (a type of zero point adjustment).

The quantitative analysis multi-strip immunochromatographic analysis method utilizing competitive immunoassay of the present invention provides a quantitative measurement method of immunochromatographic analysis utilizing competitive immunoassay that has not been mentioned in the above-mentioned prior arts.

(1) Using a multi-strip, a calibration curve is obtained based on the signal intensity of the test line according to the concentration of the standard substance, and the concentration of the target substance is calculated using this.

(2) When the concentration of the target substance increases to a significant level on the calibration curve of the standard substance: When the detection ligand of the target substance-detection ligand complex binds to the capture ligand of the control line and saturates it, the label signal of the control line (first) detection ligand does not change, so the concentration above that cannot be inferred;

By designing the site where the capture ligand of the control line (first) binds to be the target substance of the target substance-detection ligand complex, the high concentration of target substance breaks the triple complex of detection ligand-target substance-capture ligand, causing the labeled detection ligand to fall off and weaken the signal (hook phenomenon).

(3) By comparing the signal intensity patterns of the test line and control line (first) according to the calibration curve of the above standard material with the signal intensity pattern of the sample target material, it is possible to determine how far the target material is from the point where it is saturated with the detection ligand.

In this way, when the target substance is excessively abundant and exceeds the maximum value of the range measured by the calibration curve (dynamic range), it can be differentiated by designing a calibration curve using multiple strips and a different configuration of the immunochromatographic device (test line and control line). A more detailed explanation of this is as follows.

The multi-strip immunochromatographic analysis method of the present invention, when quantifying (or measuring) a target substance, first measures the signal intensity of a standard substance at various concentration levels (e.g., S0, S1, S2, S3, S4, S5) using several strips to obtain a calibration curve, and then quantifies the target substance of a sample using this. 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 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 calibration curve to determine its concentration.

In a typical competitive immunoassay-based concentration measurement, the signal intensity of the test line is high when the target substance concentration is low, and as the target substance concentration increases, the signal intensity decreases and eventually disappears. This point can be seen as the state where the target substance saturates the detection ligand, and the signal value exceeds the maximum value of the dynamic range (the width between the maximum and minimum values of the signal measurement amount) and disappears without further change even as the target substance concentration increases.

This phenomenon is commonly experienced when measuring using enzyme-linked immunosorbent assay (ELISA) equipment in a central instrument analysis laboratory. That is, when the concentration of the target substance in the sample is significantly high, the detection ligand is completely saturated with the target substance, cannot react with the standard substance immobilized on the bottom of the experimental well plate, and is removed along with the target substance during the washing process. Therefore, it is impossible to provide any information about whether the initial amount (or concentration) of the target substance is close to or significantly beyond the maximum value of the signal value dynamic range. Of course, even in such cases, one method, if any, is to dilute the sample to lower the concentration of the target substance and re-measure.

Meanwhile, in a competitive immunochromatographic assay (competitive ICA) such as the present invention, in this assay method where there is no separate washing process in the analysis experiment process, an excessive amount of target material saturates the detection ligand, leaving no signal value on the test line where the standard material is fixed, while at the same time providing significant additional information depending on the composition of the first control line. If it is assumed that the capture ligand that reacts with the detection ligand of the target material-first detection ligand conjugate and captures the conjugate is fixed to the first control line. In this case, once the detection ligand of the conjugate reacts with the capture ligand of the first control line in the form of target material-first detection ligand-capture ligand, the signal value that has become maximum no longer changes.

However, if the capture ligand that reacts with the target substance of the target substance-first detection ligand conjugate and captures the conjugate is fixed to the first control line, the situation changes. First, when the target substance of the conjugate reacts with the capture ligand of the first control line, as the amount of the target substance increases, the signal value reaches the maximum in the form of first detection ligand-target substance-first capture ligand. However, after that, as an excessive concentration of the target substance is placed between the triple structures, separation occurs in the form of first detection ligand-target substance, target substance, target substance-first capture ligand (Hook effect), and as the concentration increases, the signal intensity actually decreases.

