KR20180023563A - Method for diagnosing Rheumatoid Arthritis based on lateral flow assay using anti-CCP antibody and Rheumatoid Factor as marker - Google Patents

Abstract

Disclosed in the present invention is a method for diagnosing rheumatoid arthritis based on lateral flow immunoassay using anti-CCP antibodies and rheumatoid factors as markers. The method according to the present invention enables the simultaneous detection of two markers, thereby enabling faster and more accurate diagnosis.

Description

Methods for diagnosing rheumatoid arthritis using an anti-CCP antibody and a rheumatoid factor based on lateral flow immunoassay [

It is a point of care test (POCT) based diagnosis of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic, progressive systemic inflammatory disease that mainly involves the involvement of the synovial membrane and destroys the bone and cartilage. RA occurs in 0.5% to 1% of adults worldwide and is similar across all races and peoples, but women are 2-3 times more likely to be men than men. Although RA can occur in all ages, it is a disease that is prominent at ages between 40 and 60 years.

In the diagnosis of RA, there are no so-called gold standards such as biochemical markers, pathologic findings, and imaging findings that can be recognized by anyone. Since 1987, rheumatoid factor has been used as a serological test in the diagnosis of rheumatoid arthritis in the American College of Rheumatology (ACR). The rheumatoid factor is an autoantibody that binds to the Fc region of immunoglobulin IgG and is elevated in about 80% of patients with rheumatoid arthritis, but is not as specific for arthritis as other autoimmune diseases, chronic inflammation, malignant tumors, There is a disadvantage that also appears. Other autoantibodies found in patients with rheumatoid arthritis include autoantibodies to RA33 and Sa, p68, calpastatin, and perinuclear factor, but serologic tests for them have not been developed yet.

The American and European Rheumatologists Association added an anti-CCP antibody test as a serologic test in the revised rheumatoid disease classification in 2010. Anti - CCP (Cyclic Citrullinated Peptide) antibodies are autoantibodies that react with antigenic determinants containing citrulline residues and are a class of anti - fillagrin antibodies. Recently, a second-generation anti-CCP antibody having a specificity of 80 to 100% with similar sensitivity to rheumatoid factor has been developed and used. Anti-CCP antibodies are associated with high diagnostic specificity and functional status of patients, and have been reported to be associated with joint damage in patients with premature rheumatoid arthritis.

The existing method is as follows.

The anti-CCP antibody test is mainly performed using a microplate ELISA (ezyme-linked immunosorbent assay) and an automated instrument. A rapid kit of Euro-diagnosis's CCPoint product is commercialized by lateral flow analysis. The microplate ELISA assay tests the anti-CCP antibody by reacting the enzyme-conjugated antibody after reacting the sample on a CCP coated plate. Chemiluminescent microparticle immunoassay and electrochemiluminescence immunoassay methods are used for automated methods (see table below).

<Microplate ELISA Assay>

<Automated analysis method>

In the rheumatoid factor assay, RF-IgM assays use aggregation reactions of human or animal IgG-coated particles (eg, latex, sheep erythrocytes). Latex agglutination can be highly sensitive, but highly false, and non-specific agglutination appears in normal subjects. At present, automated assays that can be quantified using the nephelometry / turbidimetry method are most widely used (see table below).

<RF test method>

The cause of rheumatoid arthritis is unclear, and genetic, environmental, and opportunistic factors are thought to be involved. There is no definite way to diagnose rheumatoid arthritis, especially POCT (point of care treatment) based detection method has not been developed to date.

The present invention provides an in vitro fluorescent immunoassay method capable of simultaneous quantification of anti-CCP antibodies and rheumatoid factors in a single kit to diagnose rheumatic diseases.

The present invention provides a method for diagnosing rheumatoid arthritis using an anti-CCP antibody and a rheumatic factor based on lateral flow systems, and a kit and system for use in the method.

