JP7651402B2 - A method for determining the incidence of any one disease selected from the group consisting of hypertension, cardiomyopathy, and atrial fibrillation, a test reagent for determining the incidence of said disease, and a test kit for determining the incidence of said disease - Google Patents

Description

The present invention relates to a method for determining the incidence of any one disease selected from the group consisting of hypertension, cardiomyopathy, and atrial fibrillation, a test reagent for determining the incidence, and a test kit for determining the incidence.

Traditionally, free immunoglobulin light chains (FLC), consisting of kappa and lambda chains, have been used to diagnose blood diseases such as myeloma and amyloidosis. In myeloma, depending on the type of cell that develops, tumors that produce the immunoglobulin light chain kappa chain have an increased kappa chain and an elevated kappa/lambda chain ratio, whereas tumors that produce the immunoglobulin light chain lambda chain have an increased lambda chain and a decreased kappa/lambda chain ratio.

It has also been reported that FLC is a useful indicator for diseases other than bone marrow tumors. For example, Patent Document 1 proposes a method for detecting at least one disease selected from cardiac failure, cardiomyopathy, myocarditis, and acute myocardial infarction using one or more values of FLC kappa chain value, lambda chain value, and kappa chain/lambda chain ratio as indicators. Patent Document 2 describes a method for measuring diabetes using FLC kappa chain value, lambda chain value, or kappa chain/lambda chain ratio. Non-Patent Document 1 proposes the use of cFLC (polyclonal combined immunoglobulin free light chain: the sum of FLC kappa chain content and FLC lambda chain content) as an indicator of the risk of developing cardiovascular disease in type 2 diabetes patients. Non-Patent Document 2 shows that the kappa chain/lambda chain ratio increases as the stage of chronic kidney disease (CKD) progresses.
Furthermore, Patent Document 1 and Non-Patent Document 3 report methods for detecting FLC kappa chain and lambda chain levels with higher sensitivity.

Patent No. 4950879 International Publication No. 2017/170847

Diabetes care, 2014, 37, p2028-2030 Clin J Am Soc Nephrol. 2008 November; 3(6): 1684-1690. J Immunol Methods. 2013, May 31; 391(1-2):1-13

Currently, hypertension is diagnosed by measuring blood pressure, but blood pressure often fluctuates transiently, and blood pressure must be monitored for a certain period of time before a diagnosis of hypertension can be made.

In addition, cardiomyopathy is diagnosed using imaging diagnostics such as echocardiography and MRI, but these imaging diagnostics are not usually included in health checkups, making it difficult to screen for cardiomyopathy.
Atrial fibrillation can be definitively diagnosed by electrocardiogram, but cannot be definitively diagnosed without an electrocardiogram. Furthermore, in paroxysmal atrial fibrillation, the electrocardiogram is normal when not experiencing an attack.

The present invention aims to provide a method for determining the prevalence of hypertension, cardiomyopathy, or atrial fibrillation using at least one sample selected from the group consisting of urine and saliva, a test reagent for determining the prevalence of the above diseases, and a test kit for determining the prevalence of the above diseases.

The present invention includes the following embodiments.
Item 1. A method for determining whether a subject suffers from any one disease selected from the group consisting of hypertension, cardiomyopathy, and atrial fibrillation, comprising the following steps (1) and (2): (1) obtaining a measurement value of at least one type selected from the group consisting of a kappa chain and a lambda chain of free immunoglobulin light chains in at least one sample selected from the group consisting of urine and saliva collected from the subject, and (2) comparing at least one measurement value selected from the group consisting of the kappa chain measurement value, the lambda chain measurement value, and a measurement value obtained by adding the kappa chain measurement value and the lambda chain measurement value obtained in step (1) with a corresponding standard value, indicating that the subject suffers from any one disease selected from the group consisting of hypertension, cardiomyopathy, and atrial fibrillation when the measurement value is higher than the corresponding standard value.
Item 2. The method according to Item 1, wherein the cardiomyopathy is dilated cardiomyopathy or hypertrophic cardiomyopathy.
Item 3. A test reagent for carrying out the method according to Item 1 or 2, in which a measurement value of a kappa chain of a free immunoglobulin light chain is obtained in the step (1), the test reagent comprising a free immunoglobulin light chain kappa chain antibody.
Item 4. A test reagent comprising a free immunoglobulin light chain lambda chain antibody for carrying out the method according to Item 1 or 2, in which a measured value of a lambda chain of a free immunoglobulin light chain is obtained in the step (1).
Item 5. A test kit for carrying out the method according to Item 1 or 2, in which measurement values of free immunoglobulin light chain kappa chain and lambda chain are obtained in the step (1), comprising a free immunoglobulin light chain kappa chain antibody and a free immunoglobulin light chain lambda chain antibody.
Item 6. A test kit for carrying out the method according to Item 1 or 2, in which a measurement value of a free immunoglobulin light chain kappa chain is obtained in the step (1), comprising a free immunoglobulin light chain kappa chain capture antibody and a free immunoglobulin light chain kappa chain detection antibody.
Item 7. A test kit for carrying out the method according to Item 1 or 2, in which a measurement value of a free immunoglobulin light chain kappa chain is obtained in the step (1), comprising a free immunoglobulin light chain lambda chain capture antibody and a free immunoglobulin light chain lambda chain detection antibody.
Item 8. A test kit for carrying out the method according to Item 1 or 2 in which a measurement value of a free immunoglobulin light chain kappa chain is obtained in the step (1), the test kit comprising a free immunoglobulin light chain kappa chain capture antibody, a free immunoglobulin light chain kappa chain detection antibody, a free immunoglobulin light chain lambda chain capture antibody, and a free immunoglobulin light chain lambda chain detection antibody.