The multi-strip immunochromatographic analysis method of the present invention is a method in which a target substance-first detection ligand complex formed by saturating the labeled first detection ligand (10) with the target substance (9) is not captured by the standard substance (13) in the test line (3) and thus no detection signal appears, and a detection signal appears when the target substance is captured by the capture ligand (14) through the target substance in the first control line (4).

(a) If there is no difference compared to the detection signal of the first control line by the saturated standard substance-first detection ligand, the amount of the target substance is judged to be a value estimated from the calibration curve by the standard substance.

(I) If the detection signal of the first control line by the saturated standard substance-first detection ligand is weakened or disappears compared to the detection signal of the first control line by the saturated standard substance-first detection ligand, it is characterized in that the amount of the target substance is judged to be much larger than the value estimated from the calibration curve by the standard substance.

In addition, the multi-strip immunochromatography analysis method of the present invention is characterized by measuring the signal intensity of the standard material (13) at various concentrations on the test line of the multi-strip, obtaining a calibration curve according to the concentration using the signal intensity, and using the calibration curve to quantify the target material (9).

In addition, the multi-strip immunochromatography analysis method of the present invention is characterized by repeatedly measuring the signal intensity of the target substance (9) and determining the concentration based on the calibration curve using the average value.

The quantitative analysis multi-strip immunochromatography analysis device utilizing the competitive immunoassay of the present invention is

In the case where the target substance-first detection ligand complex formed by the labeled first detection ligand (10) being saturated with the target substance (9) is not captured by the standard substance (13) in the test line (3) and thus no detection signal appears, and the target substance is captured by the capture ligand (14) through the target substance in the first control line (4) and thus a detection signal appears,

(a) If there is no difference compared to the detection signal of the first control line by the saturated standard substance-first detection ligand, the amount of the target substance is judged to be a value estimated from the calibration curve by the standard substance.

(I) A quantitative analysis multi-strip immunochromatography analysis device utilizing competitive immunoassay to determine that the amount of the target substance is much greater than the value estimated from the calibration curve by the standard substance when the detection signal of the first control line by the saturated standard substance-first detection ligand is weakened or disappeared,

The above analysis device is sequentially connected with a sample pad (1), a conjugate pad (2), a detection pad (6), and an absorption pad (8).

The above sample pad (1) is injected with a sample containing the target substance (9) or the above standard substance (13);

The above-mentioned conjugate pad (2) includes a first detection ligand (10) and a second detection ligand (11) to which a label (12) is conjugated;

The above detection pad (6) has the inspection line (3), the first contrast line (4) and the second contrast line (5):

The above test line (3) has the standard substance (13) fixed to it that binds to the first detection ligand (10) that does not bind to the target substance (9),

The above first contrast line (4) is fixed with the capture ligand (14) that binds to the target material (9) of the target material-first detection ligand conjugate and captures the conjugate,

The second anti-corrosion line (5) above has an external substance (15) fixed thereon that binds to the second detection ligand (11);

The above absorbent pad (8) is characterized by absorbing the developing liquid of the specimen to provide driving force.

In addition, the above multi-strip immunochromatography analysis device is characterized as a device that displays a signal by using any one of a fluorescent dye, gold colloid, latex particles, colored polystyrene microparticles, or enzyme as a means of displaying a signal to a label (12).

In addition, the multi-strip immunochromatography analysis device is characterized in that the device measuring the signal of the label (12) combined with the standard material (13) or the target material (9) is a CCD or CMOS.

In addition, the above multi-strip immunochromatography analysis device is characterized in that the target substance (9) is any one of protein, antigen, antibody, DNA, RNA, PNA, or aptamer.

In addition, the multi-strip immunochromatography analysis device is characterized in that the sample is any one of blood, urine, saliva, spinal fluid, cell culture fluid, or microbial culture fluid.

The purpose of the present invention is to measure the marker concentration value for in vitro diagnostics (IVDs) of human diseases using a quantitative analysis multi-strip immunochromatography analysis device utilizing the above competitive immunoassay.