In one embodiment, the invention provides a method of treating a patient suffering from rheumatoid arthritis, comprising: providing a sample from a subject requiring diagnosis of rheumatoid arthritis; Loading the sample into a lateral flow strip comprising an anti-CCP antibody and a rheumatoid factor detection reagent, resulting in the formation of a complex of the anti-CCP antibody and rheumatoid factor and the respective detection reagent in the sample, followed by detection of the complex Wherein the second complex of the complex and the detection antibody is formed, and then detecting a signal emitted by the second complex, wherein the second complex of the complex and the detection antibody is subsequently detected, Detection method.

In one embodiment according to the present application, the sample is whole blood, serum or plasma.

In one embodiment according to the present application, the detection is a quantitative or qualitative analysis.

The detection method according to the present invention can be useful for diagnosis of rheumatoid arthritis.

The present invention discloses a method for diagnosing rheumatoid arthritis using anti-CCP antibodies and a rheumatic factor based on lateral flow immunoassay for the diagnosis of rheumatoid arthritis. The method according to the present invention enables the simultaneous detection of two markers, enabling faster and more accurate diagnosis. In particular, the lateral flow detection technique enables rapid and precise quantification.

1 is a diagrammatic representation of a strip used in a lateral flow system in accordance with an embodiment of the present invention.
Figure 2 schematically illustrates a cartridge housing in which the strip according to the present invention is mounted and used.
FIG. 3 is a graph showing the results of quantitation of anti-CCP by the method according to the present invention using 3 RA confirmed patient serum (n = 44) and healthy human serum (n = 32) as a sample.
FIG. 4 is a graph showing the results of quantifying RF IgM using the method according to the present invention, using serum of RA (n = 46) and healthy human serum (n = 44) as a sample.
FIG. 5 shows the result of analyzing the anti-CCP antibody standard material on a single strip in which the capturing substance for testing the anti-CCP antibody and RF IgM was fixed on the test lines 1 and 2 of the strip. FIGS. 5 And the results of the standard curve analysis. As shown in Table 1, fluorescence of the anti-CCP antibody reference material in the test 1 line was quantified and a standard graph was generated. As a result, the pattern was increased depending on the concentration of the reference material. The reactivity was 0.6 or less, indicating no difference in reactivity. Based on this, the curve fitting of the standard quantitative curve shows the correlation r-sq. > 0.99.
Figure 6 shows the results of analysis of RF standard material in a single strip on which capturing material for testing anti-CCP antibody and RF IgM was immobilized on the test lines 1 and 2 of the strip. Figures 6a and 6b show the reactivity And standard curve analysis results. The fluorescence quantification value for the RF standard material in the test 1 and the test 1 line was not more than the test 1 / control ratio 0.7. On the other hand, the reactivity with the RF target factor in the test 2 line showed a pattern of reactivity increase depending on the standard material. Based on this, the curve fitting of the standard quantitative curve shows the correlation r-sq. > 0.99.

The present invention relates to a method for detecting rheumatoid arthritis markers in Invitro using lateral flow analysis.

Markers employed in the method according to the present invention are anti-CCP antibodies and rheumatoid factor (RF). As shown in FIG. 3, the marker was found to be increased in patients with rheumatoid arthritis.

Rheumatoid arthritis is a chronic progressive systemic autoimmune disease, distinct from degenerative arthritis, a non-inflammatory chronic arthritis characterized primarily by cartilage damage due to aging.

The cause of rheumatoid arthritis is unclear, and genetic, environmental, and opportunistic factors are thought to be involved. There is no definite way to diagnose rheumatoid arthritis so far. Especially, the point of POCT care treatment based detection method has not been developed until now.

This is a method that can simultaneously detect both anti-CCP antibodies and rheumatoid factors at the quantitative and qualitative level as a rheumatoid arthritis marker.

Biological samples used in the method according to the present invention include whole blood, plasma or serum. It is not particularly limited as long as the marker can be detected.