A method for measuring the incidence of any one disease selected from the group consisting of hypertension, cardiomyopathy, and atrial fibrillation.The incidence of the disease can be measured.

1. Explanation of Terms First, the terms used in the present specification, claims, and abstract will be explained.

In this specification, "hypertension" refers to a state in which the systolic blood pressure measured at rest is 140 mmHg or more and/or the diastolic blood pressure is 90 mmHg or more, and said blood pressures are maintained for a certain period of time or more. Hypertension may include essential hypertension and secondary hypertension. Preferably, the hypertension is essential hypertension.

Essential hypertension includes hypertension caused by excessive salt intake, obesity, excessive alcohol consumption, mental stress, autonomic nervous system dysregulation, lack of exercise, lack of vegetables and fruits (minerals such as potassium), smoking, and genetic factors.

Secondary hypertension includes hypertension caused by heart disease, renal disease, primary aldosteronism, pheochromocytoma, etc. However, it is preferable that the hypertension disclosed in this specification does not include hypertension associated with heart failure, myocarditis, or cardiomyopathy.

The definition of "cardiomyopathy" in this specification follows the Japanese Circulation Society/Japanese Heart Failure Society Joint Guidelines for the Treatment of Cardiomyopathy (2018 revised edition; published March 29, 2019, updated June 28, 2019). According to the guidelines, cardiomyopathy may include dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and arrhythmogenic right ventricular cardiomyopathy. Preferably, cardiomyopathy is dilated cardiomyopathy or hypertrophic cardiomyopathy. Preferably, cardiomyopathy does not include cases that develop heart failure, myocarditis, or hypertension.

The diagnostic criteria for "atrial fibrillation" in this specification are in accordance with the Joint Guidelines for Cardiac Arrhythmia Drug Treatment of the Japanese Circulation Society/Japanese Society of Heart Rhythm Electrocardiology (2020 revised edition; published March 13, 2020). Preferably, cardiomyopathy does not include cases of heart failure, myocarditis, or hypertension.

In this specification, the term "subject" refers to an individual to which the measurement method according to the present invention described below is applied.

In this specification, the term "individual" is not particularly limited, and examples thereof include mammals such as humans, mice, rats, dogs, cats, rabbits, cows, horses, goats, sheep, and pigs. Preferably, the individual is a human, dog, or cat, and more preferably, a human. In addition, the individual includes both individuals with and without a disease (healthy individuals). The individual may be of any age or sex (male or female).

In this specification, the "specimen" is at least one selected from the group consisting of urine and saliva. Preferably, it is urine.

In this specification, "urine" is not particularly limited. Urine may be any of random urine, first urine in the morning, hourly urine, pooled urine, catheter urine, and suprapubic needle aspirate urine.

Furthermore, the urine sample may be fresh or may have been preserved. When the urine sample is preserved, it may be preserved at room temperature, in a refrigerated environment, or in a frozen environment, but is preferably preserved in a frozen environment. The urine sample may also contain a preservative such as toluene, xylene, hydrochloric acid, neutral formalin, or sodium azide.

In this specification, "saliva" is not limited as long as it is collected from the oral cavity of a subject. Saliva may be collected by collecting naturally secreted saliva, or may be collected from the salivary glands. When collected, saliva may be mixed with a buffer solution, a surfactant, etc. to reduce viscosity. It may also be mixed with a preservative such as sodium azide to prevent spoilage. The saliva sample may be fresh or may have been preserved. When preserving a saliva sample, it may be stored at room temperature, in a refrigerated environment, or in a frozen environment, but is preferably frozen.

In this specification, free immunoglobulin light chains (FLC) are also referred to as immunoglobulin free L chains or Bence Jones proteins. Free immunoglobulin light chains are monomers, dimers, etc. of immunoglobulin light chains that are not associated with immunoglobulin heavy chains. Immunoglobulin light chains are classified into two types, kappa (κ) chains and lambda (λ) chains, based on differences in antigenicity. In the following, free immunoglobulin light chain kappa chains may be referred to as FLC kappa chains or simply kappa chains. Similarly, free immunoglobulin light chain lambda chains may be referred to as FLC lambda chains or simply lambda chains.

The "measured value of kappa chain" refers to a value reflecting the concentration of free immunoglobulin light chain kappa chain in a sample, and may be either a molar concentration or a mass ratio per a fixed volume of sample (mass/volume), but preferably mass/volume. The "measured value of lambda chain" refers to a value reflecting the concentration of free immunoglobulin light chain lambda chain in a sample, and may be either a molar concentration or a mass ratio per a fixed volume of sample (mass/volume), but preferably mass/volume. In addition to the above, the value reflecting the concentration may be the intensity of a signal such as fluorescence or luminescence.