In addition, in the use of the above multi-strip immunochromatography analysis device, the human disease is an infectious viral disease including a coronavirus or influenza virus, and the marker concentration value is characterized as a value that quantitatively measures the concentration of a viral antigen or antibody to the virus as a disease marker of a patient infected with the infectious virus.

In addition, in the use of the above multi-strip immunochromatography analysis device, the human disease is sepsis of a patient complaining of systemic inflammatory response syndrome (SIRS), and the marker concentration value is characterized by being a value for quantitatively measuring the concentration of PCT (procalcitonin) and CRP (C-reactive protein) as disease diagnostic markers for the analysis of the above sepsis.

In addition, in the use of the above multi-strip immunochromatography analysis device, the human disease is myocardial infarction, and the marker concentration value is characterized by being a value for quantitatively measuring the concentration of troponin I, troponin T, and creatin kinase-MB (CK-MB) as disease diagnosis markers of a patient who has suffered myocardial infarction.

In addition, in the use of the above multi-strip immunochromatography analysis device, the human disease is a respiratory disease or heart failure disease of a patient complaining of acute respiratory distress, and the marker concentration value is characterized by being a value for quantitatively measuring the concentration of Brain natriuretic peptide (BNP) and N-terminal prohormone of brain natriuretic peptide (NT-proBNP) as a differential diagnostic marker for differential diagnosis of respiratory disease and heart failure disease of a patient complaining of acute respiratory distress.

In addition, in the use of the above multi-strip immunochromatography analysis device, the human disease is a neurological disease of a patient whose spontaneous circulation has returned after cardiac arrest, and the marker concentration value is characterized by being a value that quantitatively measures the concentration of S100B, neuron-specific enolase (NSE), as a prognostic marker of the neurological disease of a patient whose spontaneous circulation has returned after cardiac arrest.

In addition, in the use of the above multi-strip immunochromatography analysis device, the human disease is a malignant and benign tumor, and the marker concentration value is characterized by being a value for quantitatively measuring the concentration 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.) for predicting the treatment effect of the tumor, for follow-up observation of the tumor, or for screening the tumor.

Conventional single-strip competitive immunochromatographic analyzers have improvements, such as the ability to observe the test line response due to the detection ligand, which decreases as the amount of the target substance increases, the first reference line response due to the detection ligand, which increases as the amount of the target substance increases, and the second reference line response to confirm the operation of the device, including the development of the sample solution. However, these analytical devices could not obtain a calibration curve according to various concentrations of a standard substance, and in particular, could not estimate it when measuring an excessive concentration of the target substance.

According to the quantitative analysis multi-strip immunochromatography assay kit utilizing the competitive immunoassay of the present invention, a calibration curve can be obtained using standard substances of various concentrations to measure the concentration of a target substance, and a method and device are provided for detecting a case in which the amount of the target substance in a sample is significantly high enough to exceed the measurement range of the calibration curve. In addition, the present invention can be used to measure a target substance in a human-derived material (e.g., blood) for in vitro diagnosis of human diseases.

Therefore, the multi-strip immunochromatography assay kit of the present invention can provide a quantitative measurement device for biological substances that can be rapidly tested on site, replacing the existing single-strip immunochromatography assay method that was difficult to quantitatively test. In addition, the present invention has the advantage of confirming the reproducibility of quantitative results by repeatedly measuring the sample, and reducing the error (variance) due to analysis by using an average value instead of a single value for the test value used in the post-analysis.

In addition, the multi-strip immunochromatography assay kit of the present invention has the effect of drastically reducing the testing time, testing cost, and equipment cost compared to devices that require a central laboratory, such as enzyme-linked immunosorbent assay (ELISA) commonly used for quantitative measurement.

Figure 1 is a conceptual diagram of a unit strip immunochromatography device of the present invention for measuring a target substance. Panel A shows the structure, and Panel B shows the approximate change in signal value according to the concentration of the target substance.

Figure 2 is an explanatory diagram of substances captured on the test line, first reference line, and second reference line fixed to the multi-strip immunochromatography of the present invention when attempting to obtain a calibration curve for standard substances of various concentrations.

