KR102047978B1 - Method for diagnosing rheumatoid arthritis based on lateral flow assay using anti-CCP antibody and Rheumatoid Factor - Google Patents

Abstract

The present application is a lateral flow-based in vitro fluorescence immunoassay that can simultaneously quantify anti-CCP antibodies and rheumatoid factors in a single kit for diagnosing rheumatoid arthritis.

Description

Method for diagnosing rheumatoid arthritis based on lateral flow assay using anti-CCP antibody and Rheumatoid Factor}

Point-of-care test (POCT) based rheumatoid arthritis diagnosis technology.

Rheumatoid Arthritis (RA) is a chronic progressive systemic inflammatory disease that primarily invades the joint's synovial membrane and destroys bone and cartilage. RA occurs in 0.5% to 1% of adults worldwide and is similar in all races and ethnic groups, but women are 2-3 times more likely than men. RA is a disease that can appear at any age, but especially at ages between 40 and 60 years.

In the diagnosis of RA, there is no so-called gold standard such as biochemical markers, pathological findings, or imaging findings that can be recognized by anyone. Rheumatoid factor has been used as a serological test in rheumatic diagnosis since 1987 according to the American College of Rheumatology (ACR) classification criteria. Rheumatoid factor is an autoantibody that binds to the Fc region of immunoglobulin IgG and is increased in about 60-80% of patients with rheumatoid arthritis, but has no specific specificity for arthritis, other autoimmune diseases, chronic inflammation, malignancy, There are also disadvantages to healthy older adults. Other autoantibodies found in patients with rheumatoid arthritis include RA33 and autoantibodies against Sa, p68, calpastatin, and perinuclear factor, but serological tests have not yet been developed.

The American and European Rheumatic Society added anti-CCP antibody tests as serological tests in the Rheumatoid Arthritis Diagnostic Classification revised in 2010. Anti-CCP (Cyclic Citrullinated Peptide) antibodies are autoantibodies that respond to epitopes containing citrulline residues and are a class of anti-fillagrin antibodies. Recently, anti-CCP antibody assays with similar specificities (90-98%) with similar sensitivity to rheumatoid factor tests have been developed and used.

Anti-CCP antibody tests have been associated with high diagnostic specificity and functional status of patients, as well as joint damage in patients with early rheumatoid arthritis.

Conventional methods for the diagnosis of rheumatoid arthritis are as follows. Anti-CCP antibody test is mainly used for microplate ELISA (ezyme-linked immunosorbent assay) and automated equipment. As a lateral flow analysis method, a rapid kit of the CCPoint product of Euro-diagnosis is commercialized. The microplate ELISA assay tests anti-CCP antibodies by diluting samples on CCP-coated plates and reacting enzyme-linked antibodies. Automated assays include chemiluminescent microparticle immunoassay and electrochemiluminescence immunoassay methods.

Assays using rheumatoid factor mainly use aggregation of human or animal IgG-coated particles (eg latex, sheep erythrocytes). Latex agglutination is a very sensitive method, but it can be highly false positive, and nonspecific agglutination occurs in normal individuals. Currently, automated methods that allow quantitative testing using nephelometry / turbidimetry methods are most widely used. These tests are mostly used to measure IgM type rheumatoid factor. In addition, the IgG-type rheumatoid factor test, which is known to be correlated with the activity of rheumatoid arthritis, is mainly used for the analysis of enzyme immunoassay.

The cause of rheumatoid arthritis is not clear, and it is thought that genetic, environmental, and opportunistic factors are involved, and there is no reliable method to date. Recently, a POCT product for qualitative analysis has been developed that simultaneously tests antibodies against rheumatoid factor and mutated citrullinated vimentin (MCV). Detection methods have not been developed to date.

The present application is to provide a lateral flow-based in vitro fluorescence immunoassay method that can quantify anti-CCP antibody and rheumatoid factor simultaneously in a single kit for diagnosing rheumatoid arthritis disease.

In order to solve the above problems, the present invention comprises the steps of providing a sample from a subject requiring a diagnosis of rheumatoid arthritis (RA);

The sample was provided with an IgG detector for detecting an anti-CCP antibody and a rheumatoid factor IgG (RF IgG) and an IgM detector for detecting a rheumatoid factor IgM (RF IgM). Forming a target-detector complex of a target and a detector in a sample;

Reacting the non-labeled IgG Fc fragment with the IgG detector that did not form the first complex with the detection object to form a complex of the unlabeled IgG Fc fragment and the IgG detector in the sample;

Forming a second complex of the first complex and the capture on a lateral flow strip to which the CCP antibody capture and the IgG Fc fragment, which is a rheumatoid factor capture, are immobilized;

It provides a method for the simultaneous detection of lateral flow-based rheumatoid arthritis markers in the in vitro comprising measuring a signal emitted from the second complex.