In this specification, the measured value of the concentration of kappa chains may be referred to as the "kappa chain measurement value," and the measured value of the concentration of lambda chains may be referred to as the "lambda chain measurement value." In addition, the sum of the kappa chain measurement value and the lambda chain measurement value may be referred to as the "kappa chain + lambda chain measurement value." The "+" in the above means "plus."

In this specification, the term "reference value" refers to a reference value corresponding to each measurement determined based on the kappa chain measurement value, lambda chain measurement value, or kappa chain + lambda chain measurement value in a sample from an individual not suffering from a disease such as hypertension, cardiomyopathy, atrial fibrillation, heart failure, myocarditis, diabetes, etc. (hereinafter referred to as an "unaffected individual"). Preferably, the unaffected individual is a healthy individual.

For example, when the kappa chain measurement value in a sample taken from a subject is the subject of evaluation, the "corresponding reference value" is a reference value determined based on the kappa chain measurement value in a sample from an unaffected individual. When the lambda chain measurement value in a sample taken from a subject is the subject of evaluation, the "corresponding reference value" is a reference value determined based on the lambda chain measurement value in a sample from an unaffected individual. When the kappa chain + lambda chain measurement value in a sample taken from a subject is the subject of evaluation, the "corresponding reference value" is a reference value determined based on the kappa chain + lambda chain measurement value in a sample from an unaffected individual. There may be one or more unaffected individuals for determining the reference value.

As another aspect of the "corresponding reference value", the reference value can be obtained as follows. For example, kappa chain measurement value, lambda chain measurement value, or kappa chain + lambda chain measurement value of a sample taken from an individual suffering from any one of hypertension, cardiomyopathy, and atrial fibrillation (hereinafter referred to as "affected individual") is obtained. In this case, it is preferable that there are multiple samples for each disease. Based on the respective measurements obtained from these multiple samples and the measurements obtained from multiple samples taken from non-affected individuals, the value that can most accurately classify each disease as positive (hereinafter simply referred to as "positive") or negative (hereinafter simply referred to as "negative") can be set as the "reference value". Here, the "value that can most accurately classify" can be appropriately set based on indicators such as sensitivity, specificity, positive predictive value, and negative predictive value depending on the purpose of the test.

Another aspect of the "corresponding reference value" is as follows.
For example, kappa chain measurement values, lambda chain measurement values, or kappa chain + lambda chain measurement values are obtained from a sample taken from an affected individual suffering from any one of hypertension, cardiomyopathy, and atrial fibrillation. In this case, there are multiple samples for each disease. The highest value for each disease among the groups of measurement values obtained from these multiple samples may be used as the reference value. Alternatively, the lowest value among the groups of measurement values of samples taken from multiple non-affected individuals may be determined as the reference value. For example, when it is desired to reduce false positives as much as possible, the reference value can be suitably used.

In addition, the lowest value for each disease among a group of measurement values obtained from samples taken from multiple diseased individuals may be used as the reference value. Alternatively, the highest value among a group of measurement values for samples taken from multiple non-diseased individuals may be determined as the reference value. For example, when it is desired to reduce false negatives as much as possible, such a reference value can be preferably used.

These reference values may be determined when the kappa chain/lambda chain ratio measurement value and the lambda chain/kappa chain ratio measurement value in the subject's sample are obtained from the subject, or may be determined in advance.

The term "healthy individual" is not particularly limited. It preferably refers to a human or non-human mammal as described in the "Individual" section, who shows no abnormal data in biochemical tests, blood tests, urine tests, serum tests, physiological tests, etc. The age and sex of a healthy individual are not particularly limited.

"Multiple specimens" refers to two or more, preferably five or more, and more preferably ten or more biological samples. These may be specimens collected from different individuals, or multiple specimens collected at different times from the same individual.

A "multiple values" is 2 or more, preferably 5 or more, more preferably 10 or more kappa chain/lambda chain ratio measurements, lambda chain/kappa chain ratio measurements, kappa chain-lambda chain measurements, lambda chain-kappa chain measurements, kappa chain+lambda chain ratio measurements, kappa chain concentrations, or lambda chain concentrations.
A "plurality of individuals" refers to two or more individuals, preferably five or more individuals, and more preferably ten or more individuals.

The individual from which each measurement is taken to determine the reference value does not necessarily have to be the same species, age, sex, etc. as the subject, but it is preferable that they are of the same species. In addition, it is preferable that the individual is of the same age and/or sex as the subject.

In the present invention, the "free immunoglobulin light chain kappa chain antibody" is also described as "anti-kappa chain antibody". There is no limitation on the free immunoglobulin light chain kappa chain antibody as long as it specifically binds to the kappa chain, and any of polyclonal antibodies, monoclonal antibodies, and fragments thereof (e.g., Fab, F(ab) ₂ , etc.) obtained by immunizing animals other than humans with the kappa chain or a part thereof as an antigen can be used. However, it is preferable to remove at least the hypervariable region from the antigen. Furthermore, the class and subclass of immunoglobulin are not particularly limited. Furthermore, it may be a chimeric antibody. Furthermore, it may be an scFv, etc. Furthermore, as the anti-kappa chain antibody, for example, the antibodies described in Patent Document 2, Non-Patent Document 3, Nakano T, and Nagata A. [J Immunol Methods. 2003 Apr 1;275(1-2):9-17. doi: 10.1016/s0022-1759(02)00512-4.], and commercially available products such as anti-Kappa light chain antibody [RM103] (ab190484) (Abcam) can also be used.