Panel A shows the case without a standard (S ₀ ), panel B shows the case with low-concentration standards (S ₁ -S ₂ ), panel C shows the case with high-concentration standards (S ₃ -S ₄ ), and panel D shows the case with the highest-concentration standard (S ₅ ).

Figure 3 illustrates a reaction that may appear in the first control line when a target substance of a concentration higher than the maximum is injected into a multi-strip immunochromatography. Panels A and B are cases of the present invention, and panel C is a general case for the control.

Panel A shows the case where the target substance (sample W) is injected at a concentration that does not exceed the dynamic range, and the highest first contrast signal value is measured.

Panel B shows a case where an excessive concentration of target material (sample X) outside the dynamic range is injected, and a first reference line signal value lower than the maximum concentration is measured.

Panel C shows a case where an excessive concentration of target material (sample X) outside the dynamic range is injected, and the highest first contrast signal value remains unchanged.

Figure 4 is a schematic diagram of a multi-strip immunochromatography analysis device of the present invention. Its configuration can be divided into a part for obtaining a calibration curve using standard substances (Standards) of various concentrations, and a part for repeatedly measuring the target substance of the sample. In particular, in the sample example, it shows the changes that occur when a target substance (sample W) with a high concentration that does not exceed the dynamic range and a target substance (sample X) with an excessive concentration that exceeds the dynamic range are injected.

Figure 5 is a representative calibration curve for platelet factor 4 (PF-4), a target substance present in human serum, which can be obtained through a competitive immunoassay such as the multi-strip immunochromatography of the present invention.

Hereinafter, specific details for carrying out the present invention will be described in detail with reference to the drawings.

First, immunochromatographic analysis can be considered qualitative and quantitative in terms of the precision with which it measures target substances. Qualitative tests, such as those found in pregnancy test kits or rapid COVID-19 diagnostic kits, have a control line that indicates normal functioning, along with a test line. This test line 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.

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 where the amount present varies significantly, such as pregnancy tests or COVID-19 infections, where qualitative analysis alone is sufficient.

To date, research efforts aimed at developing 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 analyte concentration on the test line and label signal measurements, as well as non-optical measurement methods (e.g., magnetic immunoassay; MIA). Furthermore, devices (e.g., microfluidics) to maintain a constant flow rate to reduce variability in capillary pumping of sample solutions have been developed. Such technical research has significantly improved the instrumental aspects of quantitative measurement in immunochromatographic analysis.

However, in immunoassays based on antigen-antibody reactions, such as immunochromatography, in addition to device improvements like automation, it is essential to first perform calibration using standard substances. This is a natural process, similar to a kind of "zero adjustment." In other words, at the time of the experiment, standard substances of various concentrations must be prepared, 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. This is because the signal value in measurements using these immunoassays can vary significantly depending on not only the physical conditions such as atmospheric pressure, temperature, and humidity at the time of measurement, but also the experimenter's habits and the manufacturing date (lot number) of the analysis device.

FIG. 1 is a perspective view (panel A) 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 detection pad (6) including a test line (3) and first and second control lines (4, 5), and an absorbent pad (8) on an adhesive plastic support (7). 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. In addition, the sample pad 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.

A sample dropped onto a sample pad passes through a conjugate pad (2) including a first conjugate composed of a first detection ligand (10)-label (12) that reacts with the target substance of the sample and a second conjugate composed of a second detection ligand (11)-label (12) that does not react with the target substance of the sample, and the labeled conjugates move to a detection pad (6) that indicates the detection result.

The above detection pad includes a test line (3), a first reference line (4), and a second reference line (5) which are positioned spaced apart from each other, and a standard substance (13) is fixed to the test line (3) so that an empty first detection ligand (10) that has not bound to a target substance in a sample is captured. The first reference line (4) has a capture ligand (14) fixed thereto which is designed to react with a target substance portion of a first detection ligand-target substance conjugate formed by the first detection ligand (10) reacting with a target substance (9) to form a conjugate, thereby capturing the conjugate. In addition, the second reference line has a foreign substance (15) fixed thereto to which a second detection ligand (11) that does not react with a target substance under any circumstances binds.