In another aspect, the present invention comprises the steps of providing a sample from a subject requiring a diagnosis of rheumatoid arthritis (RA); IgG sample for detecting anti-CCP antibody and rheumatoid factor IgG (RF IgG), IgM detector and control for detecting Rheumatoid Factor IgM (RF IgM) Providing an antibody detector to form a target-Detector complex of the detection target and the detector;

Reacting the non-labeled IgG Fc fragment with the IgG detector that did not form the first complex with the detection object to form a complex of the unlabeled IgG Fc fragment and the IgG detector in the sample;

Forming a second complex of the first complex and the capture on a lateral flow strip to which an anti-ccp antibody capture CCP, a rheumatoid factor capture IgG Fc fragment, and a control protein are immobilized; And

It provides a method for the simultaneous quantitative testing of lateral flow-based rheumatoid arthritis markers in the in vitro, including measuring the signal emitted from the second complex.

Finally, the present invention is a method for detecting the lateral flow-based rheumatoid factors RF IgG, RF IgM and anti-ccp antibody,

Among the IgG detectors for detecting RF IgG which is an anti-CCP antibody and rheumatoid factor in a sample, an unreacted IgG detector that does not react with the anti-CCP antibody and rheumatoid factor IgG (RF IgG) is reacted with an unlabeled IgG Fc fragment. Suppresses cross-reaction between IgG Fc fragment located on the lateral flow strip as a reactive IgG detector and rheumatoid factor trap, and the IgG detector and the IgM detector are detected through different optical systems. Provides a method for detecting rheumatic factors based on lateral flow.

The present application discloses a method for diagnosing rheumatoid arthritis using a lateral flow immunoassay-based anti-CCP antibody and rheumatoid factor for the diagnosis of rheumatoid arthritis. The method according to the present invention allows simultaneous detection of three markers of IgM rheumatoid factor (RF IgM), IgG rheumatoid factor (RF IgG) and anti-CCP antibody, allowing for faster and more accurate diagnosis. In particular, in order to suppress the cross-reaction between the non-binding anti-human IgG and the RF trapping IgG Fc fragment in the sample, a non-labeled IgG Fc additive was added to quickly and accurately detect the anti-CCP antibody and the rheumatoid factor IgG (RF IgG). Can be quantified.

FIG. 1 is a schematic diagram of binding a labeled anti-human IgG detector in a detection solution that remains unreacted with human IgG in a sample after mixing a sample and a detection solution by adding a non-labeled IgG Fc. The four conjugated detectors in the detection solution were TRF (T) conjugated anti-human IgG (IgG-T Detector), anti-control antibody (Control-T Detector), and Fluorescent (F) conjugated anti-human IgM (IgM), respectively. -F Detector) and anti-control antibody (Control-F Detector).
FIG. 2 is a schematic diagram of a cartridge in which CCP peptide and IgG Fc are immobilized with control (ex. Chicken IgY, streptavidin, KLH, Rabbit IgG) and test 2 lines on nitrocellulose membrane, respectively.
3 is a schematic diagram of IgM RF detection using a fluorescence optical system and loading IgG RF and anti-CCP using a time resolved fluorescence optical system after loading a non-labeled sample mixture into a cartridge.
4 is a schematic diagram showing the effect of cross-reaction between labeled anti-human IgG in detection solution and RF capture (IgG Fc) immobilized on nitrocellulose membrane when the non-labeled IgG Fc is added to the sample and the detection solution mixture. .
5 is a schematic diagram of a standard curve of anti-CCP through T-optics.
6 is a schematic diagram of a standard curve of IgM RF through F-optics.
7 is a schematic diagram of the standard curve of IgG RF through T-optics.
FIG. 8 shows the results of three commercial products and clinical sensitivity for each of IgM, IgG RF and anti-CCP detected through triple detection.
9 is a single, double, triple detection number for the 30 cases of rheumatoid disease serum through triple detection method.
FIG. 10 is a comparison table of clinical sensitivity and determination method for triple detection method and two commercial product combinations (anti-CCP product and RF product), and the sensitivity setting in the aspect determination criteria of rheumatoid patient serum, and the negative determination criteria of control serum. The specificity is set at.
FIG. 11 is a schematic diagram of a cartridge having CCP peptide, IgG Fc immobilized with control (ex. Chicken IgY, streptavidin, KLH, Rabbit IgG), IgG Fc fragment and test 2 lines on nitrocellulose membrane, respectively.
FIG. 12 is a result of detecting IgM RF using a fluorescence optical system after loading a sample mixture containing a non-label into the cartridge through the cartridge of FIG. .

The present invention provides a sample derived from a subject in need of a diagnosis of rheumatoid arthritis (RA); an IgG detector for detecting an anti-CCP antibody and rheumatoid factor IgG (RF IgG) that are targets on the sample. And providing an IgM detector for detecting Rheumatoid Factor IgM (RF IgM) as a target to form a target-Detector complex of the target and the detector in the sample; Reacting the non-labeled IgG Fc fragment with the IgG detector that failed to form the first complex with the detection target to form a complex of the unlabeled IgG Fc fragment and the IgG detector in the sample; anti-CCP antibody capture Forming a second complex of the first complex and the trap on the lateral flow strip having the phosphorus CCP and the IgG Fc fragment, which is a rheumatoid factor trapper; measuring a signal emitted from the second complex; A method for the simultaneous detection of lateral flow-based rheumatoid arthritis markers in.