In the present specification, a "free immunoglobulin light chain lambda chain antibody" is also described as an "anti-lambda chain antibody". There are no limitations on the free immunoglobulin light chain lambda chain antibody as long as it specifically binds to the lambda chain, and any of polyclonal antibodies, monoclonal antibodies, and fragments thereof (e.g., Fab, F(ab) ₂ , etc.) obtained by immunizing animals other than humans with a kappa chain or a part thereof as an antigen can be used. However, it is preferable to remove at least the hypervariable region from the antigen. Furthermore, the class and subclass of immunoglobulin are not particularly limited. Furthermore, it may be a chimeric antibody. Furthermore, it may be an scFv or the like. Furthermore, as the anti-lambda chain antibody, for example, the antibodies described in Patent Document 2, Non-Patent Document 3, and Nakano T and Nagata A. [J Immunol Methods. 2003 Apr 1;275(1-2):9-17. doi: 10.1016/s0022-1759(02)00512-4.], and commercially available products such as anti-Lambda Light chain antibody [2G9] (ab1943) (Abcam) can also be used.

In this specification, a "capture antibody" is an antibody used for the purpose of capturing an antigen in an immunoassay method or the like. A "detection antibody" is an antibody used for the purpose of detecting an antigen, and is preferably an antibody labeled with a labeling substance as described below.

2. Method for Measuring Concentrations of FLC Kappa Chains and FLC Lambda Chains In this section, a method for obtaining measured values of FLC kappa chains and FLC lambda chains will be described in "3. Method for Measuring Disease Incidence" below.

The measured values of FLC kappa chain and FLC lambda chain can be obtained by an immunoassay method using the anti-kappa chain antibody or anti-lambda chain antibody described in "1. Explanation of Terms" above. The immunoassay method can be performed using a known ELISA method, immunochromatography method, or the like. Alternatively, as a more sensitive method, a competitive ELISA method can be used. The measured values of FLC kappa chain and FLC lambda chain can be obtained using two independent measurement systems or one measurement system.

When using a measurement system based on the ELISA method, an anti-kappa chain antibody or anti-lambda chain antibody for antigen capture can be immobilized in advance on a solid phase such as a microplate, fluorescent beads, or magnetic beads, and a complex can be formed between the immobilized anti-kappa chain antibody or anti-lambda chain antibody and the kappa chain or lambda chain in the sample. The concentration of kappa chain or lambda chain contained in the sample can be measured by detecting the complex immobilized on the solid phase or the complex formed on the solid phase by a method known in the art. In addition, in this method, a complex can be formed between the anti-kappa chain antibody for antigen capture or the anti-lambda chain antibody for antigen capture and the kappa chain or lambda chain in the sample first, and then the complex can be immobilized on the solid phase.

The method of immobilizing the anti-kappa chain antibody or anti-lambda chain antibody for antigen capture on the solid phase is not particularly limited. It can be performed directly or indirectly via another substance using a known method. An example of direct binding is physical adsorption. Specifically, each capture antibody can be directly physically bound to a microplate using an immunoplate or the like. Alternatively, the anti-kappa chain antibody or anti-lambda chain antibody for antigen capture can be indirectly immobilized on the solid phase. The indirect immobilization of the capture antibody on fluorescent beads, magnetic beads, etc. can be performed according to the method described in, for example, Patent Document 2 or Non-Patent Document 3.

The shape of the solid phase is not particularly limited, and examples of the solid phase include microplates, microtubes, test tubes, beads, membranes, and paper. The material of the solid phase is not particularly limited, and for example, polystyrene, polypropylene, and the like can be used for microplates, microtubes, test tubes, and the like. In the case of beads, polystyrene Xmap (registered trademark) beads (Luminex) and MagPlex (registered trademark) microspheres (Luminex) and the like can be used.

In this method, the solid phase may be washed following the formation of the complex. When washing, PBS containing a surfactant or the like may be used.

In this method, the detection of the complex can be performed using a detection anti-kappa chain antibody labeled with a labeling substance or a detection anti-lambda chain antibody labeled with a labeling substance in the case of a normal ELISA method, or can be performed using an unlabeled anti-kappa chain antibody or an anti-immunoglobulin antibody labeled with a labeling substance that can bind to an unlabeled anti-lambda chain antibody, but it is preferable to use a labeled detection anti-kappa chain antibody or a labeled detection anti-lambda chain antibody. In addition, it is preferable that the epitope in the antigen of the anti-kappa chain antibody or anti-lambda chain antibody used for detection is different from the epitope in the antigen of the anti-kappa chain antibody or anti-lambda chain antibody used for antigen capture.

When measuring the concentration of FLC kappa chain or FLC lambda chain by competitive ELISA, the FLC kappa chain or FLC lambda chain in the sample can be contacted with a capture anti-kappa chain antibody or anti-lambda chain antibody in the presence of a competitor labeled with a labeling substance. The competitor is a peptide or protein having an epitope recognized by the same capture antibody as the FLC kappa chain or FLC lambda chain in the sample. The competitor may be isolated from an individual, or may be synthesized by genetic engineering or chemical means.