In one embodiment according to the present invention, plasma or serum is used.

In the method according to the invention the marker is detected by means of said chromatographic analysis, in particular by lateral flow analysis.

Lateral flow analysis is a method of quantitatively or qualitatively examining a target analyte, e.g., a particular nucleic acid or protein, contained in a sample, for example, by using oligonucleotides or specific antibodies and / or antigens that hybridize to a nucleic acid of constant sequence A strip comprising a nitrocellulose membrane (developing medium) bonded at a specific location is used. The strip is used for the detection of a specific nucleic acid or protein in a sample through a sequence-specific hybridization reaction or an antigen-antibody reaction by moving the sample by a chromatographic method. For example, reference may be made to those described in Korean Patent Laid-Open Nos. 2003-0065341, 2011-0007699, 2011-0127386, and 1149357.

In one embodiment according to the present invention, the strip includes a free pad, a sample separation pad, a developing medium, and an absorbent pad, which are described above with reference to FIG. The free pads and the absorbent pads are positioned at both ends of the support so that they do not overlap with each other or overlap each other, the development medium is positioned between the sample separation pad and the absorption pad, and the amount of the developing medium Each of the ends is in contact with one end of the absorbent pad and one end of the sample separation pad. The test line is provided with a captor capable of capturing the anti-CCP antibody as a rheumatoid arthritis marker and the rheumatoid factor have. When the sample develops, the anti-CCP antibody and the rheumatoid factor in the sample specifically bind to the captor to form a complex, and the complex is detected by a detection antibody labeled with a fluorescent substance.

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

Referring to Fig. 2, the cartridge for lateral flow analysis generally comprises a base member; And a cover member to be engaged with the base member, wherein the base member includes a strip accommodating portion for accommodating a strip used for the lateral flow analysis, and the cover member, when stuck with the base member, A detection window and a detection antibody introduction port formed at a portion corresponding to the detection window and a sample introduction port.

The lateral flow analysis for the detection of rheumatoid arthritis markers according to the present invention has the following characteristics. If a whole blood sample can be used without using a conjugation pad on the strip, a bonding pad with a whole blood pad (or sample separation pad) or a general pad bonded to the strip is used. In order to improve the analytical sensitivity, the sample and the detection antibody are separately dripped onto a single strip, and a corresponding inlet is formed in the housing. The samples required for the analysis are whole blood, plasma, and serum. The maximum amount of sample available is 20ul, minimum 5ul. Quantification is possible using a fluorescently labeled detection antibody, and the fluorescence signal is detected and quantified through a laser induced surface fluorescence detection device.

Example

Example  One: Anti-CCP antibody and IgM -RF simultaneous detection Lateral flow  analysis

This method is a lateral flow fluorescence immunoassay which simultaneously detects anti-CCP antibody and IgM-RF. This method is designed to bind an autoantibody present in analytical samples (whole blood, plasma, serum) with the Fc portion of IgG in two capture lines and anti-human IgG. This method does not require additional washing steps. After completion of the reaction within 15 minutes, the CCP conjugator capable of binding to the detection antibody-anti CCP antibody and the fluorescence of the fluorescent material combined with the anti-human IgM- The amount was quantified.

To examine RF, Fc of immunoglobulin G was immobilized on NC (HF90, HF135 & HF180) membranes, and anti-human IgM and fluorophore were used as a detection antibody.

In order to examine the anti-CCP antibody, two or four kinds of peptides used in the capture antigens (patent EP 1 456 662 B1, EP 2 058 663 A1) were used in combination, and the detection antibodies were immunoglobulin G Were used. Anti-human IgG was immobilized on the NC membrane, conjugated with cyclic citrullinated peptide, Biotin, or BSA as a detection antibody, and then conjugated with phosphors. Immediately after the introduction of the serum, plasma or whole blood of the RA patient into the sample inlet of the cartridge, the buffer solution containing the detection antibody was introduced into the detection antibody introduction port and the lateral flow immunity reaction proceeded for 12 minutes. The amount of fluorescence was quantified using a laser induced surface fluorescence detector.