Lateral flow analysis is a method of quantitatively or qualitatively examining a target analyte, eg, a particular nucleic acid or protein, contained in a sample, e.g., an oligonucleotide or a specific antibody and / or antigen that hybridizes to a nucleic acid of a constant sequence. Strips containing nitrocellulose membranes (developing media) that are bonded to specific locations are used. Strips are used for the detection of specific nucleic acids or proteins in a sample through chromatographic methods to transfer the sample and through sequence specific hybridization or antigen antibody reactions. For example, reference may be made to those disclosed in Korean Unexamined Patent Publication Nos. 2003-0065341, 2011-0007699, 2011-0127386, and Registration No. 1149357.

The strip includes a prepad, a sample separation pad, a development medium, and an absorbent pad, which can be located on a support. The prepad and the absorbent pad are respectively positioned at both ends of the support so as not to overlap or overlap each other, the development medium is located between the sample separation pad and the absorption pad, and the amount of the development medium Each end is in contact with one end of the absorbent pad and one end of the sample separation pad, and the test line can be attached with a capturer capable of capturing the anti-CCP antibody and rheumatoid factor, a rheumatoid arthritis marker. have. Before the sample is developed, the sample and the detection buffer solution containing the detector are reacted so that the anti-CCP antibody and rheumatoid factor in the sample specifically bind to the detector to form a complex, and the sample containing the complex is developed. The complex may be specifically detected by a detection antibody labeled with a fluorescent substance by specifically binding to the trap.

In addition, the lateral flow analysis may include a strip for developing a sample as described above and detecting a reactant, and a housing or a cartridge on which the strip is mounted.

Whole blood / plain pad may be used for the strip, and the plain pad may be a bonding pad bonded to a conjugation pad.

The conjugate pad may be coupled to the conjugation pad.

In addition, the conjugation pad suppresses cross-reaction of unreacted IgG detectors that do not react with the anti-CCP antibody and rheumatoid factor IgG (RF IgG) among IgG detectors for detecting anti-CCP antibody and rheumatoid factor IgG (RF IgG). In order to unlabel the IgG Fc fragment, the detector can be immobilized.

Rheumatoid arthritis herein is a chronic progressive systemic autoimmune disease, distinguished from degenerative arthritis, which is a non-inflammatory chronic arthritis mainly characterized by cartilage damage due to aging. The cause of rheumatoid arthritis is not clear, and it is thought that genetic, environmental and opportunistic factors are involved, and there is no reliable diagnosis to date, especially POCT (point of Care test-based detection methods have not been developed to date. In the present application, anti-CCP antibody and rheumatoid factor (IgM type, IgG type RF), as rheumatoid arthritis markers, can be detected simultaneously at both quantitative and qualitative levels.

Subject-derived samples for use in the methods according to the invention include whole blood, plasma or serum. The detection of the marker is not particularly limited. In embodiments according to the present invention plasma or serum may be used.

Anti-CCP antibodies are autoantibodies that respond to epitopes containing citrulline residues and may correspond to one type of antifillagrin antibody.

Rheumatoid factor (RF) is an autoantibody that binds to the Fc portion of IgG, most of which belongs to IgM (RF IgM), but also includes IgG (RF IgG) or IgA (RF IgA).

The detection antibody is an antibody that specifically reacts to the detection object, and anti-human IgG or anti-human IgM may be used as the detection antibody.

Anti-human IgG, a detection antibody for detecting anti-CCP antibody and rheumatoid factor IgG (RF IgG), has a strong cross reaction with Fc fragment, an RF capture. As a result, the presence of IgG RF in the sample binds to the detection antibody, but the remaining non-human IgG binds to the Fc fragment, which is an RF capture. In addition, the detection antibody may vary depending on the amount of anti-CCP antibody and IgG RF in the sample, and thus the amount of non-binding anti-human IgG may be changed. You will not be able to. Therefore, in order to develop a cartridge that simultaneously analyzes anti-CCP antibody, IgM and IgG rheumatoid factor, it is a major challenge to eliminate the cross-reaction between the trapping Fc fragment to test anti-human IgG and rheumatoid factor.

Anti-human IgM detection antibodies can not only analyze IgM RF but also respond to IgM type anti-CCP antibodies. Currently, some studies on subclasses of anti-CCP antibodies have been reported, but the clinical evidence is insufficient. For the present invention, an anti-CCP antibody was analyzed using an anti-CCP assay cartridge, and a sensitivity difference was confirmed in an anti-CCP antibody analysis with an IgG type. As reported by Aboukhamis I (2010), the IgG-type anti-CCP antibody showed higher specificity for rheumatoid diagnosis, and the anti-CCP reactivity of the IgM type was very insignificant. Therefore, the anti-CCP sensitivity of the IgM type was not considered when analyzing anti-CCP antibodies using two detection antibodies, anti-human IgG / M.