The labeling substance used for detection, which is labeled to the anti-kappa chain antibody or anti-lambda chain antibody, the labeled anti-immunoglobulin antibody, or the competitor, is not particularly limited as long as it generates a detectable signal. For example, fluorescent substances, radioisotopes, enzymes, etc. are included. Examples of enzymes include alkaline phosphatase, peroxidase, etc. Examples of fluorescent substances include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, and Alexa Fluor (registered trademark), and fluorescent proteins such as GFP, etc. Examples of radioisotopes include ¹²⁵ I, ¹⁴ C, and ³² P. Among them, alkaline phosphatase or peroxidase is preferred as the labeling substance.

The anti-kappa chain antibody or anti-lambda chain antibody used for detection can be obtained by labeling each antibody with the above-mentioned labeling substance using a labeling method known in the art. Labeling may also be performed using a commercially available labeling kit. The labeled immunoglobulin antibody may be labeled using the same method as that used for labeling the anti-kappa chain antibody or anti-lambda chain antibody, or a commercially available antibody may be used.

In this method, the concentration of FLC kappa chain or FLC lambda chain contained in a sample can be measured by detecting a signal generated by the labeling substance of the detection antibody contained in the complex. Here, "detecting a signal" includes qualitatively detecting the presence or absence of a signal, quantifying the signal strength, and semi-quantitatively detecting the signal strength. Semi-quantitative detection means indicating the signal strength in stages, such as "no signal generated," "weak," "medium," and "strong." In this method, it is preferable to detect the signal strength quantitatively or semi-quantitatively.

A known method can be used to detect the signal. In this method, a measurement method can be appropriately selected according to the type of signal derived from the labeling substance. For example, when the labeling substance is an enzyme, the signal, such as light or color, generated by reacting the enzyme with a substrate can be measured using a known device such as a fluorometer, luminometer, or spectrophotometer.

The substrate for the enzyme can be appropriately selected from known substrates depending on the type of the enzyme. For example, when alkaline phosphatase is used as the enzyme, the substrate can be a chemiluminescent substrate such as CDP-Star (registered trademark) (disodium 4-chloro-3-(methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)trixylo[3.3.1.13,7]decane]-4-yl)phenyl phosphate), or a chromogenic substrate such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), disodium 5-bromo-6-chloro-indolyl phosphate, or p-nitrophenyl phosphate. When the labeling substance is peroxidase, the substrate can be tetramethylbenzidine (TMB), etc.

When the labeling substance is a radioisotope, the radiation as a signal can be measured using a known device such as a scintillation counter. When the labeling substance is a fluorescent substance, the fluorescence as a signal can be measured using a known device such as a fluorescent microplate reader or the Luminex (registered trademark) system (Luminex). The excitation wavelength and the fluorescence wavelength can be appropriately determined depending on the type of fluorescent substance used.

The signal detection result can be used as a measurement value of the kappa chain or a measurement value of the lambda chain. For example, when the signal strength is quantitatively detected, the measurement value of the signal strength itself or a value calculated from the measurement value of the signal strength can be used as the measurement value of the kappa chain or the measurement value of the lambda chain.

When using a measurement system based on the immunochromatography method (also called lateral flow immunoassay), for example, a capture antibody is immobilized in advance at a predetermined position (detection position) on a solid phase such as a membrane or paper, such as a nitrocellulose filter. In addition, a detection antibody is impregnated at a position (detection antibody application position) on the solid phase on which the capture antibody is immobilized, which is different from the immobilization position of the capture antibody. The detection antibody is not immobilized on the solid phase. Measurement samples such as a specimen, a dilution of the specimen, and a supernatant obtained by centrifuging the specimen are applied on the detection antibody application position or on the distal side of the detection position across the detection antibody application position. When the measurement sample is applied, the measurement sample permeates into the solid phase. If the measurement sample contains an antigen that binds to the detection antibody, the antigen in the measurement sample and the detection antibody contained in the detection antibody application position form a complex and permeate into the solid phase and move to the detection position. If the measurement sample contains an antigen, the capture antibody captures the complex of the antigen and the detection antibody at the detection position. The detection antibody used in the immunochromatography method is labeled with a metal nanoparticle such as gold nanoparticle as a labeling substance. Therefore, the metal nanoparticles on the detection antibody that have aggregated due to the antigen-antibody reaction exhibit a color. When gold nanoparticles are used, the complex of the antigen captured by the capture antibody and the detection antibody generally exhibits a red color. The color of the metal nanoparticles can be qualitatively observed visually, but the signal intensity of the color can also be measured using an immunochromatographic reader or the like. The measured value of the color signal intensity itself or a value calculated from the measured value of the signal intensity can be used as the measured value of the kappa chain or the measured value of the lambda chain.

Here, the color of the metal nanoparticles can be improved by adsorbing a metal ion different from the metal used to label the detection antibody onto the metal nanoparticles, thereby causing the detection antibody to exhibit a different color tone. For example, if the labeled metal nanoparticles are gold nanoparticles, silver ions can be adsorbed onto them.