Here is a rough overview of the signal value changes according to the concentration of the target substance in the competitive immunochromatography of the above unit strip (Panel B). First, the signal value change of the test line shows a typical curve (calibration curve) of a competitive immunoassay, decreasing and then gradually disappearing as the concentration of the target substance increases. Next, looking at the signal value change of the first control line, as the concentration of the target substance increases, the signal value increases until it reaches the maximum at some point and then gradually decreases thereafter. This is because the triple structure of the first detection ligand-target substance-first capture ligand at the first control line showed the highest signal value, but due to the added target substance, separation occurs in the form of the first detection ligand-target substance, target substance, target substance-first capture ligand (Hook effect), so that the signal intensity actually decreases as the concentration increases. Finally, since the amount of the second detection ligand (11) in the conjugate pad (2) is constant, the signal value exhibited by the second detection ligand captured by the external substance (15) of the second control line is also constant. In addition to confirming the operation of the analysis device of the present invention, this signal value can also be used to standardize the signal value exhibited by each unit strip. It goes without saying that such standardization of strip signals is important for quantitative analysis.

FIG. 2 illustrates obtaining a calibration curve using signal values of standard substances at various concentrations using the multi-strip immunochromatography of the present invention. Panel A represents the case where there is no standard substance (S ₀ ), Panel B represents the case where there is a low-concentration standard substance (S ₁ -S ₂ ), Panel C represents the case where there is a high-concentration standard substance (S ₃ -S ₄ ), and Panel D represents the case where there is the highest concentration standard substance within the dynamic range (S ₅ ). In the case where there is no standard substance (S ₀ ), all detection ligands are captured by the standard substance of the test line, and in the case where there is a low-concentration standard substance (S ₁ -S ₂ ), a large portion of the detection ligands are bound to the standard substance of the test line in an empty state, and a small portion of the detection ligands are bound to the standard substance in a filled state (detection ligand-standard substance complex) and captured by the capture ligand of the first reference line. On the other hand, in the case of a high concentration standard substance (S ₃ -S ₄ ), a small portion of the detection ligand binds to the standard substance of the test line in an empty state, and a large portion of the detection ligand binds to the standard substance and is filled (detection ligand-standard substance complex) and is captured by the capture ligand of the first reference line. Finally, in the case of the standard substance with the highest concentration within the dynamic range (S ₅ ), most of the detection ligand binds to the standard substance and is filled (detection ligand-standard substance complex) and is captured by the capture ligand of the first reference line.

Figure 3 compares the reactions that can appear in the first control line when a target substance exceeding the maximum concentration is injected into the multi-strip immunochromatography of the present invention (Panels A and B) with the general case (Panel C). Panel A is a case where a target substance (sample W) is injected at a concentration that does not exceed the dynamic range, and the highest first control line signal value is measured. That is, most of the first detection ligands are saturated with the target substance, and the first detection ligand-target substance is captured by the capture ligand of the first control line via the target substance. Panel B is a case where a target substance (sample X) at an excessive concentration that exceeds the dynamic range is injected, and a first control line signal value lower than the maximum is measured. That is, the triple structure formed in the first contrast line, the first detection ligand-target substance-capture ligand, is split into the form of the first detection ligand-target substance, target substance, target substance-capture ligand by the added target substance, so that the first detection ligand is swept away, and the signal in the first contrast line becomes much weaker. Panel C is a case where an excessive concentration of target substance (sample X) that is out of the dynamic range is injected, and the maximum signal value of the first contrast line does not change. What makes panel C different from panel B is that the capture ligand of the first contrast line binds to the first detection ligand, not to the target substance portion of the first detection ligand-target substance. In other words, even when an excessive concentration of target substance is injected, the first detection ligand-capture ligand bond of target substance-first detection ligand-capture ligand cannot be broken.