 The detector may bind a label to a detection antibody, and more preferably, the label may include nanoparticles or fluorescent particles.

The nanoparticles may be selected from the group consisting of metal nanoparticles, magnetic nanoparticles, silica nanoparticles, and latex nanoparticles.

Fluorescent particles are particles which emit light when a light source is provided, and Alexa Fluor 350, 405, 430, 488, 500, 514, 633, 647, 660, 680, 700, cy3, cy5, cy7, Rupy (tris ( 2,2-bipyridyl) ruthenium (II)), fluoresein Isothiocyanate (FITC), rhodamine 6G (rhodamine 6G), rhodamine B (rhodamine B), 6-carboxy-tetramethyl-rhodamine (TAMRA), Texas red ), DAPI (4,6-diamidino-2-phenylindole) and coumarin, but not limited to any one selected from the group consisting of.

The fluorescent particles may be time-resolved fluorescence (TRF). The time-divided fluorescent particles may include a complex compound of a lanthanide series element. The lanthanide element may include europium (III), terbium (III), samarium (III), dysprosium, and / or combinations thereof, and more specifically, europium. It may be a complex compound.

In order to detect anti-CCP antibody and rheumatoid factor at the same time, the detector may be detected using different optical systems, and for this purpose, ordinary fluorescent particles and time-division fluorescent particles may be used simultaneously.

In the present invention, the general fluorescent particles may refer to fluorescent particles that are not time division fluorescent particles.

In the present invention, the optical system is for detecting a fluorescence signal generated from a sample to be measured. The optical system may include an illumination optical system for illuminating the measurement sample and a detection optical system for detecting a fluorescence signal generated in the measurement sample by the excitation light.

The optical system may detect a fluorescent signal generated from ordinary fluorescent particles or time division fluorescent particles.

Specifically, the detector may be a TRF (T) conjugated anti-human IgG (IgG-T Detector), an anti-control antibody (Control-T Detector), a fluorescent (F) conjugated anti-human IgM (IgM-F Detector), or anti-control It may be an antibody (Control-F Detector).

 Since anti-human IgG and anti-human IgM used as detection antibodies respond to both RF capture IgG Fc, IgM RF and IgG RF reactivity cannot be distinguished. Therefore, in the present invention, a dual optical system can be used to inspect each. Anti-human IgG was used for conjugation with Europium (Eu) bead, and anti-human IgM was conjugated with fluorescence dye. Since the lanthanide compound Eu does not perform the fluorescence process, it is Eu3 + chelate. Since Eu3 + chelate has a large Stokes shift, which is a difference between hvEX (333 nm) and hvEM (613 nm), it is very easy to select an excitation filter and an emission filter. Can be. And since the lifetime of about 1000us is longer than the lifetime of general phosphors (1 ~ 10ns), it is possible to efficiently remove the fluorescence emission of other phosphors in the experiment. The present invention enables dual quantification of anti-CCP, IgG and IgM RF in a single sample by using dual optics incorporating a fluorescence optical system and a time division fluorescence optical system.

The target-detector complex of the detection target and the detector may be generated by combining an anti-CCP antibody and a rheumatoid factor (RF) as a detection target (IgG detector and IgM detector), respectively.

The first complex is an anti-ccp antibody-IgG detector combined with an anti-ccp antibody and an IgG detector. The first complex, an RF IgG-IgG detector combined with an RF IgG and an IgG detector, and an RF IgM coupled with an RF IgM and an IgM detector. It may consist of an IgM detector first complex.

Unlabeled IgG Fc was added to inhibit cross-reaction with non-binding anti-human IgG and RF capture IgG Fc fragment in the sample.

The unlabeled IgG Fc may be some fragment of Ig G Fc.

The unlabeled IgG Fc may react with an IgG detector that does not form a first complex to form a complex of an unlabeled IgG Fc fragment and an IgG detector in the sample. The complex of the unlabeled IgG Fc fragment and the IgG detector prevents the IgG detector that does not form the first complex from reacting with the IgG Fc fragment, a rheumatoid factor trapper immobilized in the strip, thereby enabling more accurate RF IgG measurement.

The unlabeled IgG Fc may be provided directly to the sample or may be immobilized on a strip. The unlabeled IgG Fc immobilized on the strip may react with an IgG detector that does not form a first complex, included in the sample. Preferably, an IgG detector that does not form the first complex may first react with an unlabeled IgG Fc prior to reacting with a capturer for detecting anti-ccp antibody and RF.

The capturer may be a ccp peptide to detect the anti-CCP antibody, and more specifically, two to four kinds of peptides used in (Patent EP 1 456 662B1, EP 2 058 663 A1) may be used in combination. Also, the capturer can be an IgG Fc fragment to detect RF.