3. Method for determining the incidence of a disease This specification discloses a method for determining the incidence of a disease selected from the group consisting of hypertension, cardiomyopathy, and atrial fibrillation in a subject (hereinafter, sometimes simply referred to as a "measurement method"). The measurement method is also a method for assisting in the diagnosis of the disease. The measurement method includes the following steps (1) and (2).

In step (1), according to the above-mentioned "2. Method for measuring the concentrations of FLC kappa chains and FLC lambda chains", a measurement value of at least one type selected from the group consisting of kappa chains and lambda chains of free immunoglobulin light chains in at least one sample selected from the group consisting of urine and saliva collected from a subject is obtained.

In step (2), at least one measurement value selected from the group consisting of the kappa chain measurement value, the lambda chain measurement value, and the sum of the kappa chain measurement value and the lambda chain measurement value obtained in step (1) is compared with a corresponding reference value. Here, when the measurement value is compared with the corresponding reference value, if the measurement value is higher than the corresponding reference value, it indicates that the subject is suffering from any one disease selected from the group consisting of hypertension, cardiomyopathy, and atrial fibrillation.

Furthermore, if at least one measurement value selected from the group consisting of the kappa chain measurement value, the lambda chain measurement value, and the sum of the kappa chain measurement value and the lambda chain measurement value obtained in step (1) is higher than the corresponding reference value, it can be determined that the subject is suffering from any one disease selected from the group consisting of hypertension, cardiomyopathy, and atrial fibrillation.

In addition, if at least one measurement value selected from the group consisting of the kappa chain measurement value, the lambda chain measurement value, and the sum of the kappa chain measurement value and the lambda chain measurement value obtained in step (1) is lower than the corresponding reference value, it can be determined that the subject does not suffer from any one disease selected from the group consisting of hypertension, cardiomyopathy, and atrial fibrillation.

In another embodiment, if at least one measurement value selected from the group consisting of the kappa chain measurement value, the lambda chain measurement value, and the sum of the kappa chain measurement value and the lambda chain measurement value obtained in step (1) is higher than the corresponding reference value, it can be determined that the subject is suffering from hypertension.

In addition, if at least one measurement value selected from the group consisting of the kappa chain measurement value, the lambda chain measurement value, and the sum of the kappa chain measurement value and the lambda chain measurement value obtained in step (1) is lower than the corresponding reference value, it can be determined that the subject does not suffer from hypertension.

In another embodiment, if at least one measurement value selected from the group consisting of the kappa chain measurement value, the lambda chain measurement value, and the sum of the kappa chain measurement value and the lambda chain measurement value obtained in step (1) is higher than the corresponding reference value, it can be determined that the subject is suffering from cardiomyopathy.

In addition, if at least one measurement value selected from the group consisting of the kappa chain measurement value, the lambda chain measurement value, and the sum of the kappa chain measurement value and the lambda chain measurement value obtained in step (1) is lower than the corresponding reference value, it can be determined that the subject does not suffer from cardiomyopathy.

In another embodiment, if at least one measurement value selected from the group consisting of the kappa chain measurement value, the lambda chain measurement value, and the sum of the kappa chain measurement value and the lambda chain measurement value obtained in step (1) is higher than the corresponding reference value, it can be determined that the subject is suffering from atrial fibrillation.

In addition, if at least one measurement value selected from the group consisting of the kappa chain measurement value, the lambda chain measurement value, and the sum of the kappa chain measurement value and the lambda chain measurement value obtained in step (1) is lower than the corresponding reference value, it can be determined that the subject does not suffer from atrial fibrillation.

4. Treatment Method When the above "3. Method for Determining the Prevalence of a Disease" suggests that a patient is suffering from any one disease selected from the group consisting of hypertension, cardiomyopathy, and atrial fibrillation, a treatment corresponding to the respective disease can be administered.
Treatment for hypertension includes limiting salt intake, potassium intake, obesity treatment, limiting alcohol consumption, exercise instruction, smoking cessation instruction, administration of antihypertensive drugs, and the like.

Treatments for cardiomyopathy include administration of beta-blockers such as Artist and Maintate; ACE (angiotensin-converting enzyme) inhibitors such as Renivace and Tanatril; angiotensin receptor antagonists such as Valsartan and Candesartan; and aldosterone receptor antagonists such as Spironolactone and Serara.

In the case of atrial fibrillation, anticoagulants such as warfarin can be administered to prevent thrombosis.

5. Test Reagent The present invention relates to a test reagent used in "3. Method for determining the onset of a disease".
The test reagent includes the following two aspects.

The test reagent of embodiment 1 contains a free immunoglobulin light chain kappa chain antibody (anti-kappa chain antibody).

The test reagent of aspect 2 contains a free immunoglobulin light chain lambda chain antibody (anti-lambda chain antibody).

The free immunoglobulin light chain kappa chain antibodies and free immunoglobulin light chain lambda chain antibodies described above in "1. Explanation of Terms" can be used.