FIG. 4 illustrates a multi-strip immunochromatography analysis device of the present invention. Its configuration can be divided into a part for obtaining a calibration curve by standard substances (Standards) of various concentrations, and a part for repeatedly measuring the target substance of the sample. In particular, in the example of the sample, it shows the changes that occur when a target substance (sample W) with a high concentration that does not exceed the dynamic range and a target substance (sample X) with an excessive concentration that exceeds the dynamic range are injected. That is, in the case of a target substance with a measurable concentration (sample W), the first detection ligand saturated with the target substance does not show any signal on the test line (3), and at the same time, it is all captured by the capture ligand of the first control line (4), showing a strong signal. At this time, there is no significant difference in the signal intensity of the first control line (4) compared to the case of the standard substance (S ₅ ) with the highest concentration within the dynamic range. This means that the concentration of the target substance is at a level that saturates the capture ligand, but is not yet high enough to break the triple structure of the first detection ligand-target substance-capture ligand. Next, when an excessively concentrated target substance (sample X) is injected, if we compare it with the case of the standard substance (S ₅ ) with the highest concentration within the dynamic range, we can see that the signal of the test line does not appear, but the signal intensity of the first reference line (4) decreases significantly. This means that the concentration of the target substance is high enough to break the triple structure of the first detection ligand-target substance-capture ligand beyond the level that saturates the capture ligand. Therefore, it can be seen that in this case (sample X), a much higher concentration of the target substance exists compared to the other cases (sample W). This can be said to be a quantitative analysis method that cannot be measured unless there is a calibration curve by a standard substance or the first reference line is not a fixed capture ligand that reacts with a standard substance other than the first detection ligand.

Figure 5 shows an example of a calibration curve for standard substances PF4 of various concentrations for quantitative measurement of human serum protein PF4 (platelet factor 4). The standard PF4 used to obtain the calibration curve consists of a total of six types (S0, S1, S2, S3, S4, S5), and the X-axis of the graph represents the concentration of the standard substance (ng/mL), and the Y-axis represents the signal intensity (unit).

(Example)

An embodiment of implementing a quantitative analysis multi-strip immunochromatography analysis kit utilizing the competitive immunoassay of the present invention is described in detail below.

The unit strip is manufactured by immobilizing protein platelet factor 4 (standard human PF4; R&D Systems, USA) as a standard substance on the test line (3) of the nitrocellulose membrane, which is the detection pad (6), mouse anti-PF4 monoclonal antibody (monoclonal anti-PF4 antibody; R&D Systems, USA) as a capture ligand (14) on the first control line, and rabbit IgG (rabbit antibody; R&D Systems, USA) as a foreign substance (15) on the second control line.

The first conjugate uses a mouse anti-PF4 polyclonal antibody (polyclonal anti-PF4 antibody; R&D Systems, USA), which is the first detection ligand (10), to which a fluorescent label (12) is conjugated.

The second conjugate uses a second detection ligand (11) that is a goat anti-rabbit IgG antibody (polyclonal anti-rabbit IgG antibody; R&D Systems, USA) conjugated with a fluorescent label (8).

When drying the first and second conjugates on a conjugate pad (Millipore, USA) (2), dilute them with phosphate buffer and use them.

A nitrocellulose membrane is attached as a detection pad (6) to a plastic support (7) containing an adhesive material, and a conjugate pad (2) and a sample pad (1) are sequentially overlapped and attached downwards, and an absorbent pad (8) is overlapped and attached upwards, and then cut with a cutter to manufacture an immunochromatography strip.

A total of 10 of these unit strips (the number can be adjusted as needed) are connected in parallel as shown in Fig. 4 to finally prepare a quantitative analysis multi-strip immunochromatography analysis device utilizing competitive immunoassay.

Dilute the standard material human PF4 in a buffer solution to prepare the concentrations of S0, S1, S2, S3, S4, and S5 in increasing order, and place them in order on the sample pads marked ‘S0’ to ‘S5’ on the ‘standard material’ strip.

For repeated measurements of a sample (diluted in buffer), prepare two (or three) equal amounts and drop them onto the sample pad of the ‘target substance’ strip. Figure 4 shows two different samples (sample W and sample X) being repeatedly administered. This is to repeatedly measure the fluorescence signal intensity of the target substance and use the average value as the final signal intensity for calibration.