The present invention comprises the steps of providing a sample from a subject in need of a diagnosis of rheumatoid arthritis (RA); IgG sample for detecting anti-CCP antibody and rheumatoid factor IgG (RF IgG), IgM detector and control for detecting Rheumatoid Factor IgM (RF IgM) Providing an antibody detector to form a target-Detector complex of the detection target and the detector;

Reacting the non-labeled IgG Fc fragment with the IgG detector that did not form the first complex with the detection object to form a complex of the unlabeled IgG Fc fragment and the IgG detector in the sample;

Forming a second complex of the first complex and the capture on a lateral flow strip in which a cyclic Citrullinated Peptide (CCP), an anti-CCP antibody capture, an IgG Fc fragment, a control protein, and a control protein are immobilized; And

The method for simultaneous quantitative testing of lateral flow-based rheumatoid arthritis markers in the in vitro comprising measuring a signal emitted from the second complex.

The control protein may be chicken IgY, but is not limited thereto unless it is inconsistent with the object of the present invention.

Providing a sample from a subject in need thereof for rheumatoid arthritis (RA) diagnosis; IgG sample for detecting anti-CCP antibody and rheumatoid factor IgG (RF IgG), IgM detector and control for detecting Rheumatoid Factor IgM (RF IgM) Providing an antibody detector to form a target-Detector complex of the detection target and the detector;

Reacting the non-labeled IgG Fc fragment with the IgG detector that did not form the first complex with the detection object to form a complex of the unlabeled IgG Fc fragment and the IgG detector in the sample;

Forming a second complex of the first complex and the capture on a lateral flow strip in which a cyclic Citrullinated Peptide (CCP), an anti-CCP antibody capture, an IgG Fc fragment, a control protein, and a control protein are immobilized; And

The method for simultaneous quantitative testing of lateral flow-based rheumatoid arthritis markers in the in vitro comprising measuring a signal emitted from the second complex.

For the quantification, a process of measuring a signal (or fluorescence amount) emitted through the second complex on the strip and analyzing the amount by a ratio value divided by the fluorescence amount of the control may be included.

The present invention is a method for detecting the side flow-based rheumatoid factor RF IgG, RF IgM and anti-ccp antibody, the anti-CCP antibody and rheumatoid in the IgG detector for detecting the RF IgG of rheumatoid factor in the sample Unreacted IgG detectors that did not react with factor IgG (RF IgG) were reacted with unlabeled IgG Fc fragments to cross-react with the unreacted IgG detectors and IgG Fc fragments located on the lateral flow strip as rheumatoid factor capturers. Suppress the above 　 The IgG detector and the IgM detector are directed to a lateral flow-based rheumatoid factor detection method in vitro, characterized in that it is detected through different optical systems.

The IgG detector may be an anti-IgG antibody conjugated with a label, or the IgM detector may be an anti-IgM antibody conjugated with a label.

The labeling material includes nanoparticles,

The nanoparticles may be any one selected from the group consisting of metal nanoparticles, magnetic nanoparticles, silica nanoparticles, and latex nanoparticles.

The labeling substance includes fluorescent particles,

The fluorescent particles are Alexa Fluor 350, 405, 430, 488, 500, 514, 633, 647, 660, 680, 700, cy3, cy5, cy7, Rupy (tris (2,2-bipyridyl) ruthenium (II) )), Fluoresein isothiocyanate (FITC), rhodamine 6G, rhodamine B, TAMRA (6-carboxy-tetramethyl-rhodamine), Texas red, DAPI (4,6-diamidino) -2-phenylindole) and coumarin, or may be time-resolved fluorescence (TRF).

 The time-resolved fluorescent particles may include europium or complexes thereof.

remind 　 Any one of an optical system for an IgG detector and an optical system for the IgM detector may be a time resolved fluorescence optical system.

<Example>

 Simultaneous Detection of Anti-CCP Antibody and IgM-RF

This method is a lateral flow fluorescence immunoassay that simultaneously examines anti-CCP antibodies and IgM-RF. This method can be designed to allow autoantibodies present in analyte (whole blood, plasma, serum) to bind CCP peptides and Fc fragments of IgG in the capture line. This method does not undergo additional washing and can be completed within 15 minutes to quantify the amount of fluorescence bound to the Fc portion of the CCP peptide or IgG using a laser-induced surface fluorescence detector.

Zeta-dividing capture of anti-CCP antibody (CCP peptide) on control line and test line 1 on the NC (HF90, HF135 & HF180) membrane and RF on capture (IgG Fc fragment) on test line 2 The cartridge was prepared by line dispensing using and drying at 37 degrees. To examine the anti-CCP antibody, the catcher used a mixture of two or four of the peptides used in (Patent EP 1 456 662B1, EP 2 058 663 A1), and the detection antibody used immunoglobulin G of the anti-human antibody. It was.