The test reagent may contain at least one or more types of anti-kappa chain antibodies or anti-lambda chain antibodies. When the anti-kappa chain antibodies or anti-lambda chain antibodies are polyclonal antibodies, they may be polyclonal antibodies obtained by immunizing one type of antigen, or may be polyclonal antibodies obtained by immunizing the same individual with two or more types of antigens in parallel. In addition, polyclonal antibodies obtained by inoculating two or more types of antigens into different animals may be mixed. When the anti-kappa chain antibodies are monoclonal antibodies, they may be monoclonal antibodies produced from one type of hybridoma, or may be monoclonal antibodies produced from two or more types of hybridomas, and may contain two or more types of monoclonal antibodies that recognize the same or different epitopes. In addition, the test reagent may contain a mixture of one or more types of polyclonal antibodies and one or more types of monoclonal antibodies.

The form of the anti-kappa chain antibody or anti-lambda chain antibody contained in the test reagent is not particularly limited, and may be in a dry or liquid state, such as an antiserum or ascites containing the respective antibody. Furthermore, the form of each antibody may be a purified antibody, an immunoglobulin fraction containing the respective antibody, or an IgG fraction containing the respective antibody, in a dry or aqueous state.

When the antiserum or ascites containing each antibody is in a dry or liquid state, it may further contain at least one of the following: a stabilizer such as β-mercaptoethanol or DTT; a protector such as albumin; a surfactant such as polyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (10) octylphenyl ether; a preservative such as sodium azide. When the antibody is in a dry or aqueous solution form of a purified antibody, an immunoglobulin fraction containing each antibody, or an IgG fraction containing each antibody, it may further contain at least one of the following: a buffer component such as phosphate buffer; a stabilizer such as β-mercaptoethanol or DTT; a protector such as albumin; a salt such as sodium chloride; a surfactant such as polyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (10) octylphenyl ether; a preservative such as sodium azide.

The anti-kappa chain antibody or anti-lambda chain antibody contained in the test reagent may be unlabeled or may be labeled with biotin or the above-mentioned labeling substance, but is preferably labeled with biotin or the above-mentioned labeling substance. The labeling substance may be one exemplified in the above section "2. Method for measuring the concentrations of FLC kappa chain and FLC lambda chain", but is preferably alkaline phosphatase. In the present invention, each antibody may be provided in a state of being immobilized on a solid phase surface or the like. The solid phase and immobilization are as exemplified in the above section "2. Method for measuring the concentrations of FLC kappa chain and FLC lambda chain". The solid phase is preferably fluorescent beads or magnetic beads.

6. Test Kit The present invention relates to a test kit including the test reagent described in "4. Test Reagent" above.
The test kit includes the following three aspects:

Aspect 1 is a test kit that includes a free immunoglobulin light chain kappa chain capture antibody and a free immunoglobulin light chain kappa chain detection antibody.

Aspect 2 is a test kit that includes a free immunoglobulin light chain lambda chain capture antibody and a free immunoglobulin light chain lambda chain detection antibody.

Aspect 3 is a test kit including a free immunoglobulin light chain kappa chain capture antibody, a free immunoglobulin light chain kappa chain detection antibody, a free immunoglobulin light chain lambda chain capture antibody, and a free immunoglobulin light chain lambda chain detection antibody.

More specifically, the test kit includes a test reagent containing an anti-kappa chain antibody and/or an anti-lambda chain antibody labeled with a labeling substance (preferably biotin) exemplified in the above section "2. Method for measuring the concentrations of FLC kappa chain and FLC lambda chain" used as a capture antibody, and a test reagent containing an anti-kappa chain antibody and/or an anti-lambda chain antibody labeled with a labeling substance (preferably alkaline phosphatase and a fluorescent substance) exemplified in the above section "2. Method for measuring the concentrations of FLC kappa chain and FLC lambda chain" used as a detection antibody. This embodiment may further include a solid phase, preferably a solid phase bound to avidin, more preferably binding beads bound to avidin, or magnetic beads. The capture antibody may also be provided in a state of being immobilized on the solid phase. Furthermore, this embodiment may include a substrate. CDP-Star (registered trademark) is preferable as the substrate.

Details of the labeling substance, solid phase, and substrate are as described in "2. Method for measuring the concentrations of FLC kappa chain and FLC lambda chain," and the descriptions therein may be incorporated herein by reference.

When the capture antibody is previously bound to the solid phase, the test kit of this embodiment may include a solid phase to which the capture antibody is immobilized, a detection antibody, and a labeling substance. The detection antibody and the labeling substance may be contained in separate containers, or may be contained in the same container. Note that when the labeling substance is an enzyme and the test kit further includes a substrate, the enzyme and the substrate must be contained in separate containers. When provided to a user, at least two of the solid phase, the detection antibody, and the labeling substance may be packaged together, or may be packaged separately.

When the capture antibody is not bound to the solid phase in advance, the test kit of this embodiment may include a solid phase, a capture antibody, a detection antibody, and a labeling substance. At least two of the capture antibody, detection antibody, and labeling substance may be contained in the same container, or may be contained in separate containers. Note that when the labeling substance is an enzyme and the test kit further includes a substrate, the enzyme and the substrate must be contained in separate containers. When provided to a user, at least two of the solid phase, capture antibody, detection antibody, and labeling substance may be packaged together, or may be packaged separately.