The detection signal, which is the result of the reaction between the test line and the first and second control lines, can be measured by a device that detects the fluorescence of the label (12) generated using a fluorescent light source. In particular, the signal values from the test line and the first control line of each unit strip can be divided by the signal value of the second control line and used to standardize each signal value. This is a factor that greatly contributes to the quantitative measurement of the target substance.

The signal intensity according to the concentration of the standard substance is selected as a calibration curve by applying a model such as a linear function, a quadratic function, a 4-parameter logistic function, or a 5-parameter logistic function to the original value or log value of the concentration and signal intensity; the signal intensity of the target substance of the sample is applied to the calibration curve to determine its concentration. That is, the concentration of the protein PF-4, which represents the fluorescence signal intensity measured in the blood sample, is determined by reading the X-coordinate corresponding to the fluorescence signal intensity of PF-4 from the calibration curve (Fig. 5).

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.

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

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

(3) Four parameter logistic model:

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

(4) Five parameter logistic model:

(Top: upper asymptotes, Bottom: lower asymptotes, EC ₅₀ : effective concentration producing 50% of the maximum response,

Slope: slope, concentration: concentration, Asymmetry: asymmetry of the sigmoid curve with respect to EC ₅₀ )

<Explanation of symbols>

1: Sample pad

2: Conjugate pad

3: Test line

4: 1st control line

5. 2nd control line

6: Detection pad

7: Support

8: Absorption pad

9: Target analyte

10: 1st detection ligand

11: 2nd detection ligand

12: Label

13: Standard material

14: Capture ligand

15: Foreign material

The quantitative analysis multi-strip immunochromatographic assay kit utilizing the competitive immunoassay of the present invention enables rapid quantitative measurement of human-derived substances such as blood, which was previously difficult to do, 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.

The use of the multi-strip immunochromatography analysis of the present invention is characterized by providing marker concentration values for in vitro diagnostics (IVDs) of specific diseases of the human body.

The above specific diseases include infectious viral diseases, systemic inflammatory response syndrome, myocardial infarction, respiratory diseases or heart failure in patients complaining of acute respiratory distress, neurological diseases in patients with recovery of spontaneous circulation after cardiac arrest, malignant and benign tumors, etc.

The above markers are respectively viral antigen and antibody; procalcitonin, C-reactive protein; troponin I, troponin T, creatin kinase-MB; Brain natriuretic peptide, N-terminal prohormone of brain natriuretic peptide; S100B, neuron-specific enolase; 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.).

Claims (15)