Anti-human IgG, a detection antibody for detecting Anti-CCP and IgG RF, was conjugated with time-resolved fluorescence (TRF) particles. Anti-human IgM, a detection antibody for detecting IgM RF, was conjugated with fluorescent molecules. When detecting a signal of a labeled detection antibody using a dual optical system, an anti-control antibody conjugated with time-resolved fluorescent particles (T) and fluorescent molecules (F) was used as a control.

In addition to the detection antibodies in the detection solution, detergents (ex. Tween-20, TritonX-100, CA630), neutral pH development solvents, and bovine serum albumin can be used.

 Immediately after introducing the serum, plasma or whole blood of the RA patient into the sample inlet of the cartridge, a detection solution containing a detection antibody (buffer solution) is introduced into the inlet, or the detection solution containing the RA patient's sample and the detection antibody is introduced in advance. After mixing, the lateral flow immune response can proceed. The amount of fluorescence can then be quantified using a laser induced surface fluorescence detector.

More specifically, the mixing ratio of the sample and the detection solution may be applied at 1: 2 to 1: 100 (v: v), and the maximum volume of the sample may be applied within 10ul. Unlabeled IgG Fc fragments are added to the sample and the detection solution to counteract the cross-reaction between labeled anti-human IgG in the detection solution and the RF capture (IgG Fc) immobilized on the nitrocellulose membrane (Figures 1 and 3).

The labeled anti-human IgG concentration and the amount of the unlabeled IgG Fc fragment were complementary to each other, and the amount of the unlabeled IgG Fc fragment increased with the increase of the detection antibody concentration.

After loading the mixed solution of the sample and the detection solution to which the unlabeled IgG Fc fragment was added to the cartridge and reacting within 15 minutes, the fluorescence value was measured using a reader equipped with dual optical systems (Fig. 2). Two control antibodies (monoconjugate conjugated to T / F optics) for detection of fluorescence signals for a total of four detection antibodies and two test antibodies for anti-CCP and RF detection. By correcting it as a fluorescence signal (test detection antibody fluorescence signal / control detection antibody fluorescence signal = ratio), the standard quantitative curve according to anti-CCP and RF concentration is prepared using the ratio value (Fig. 4-6). Based on the detection results of the triple markers in the sample, it is determined to be positive when at least one marker is positive, and negative only when anti-CCP is negative.

1. Inhibition of Cross Reaction by Addition of Unlabeled Fc Fragment

As shown in Figure 2, the control line and test line 1 capture (CCP peptide) for the anti-CCP antibody, RF capture (IgG Fc fragment) on the test line 2 using a zeta frequency divider after drying at 37 degrees The cartridge was produced.

Fluorescent nanoparticles were conjugated to a detection antibody, anti-human IgG, to prepare a detector and 0.025% was used. The fluorescence quantitative ichroma reader (boditech med.) Was used to measure the amount of detection fluorescence captured in each line in the cartridge and analyzed by the ratio value divided by the amount of fluorescence of the control. In the absence of additives (unlabeled Fc), only 100-ul of anti-human IgG, the detection antibody, was loaded into the cartridge. The cross-reaction with IgG Fc capture was very high at 7.56. It was confirmed that the cross-reaction was reduced in concentration. Cross-reaction was inhibited by masking unbound anti-human IgG by adding a non-labeled Fc fragment to the sample and a detection solution containing an anti-human IgG (see FIGS. 1 and 2). The amount of the additive may vary depending on the amount of the detection antibody, and as the concentration of the detection antibody increases, the amount of the additive may increase to suppress the cross reaction (see FIG. 3).

2. Comparison of clinical sensitivity between singe detection and commercial assays in Triple assay

As shown in Figure 2, the control line and test line 1 capture the anti-CCP antibody (CCP peptide), the test line 2 capture (IgG Fc fragment) to the line using a zeta divider and dried at 37 degrees After the cartridge was produced. A fluorescent nanoparticle (Europium chelator) was conjugated to a detection antibody, anti-human IgG, to prepare a detector was used 0.025%. The fluorescence quantitative ichroma reader (boditech med.) Was used to measure the amount of detection fluorescence captured in each line in the cartridge and analyzed by the ratio value divided by the amount of fluorescence of the control.

A total of 69 sera were used, and control sera (n = 39) including rheumatoid serum (n = 30) and non-RA serum and healthy serum (n = 39) were used in the triple detection method for simultaneous analysis with three commercial products. Clinical sensitivity / specificity (anti-CCP antibody and rheumatoid factor) were analyzed respectively (FIG. 7). Sensitivity and specificity in the commercial product was applied to the cut-off of each product, the threshold of the anti-CCP antibody and rheumatoid factor of the present invention was set through the ROC analysis based on the fluorescence ratio value.