In addition, if the kit is for performing a competitive ELISA method, it may contain a capture antibody and a competitor described in "2. Method for measuring FLC kappa chain and FLC lambda chain." In this case, the capture antibody may or may not be bound to a solid phase.

Furthermore, the test kit may have a configuration corresponding to the immunochromatography method. In the kit used for the immunochromatography method, the detection antibody labeled with a labeling substance and the capture antibody are present at different positions on the solid phase. As described above in "2. Measurement method of FLC kappa chain and FLC lambda chain", the capture antibody is immobilized at the detection position, but the detection antibody is only included at the detection antibody application position of the solid phase and is not immobilized on the solid phase. In addition to the detection antibody and the capture antibody, the solid phase may contain a control antibody for detecting the detection antibody. In addition to the solid phase, the kit corresponding to the immunochromatography method may contain a diluent containing a buffer or the like for diluting the sample.

The present invention will be described in more detail with reference to examples. However, the present invention should not be construed as being limited to the examples.

1. Methods Fifteen patients with dilated cardiomyopathy, 21 patients with hypertrophic cardiomyopathy, 16 patients with hypertension, and 29 healthy individuals were used as subjects to measure the concentrations of FLC kappa chain and FLC lambda chain in urine. The healthy individuals were those who were not suspected of having any disease, including these diseases. After urine was collected from the subjects, it was immediately frozen at -80°C and stored at -80°C until measurement.

Measurements were performed according to the method described by Nakano T, and Nagata A. [J Immunol Methods. 2003 Apr 1;275(1-2):9-17. doi: 10.1016/s0022-1759(02)00512-4.].

Using the measured values of FLC kappa chain and FLC lambda chain concentrations, the sum of the kappa chain concentration and the lambda chain concentration (kappa chain + lambda chain measurement value) and the ratio of the kappa chain concentration to the lambda chain concentration (kappa chain/lambda chain ratio) were calculated for each disease patient and healthy subject.
Statistical analysis was performed using StatFlex, using basic statistics (non-parametric methods) and comparison between two independent groups (Mann-Whitney U test).

2. Results The basic statistics and the results of the independent multi-group comparison are shown in Tables 1 to 4. The FLC kappa chain concentration and the kappa chain + lambda chain measurement values were significantly increased in patients with dilated cardiomyopathy, patients with hypertrophic cardiomyopathy, and patients with hypertension compared to healthy subjects. In addition, the FLC lambda chain was significantly increased in patients with dilated cardiomyopathy and patients with hypertrophic cardiomyopathy compared to healthy subjects. It also tended to increase in patients with hypertension. The ratio of the kappa chain concentration to the lambda chain concentration (calculated kappa chain/lambda chain ratio) did not differ significantly between healthy subjects and patients with dilated cardiomyopathy, patients with hypertrophic cardiomyopathy, and patients with hypertension.

Measurements of FLC kappa chain and FLC lambda chain obtained using urine samples have been shown to be suitable for detecting cardiomyopathy and hypertension.

Claims (7)

A method for determining whether a subject has any one disease selected from the group consisting of hypertension, dilated cardiomyopathy, and hypertrophic cardiomyopathy , comprising the following steps (1) and (2):
(1) obtaining a measurement value of at least one type of free immunoglobulin light chain selected from the group consisting of kappa chain and lambda chain in a urine sample collected from a subject; and (2) comparing at least one measurement value obtained in step (1) selected from the group consisting of the kappa chain measurement value, the lambda chain measurement value, and the sum of the kappa chain measurement value and the lambda chain measurement value with a corresponding standard value, wherein if the measurement value is higher than the corresponding standard value, it indicates that the subject is suffering from any one disease selected from the group consisting of hypertension, dilated cardiomyopathy, and hypertrophic cardiomyopathy . A test reagent comprising a free immunoglobulin light chain kappa chain antibody for carrying out the method according to claim 1 , in which a measurement value of a free immunoglobulin light chain kappa chain is obtained in the step (1). A test reagent comprising a free immunoglobulin light chain lambda chain antibody for carrying out the method according to claim 1 , in which a measurement value of a free immunoglobulin light chain lambda chain is obtained in the step (1). A test kit comprising a free immunoglobulin light chain kappa chain antibody and a free immunoglobulin light chain lambda chain antibody for carrying out the method according to claim 1 , in which measurement values of free immunoglobulin light chain kappa chain and lambda chain are obtained in step (1). A test kit comprising a free immunoglobulin light chain kappa chain capture antibody and a free immunoglobulin light chain kappa chain detection antibody for carrying out the method according to claim 1 in which a measurement value of a free immunoglobulin light chain kappa chain is obtained in step (1). A test kit comprising a free immunoglobulin light chain lambda chain capture antibody and a free immunoglobulin light chain lambda chain detection antibody for carrying out the method according to claim 1 in which a measurement value of a free immunoglobulin light chain kappa chain is obtained in the step (1). A test kit for carrying out the method according to claim 1 in which a measurement value of a free immunoglobulin light chain kappa chain is obtained in step (1), the test kit comprising a free immunoglobulin light chain kappa chain capture antibody, a free immunoglobulin light chain kappa chain detection antibody, a free immunoglobulin light chain lambda chain capture antibody, and a free immunoglobulin light chain lambda chain detection antibody.