A quantitative analysis multi-strip immunochromatography analysis method using competitive immunoassay, In the case where the target substance-first detection ligand complex formed by the labeled first detection ligand (10) being saturated with the target substance (9) is not captured by the standard substance (13) in the test line (3) and thus no detection signal appears, and the target substance is captured by the capture ligand (14) through the target substance in the first control line (4) and thus a detection signal appears, (a) If there is no difference compared to the detection signal of the first control line by the saturated standard substance-first detection ligand, the amount of the target substance is judged to be a value estimated from the calibration curve by the standard substance. (I) If the detection signal of the first control line by the saturated standard substance-first detection ligand is weakened or disappears compared to the first detection ligand, it is judged that the amount of the target substance is much larger than the value estimated from the calibration curve by the standard substance. A multi-strip immunochromatographic analysis method characterized by: In claim 1, the signal intensity of the standard material (13) at various concentrations is measured on the test line of 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 substance (9). A multi-strip immunochromatography analysis method according to claim 1, characterized in that the signal intensity of the target substance (9) is repeatedly measured and the average value is used to determine the concentration based on the calibration curve. In the case where the target substance-first detection ligand complex formed by the labeled first detection ligand (10) being saturated with the target substance (9) is not captured by the standard substance (13) in the test line (3) and thus no detection signal appears, and the target substance is captured by the capture ligand (14) through the target substance in the first control line (4) and thus a detection signal appears, (a) If there is no difference compared to the detection signal of the first control line by the saturated standard substance-first detection ligand, the amount of the target substance is judged to be a value estimated from the calibration curve by the standard substance. (I) A quantitative analysis multi-strip immunochromatography analysis device utilizing competitive immunoassay to determine that the amount of the target substance is much greater than the value estimated from the calibration curve by the standard substance when the detection signal of the first control line by the saturated standard substance-first detection ligand is weakened or disappeared, The above analysis device is sequentially connected with a sample pad (1), a conjugate pad (2), a detection pad (6), and an absorption pad (8). The above sample pad (1) is injected with a sample containing the target substance (9) or the above standard substance (13); The above-mentioned conjugate pad (2) includes a first detection ligand (10) and a second detection ligand (11) to which a label (12) is conjugated; The above detection pad (6) has the inspection line (3), the first contrast line (4) and the second contrast line (5): The above test line (3) has the standard substance (13) fixed to it that binds to the first detection ligand (10) that does not bind to the target substance (9), The above first contrast line (4) is fixed with the capture ligand (14) that binds to the target material (9) of the target material-first detection ligand conjugate and captures the conjugate, The second anti-corrosion line (5) above has an external substance (15) fixed thereon that binds to the second detection ligand (11); A multi-strip immunochromatography analysis device characterized in that the above absorbent pad (8) absorbs the developing liquid of the specimen to provide driving force. A multi-strip immunochromatography analysis device according to claim 4, characterized in that the means for indicating a signal to the label (12) is any one of a fluorescent dye, gold colloid, latex particles, colored polystyrene microparticles, or enzyme. A multi-strip immunochromatography analysis device according to claim 4, characterized in that the device for measuring the signal of the label (12) bound to the first detection ligand and the second detection ligand is a CCD or CMOS. A multi-strip immunochromatography analysis device according to claim 4, characterized in that the target material (9) is any one of a protein, an antigen, an antibody, DNA, RNA, PNA, or an aptamer. A multi-strip immunochromatography analysis device according to claim 4, characterized in that the sample is any one of blood, urine, saliva, spinal fluid, cell culture fluid, or microbial culture fluid. A multi-strip immunochromatography analysis device characterized in that it measures a marker concentration value for in vitro diagnostics (IVDs) of human diseases using the multi-strip immunochromatography analysis device of any one of claims 4 to 8. In claim 9, the human disease is a contagious viral disease including a coronavirus or influenza virus, A multi-strip immunochromatography analysis device characterized in that the above marker concentration value is a value that quantitatively measures the concentration of a virus antigen or antibody to the virus as a disease marker of a patient infected with the infectious virus. In claim 9, the human disease is sepsis in a patient complaining of systemic inflammatory response syndrome (SIRS), A multi-strip immunochromatography analysis device characterized in that the above marker concentration value is a value for quantitatively measuring the concentration of PCT (procalcitonin) and CRP (C-reactive protein) as disease diagnostic markers for the analysis of sepsis. In claim 9, the human disease is myocardial infarction, A multi-strip immunochromatography analysis device characterized in that the above marker concentration value is a value for quantitatively measuring the concentration of troponin I, troponin T, and creatin kinase-MB (CK-MB) as disease diagnosis markers of a patient who has suffered myocardial infarction. In claim 9, the human disease is a respiratory disease or heart failure disease of a patient complaining of acute respiratory distress, A multi-strip immunochromatography analysis device characterized in that the above marker concentration value is a value that quantitatively measures the concentration of brain natriuretic peptide (BNP) and N-terminal prohormone of brain natriuretic peptide (NT-proBNP) as differential diagnostic markers for differential diagnosis of respiratory disease and heart failure disease in a patient complaining of acute respiratory distress. In claim 9, the human disease is a nervous system disease of a patient whose spontaneous circulation has been restored after cardiac arrest. A multi-strip immunochromatography analysis device characterized in that the above marker concentration value is a value for quantitatively measuring the concentration of S100B, neuron-specific enolase (NSE), which is a prognostic marker for neurological diseases in patients who have returned to spontaneous circulation after cardiac arrest. In claim 9, the human disease is a malignant or benign tumor, The above marker concentration value is a multi-strip immunochromatography analysis device characterized in that it is a value for quantitatively measuring the concentration 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.) for predicting the treatment effect of the tumor, for follow-up observation of the tumor, or for screening the tumor.