<Clinical sample list>

<3 kinds of commercial products used in the present invention>

Analysis of commercial products showed similar results as previously reported (Abbas S, 2011), but the clinical sensitivity of each marker in the triple detection method showed similar levels of IgM, In case of IgG RF, each analysis showed lower sensitivity than commercial products. However, since the commercial product mainly detects IgM-type RF as an latex aggregation method, but also IgG-type or IgA-type RF, it can be difficult to directly compare with the present invention that isolates and detects IgM and IgG. have. In addition, when IgM RF and IgG RF were analyzed together, it was confirmed that the results appear with a higher level of sensitivity than the separation search result (see FIG. 8).

3. Comparison of clinical sensitivity between triple detection and two commercial tests

As shown in Figure 2, the control line and test line 1 capture the anti-CCP antibody (CCP peptide), the test line 2 capture (IgG Fc fragment) to the line using a zeta divider and dried at 37 degrees After the cartridge was produced. Detection antibody conjugated the fluorescent nanoparticles (Europium chelator) to anti-human IgG and 0.025% was used. The fluorescence quantitative ichroma reader (boditech med.) Was used to measure the amount of detection fluorescence captured in each line in the cartridge and analyzed by the ratio value divided by the amount of fluorescence of the control.

A total of 69 sera were used, and control sera (n = 39) including rheumatoid patient serum (n = 30) and non-RA serum and healthy serum (n = 39) were used for clinical analysis in the triple detection method for simultaneous analysis with each commercial product. Specific sensitivity / specificity (anti-CCP antibody and rheumatoid factor) were analyzed respectively.

Sensitivity and specificity in the product was applied to the cut-off of each product, the threshold of the anti-CCP antibody and rheumatoid factor of the present invention was set through the ROC analysis based on the fluorescence ratio value.

Positive results for each single marker, double marker, and triple marker were compared by sensitivity (see Fig. 9) for the dual and triple detection of anti-CCP and RF and the sensitivity of two commercial products. . When analyzing the sensitivity of rheumatoid patients (n = 30) using two commercial products, 83% of the anti-CCP and one or more of the RF markers were positive, up to 16% higher than the single test method. . The detection sensitivity of anti-CCP / IgM RF or anti-CCP / IgG RF and anti-CCP / IgM RF / IgG RF in the cartridge of the present invention are all 83%, which is the same as when two commercial products are used. Up to 26% more than a single test (see Figure 10).

On the other hand, rheumatoid arthritis negative serum was based on the high anti-CCP test result with a specificity of> 95% excluding low specific RF results, although 100% of both commercial anti-CCP products were used. Although the results were shown, the cartridge of the present invention showed a false positive response in one of the 39 samples, showing a specificity of 97%. This can be seen as a satisfactory level of specificity of the anti-CCP reported previously.

Claims (18)

Providing a sample from a subject in need thereof for rheumatoid arthritis (RA) diagnosis;
The sample is provided with an IgG detector for detecting an anti-CCP antibody and a rheumatoid factor IgG (RF IgG) and an IgM detector for detecting a rheumatoid factor IgM (RF IgM). Forming a target-detector complex of a target and a detector in a sample;
Reacting the non-labeled IgG Fc fragment with the IgG detector that did not form the first complex with the detection object to form a complex of the unlabeled IgG Fc fragment and the IgG detector in the sample;
Forming a second complex of the first complex and the capture on a lateral flow strip to which a cyclic Citrullinated Peptide (CCP), an anti-CCP antibody capture, and an IgG Fc fragment, a rheumatoid factor capture, are immobilized;
Measuring a signal emitted from the second complex; Simultaneous detection method of lateral flow-based rheumatoid arthritis markers in the containing. The method of claim 1, wherein the first complex
Anti-CCP Antibody-IgG Detector First Complex Combining Anti-CCP Antibody and IgG Detector,
RF IgG-IgG detector first complex, combined with RF IgG and IgG detector,
RF IgM-IgM detector first complex combined with RF IgM and IgM detector or
Simultaneous detection method of a rheumatoid arthritis marker characterized by the combination of these. The method of claim 1, wherein the second complex is a CCP-anti-CCP antibody-IgG detector second complex of the CCP and the anti-CCP antibody-IgG detector first complex,
IgG FC fragment-RF IgG-IgG detector second complex, wherein the IgG FC fragment and the RF IgG-IgG detector first complex bind to each other,
IgG FC fragment-RF IgM-IgM detector second complex, which is a combination of an IgG FC fragment and an RF IgM-IgM detector first complex, or
Simultaneous detection method of a rheumatoid arthritis marker characterized by the combination of these.
The method of claim 1, wherein the IgG detector is an anti-IgG antibody conjugated with a label, or the IgM detector is an anti-IgM antibody conjugated with a label.
The method of claim 4, wherein the labeling material comprises nanoparticles,
The nanoparticles are metal nanoparticles, magnetic nanoparticles, silica nanoparticles and latex nanoparticles, any one selected from the group consisting of rheumatoid arthritis markers characterized in that the method. The method of claim 4, wherein the labeling material comprises fluorescent particles,
The fluorescent particles are Alexa Fluor 350, 405, 430, 488, 500, 514, 633, 647, 660, 680, 700, cy3, cy5, cy7, Rupy (tris (2,2-bipyridyl) ruthenium (II) )), Fluoresein isothiocyanate (FITC), rhodamine 6G, rhodamine B, TAMRA (6-carboxy-tetramethyl-rhodamine), Texas red, DAPI (4,6-diamidino) 2-phenylindole) and coumarin or any one selected from the group consisting of time-resolved fluorescence (TRF), characterized in that the simultaneous detection of rheumatoid arthritis markers. The method of claim 6, wherein the fluorescent particles comprise europium or a complex thereof. The method of claim 1, wherein the sample is 1 to 10 µL.  The method of claim 1, further comprising determining the subject as a rheumatic high risk group when at least one of the rheumatoid factor or anti-CCP antibody is detected. Providing a sample from a subject in need thereof for rheumatoid arthritis (RA) diagnosis; IgG sample for detecting anti-CCP antibody and rheumatoid factor IgG (RF IgG), IgM detector and control for detecting Rheumatoid Factor IgM (RF IgM) Providing an antibody detector to form a target-Detector complex of the detection target and the detector;
Reacting the non-labeled IgG Fc fragment with the IgG detector that did not form the first complex with the detection object to form a complex of the unlabeled IgG Fc fragment and the IgG detector in the sample;
Forming a second complex of the first complex and the capture on a lateral flow strip in which a cyclic Citrullinated Peptide (CCP), an anti-CCP antibody capture, an IgG Fc fragment, a control protein, and a control protein are immobilized; And
Measuring a signal emitted from the second complex; Simultaneous quantitative testing method of lateral flow-based rheumatoid arthritis markers in a including. The method according to claim 10, wherein the first complex is an anti-CCP antibody-IgG detector first complex, wherein the anti-ccp antibody and the IgG detector is combined,
RF IgG-IgG detector first complex, combined with RF IgG and IgG detector,
A method for quantitative testing of rheumatoid arthritis markers, characterized in that the first combination of the RF IgM-IgM detector combined with the IgM and IgM detector. The method of claim 10, wherein the second complex is a CCP-anti-ccp antibody-IgG detector second complex, the CCP and the anti-ccp antibody-IgG detector first complex,
IgG FC fragment-RF IgG-IgG detector second complex, wherein the IgG FC fragment and RF IgG-IgG detector first complex are bound to each other,
IgG FC fragment-RF IgM-IgM detector second complex, wherein the IgG FC fragment and the RF IgM-IgM detector first complex bind to each other,
A contol antibody-control antibody detector complex or a combination thereof, wherein the contol antibody and the control antibody detector are bound to each other. As a method for detecting lateral flow-based rheumatoid factors, RF IgG, RF IgM and anti-ccp antibodies,
After reacting an anti-CCP antibody and an IgG detector for detecting RF IgG and an IgM detector for detecting RF IgM with the sample, the antibody does not react with the anti-CCP antibody and rheumatoid factor IgG (RF IgG). Unreacted IgG detectors were reacted with unlabeled IgG Fc fragments and then bound to IgG Fc fragments located on the lateral flow strips, such that the IgGs located on the lateral flow strips as unreacted IgG detectors and rheumatoid factor capturers. A method of simultaneously detecting lateral flow-based rheumatoid arthritis markers in vitro, wherein the IgG detector and the IgM detector are detected through different optical systems.
14. The method of claim 13, wherein the IgG detector is an anti-IgG antibody conjugated with a label, or the IgM detector is an anti-IgM antibody conjugated with a label.
The method of claim 14, wherein the labeling material comprises nanoparticles,
The nanoparticles are metal nanoparticles, magnetic nanoparticles, silica nanoparticles and latex nanoparticles, any one selected from the group consisting of rheumatoid arthritis markers characterized in that the method.
The method of claim 14, wherein the labeling material comprises fluorescent particles,
The fluorescent particles are Alexa Fluor 350, 405, 430, 488, 500, 514, 633, 647, 660, 680, 700, cy3, cy5, cy7, Rupy (tris (2,2-bipyridyl) ruthenium (II) )), Fluoresein isothiocyanate (FITC), rhodamine 6G, rhodamine B, TAMRA (6-carboxy-tetramethyl-rhodamine), Texas red, DAPI (4,6-diamidino) -2-phenylindole) and coumarin or any one selected from the group consisting of time-resolved fluorescence (TRF), characterized in that the simultaneous detection of rheumatoid arthritis markers.
The method for detecting and analyzing rheumatoid arthritis markers according to claim 16, wherein the time-resolved fluorescent particles comprise europium or complexes thereof.
The method for detecting and analyzing rheumatoid arthritis markers according to claim 13, wherein any one of the optical system for the IgG detector and the optical system for the IgM detector is a time resolved fluorescence optical system.

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