The results are shown in Figs. 3 to 6 and Tables 1 to 3.

The sensitivity of the sample was compared between the case where the inlet was separated from the buffer solution inlet containing the detection antibody and the case where the housing was not separated. As a result, when using a separate housing, 5 ul of serum sample was added dropwise and then 100 ul of buffer was added dropwise. When using a separate housing, 100ul was added dropwise after mixing 5ul of the sample and 100ul of the buffer. After 12 minutes, the ratio of test / control was calculated after analyzing the amount of phosphor in the test and control lines. When the ratio of mean value of negative samples + 3SD was used as the reference, the positive rate (%) of positive samples was confirmed Respectively.

The results are shown in Table 1. When the difference in sensitivity of anti-CCP test was analyzed in the case where the sample was not separated separately, the sensitivity of the serum samples to 25 samples of healthy persons and 31 samples of rheumatic patients was 56 samples, The sensitivity improvement of 12% was obtained.

[Table 1]

As a result of applying anti-CCP positive / negative control from maximum 40ul to minimum 3ul for verification of analytical sample, the difference of positive / negative control ratio was 6 times or more when using minimum 5ul and maximum 20ul as shown in Table 2 below , And reproducibility within 10%.

[Table 2]

Anti-CCP was quantified with RA serum (n = 44) and healthy human serum (n = 32). After dropping 5 ul of the sample into the inlet of the sample, 100 ul of the buffer solution was dropped immediately after 12 minutes The ratio of positive photoperiod on the test / control line was determined, and the positivity rate (%) of the positive specimen was confirmed when the ratio of the negative specimen + 3SD was used as a reference. As a result, the clinical sensitivity and specificity were 97% and 80%, respectively, as shown in FIG. 3 and Table 3 below.

[Table 3]

We also quantified ichroma RF IgM in RA patient serum (n = 46) and healthy human serum (n = 44). After dropping 100 μl of the buffer solution immediately after dropping 5 μl of the sample into the inlet of the sample, the ratio was determined by quantifying the amount of the red pigment in the test / control line after 5 minutes. When the ratio of the negative sample was + 3 SD, (%) Was confirmed. As a result, sensitivity and specificity were confirmed as shown in FIG. 4 and Table 4, and a specificity of 70% and a sensitivity of 85% were obtained. .

[Table 4]

In addition, the anti-CCP antibody reference material was analyzed in a single kit in which capture substances for testing anti-CCP antibody and RF IgM were immobilized on test lines 1 and 2. The results are shown in FIG. Reactive Assay for Standard Substance (a) and Standard Curve Assay (b) In the test 1 line, the fluorescence quantification value for the anti-CCP antibody reference material showed a reaction-dependent increase pattern depending on the standard substance, and test 2 Reactivity with the RF target factor in the line was not more than 0.6, showing no difference in reactivity. Based on this, the curve fitting of the standard quantitative curve shows the correlation r-sq. > 0.99.

In addition, RF standard substances were analyzed in a single kit in which capturing substances for testing anti-CCP antibodies and RF IgM were fixed in test lines 1 and 2. The results are shown in FIG. Reactivity analysis for reference material (a) and standard curve analysis (b) In the test 1 line, the fluorescence quantification value for the RF reference material was less than the test 1 / control ratio 0.7. On the other hand, the reactivity with the RF target factor in the test 2 line showed a pattern of reactivity increase depending on the standard material. Based on this, the curve fitting of the standard quantitative curve shows the correlation r-sq. > 0.99.

Example 2. Examination of patient specimens in the simultaneous test cartridge

Immunoglobulin G (1 mg / ml) and anti-human IgG (1 mg / ml) were immobilized on test line 2 and NC (HF90, Immobilized capture antigen can be used up to 3 mg / ml.

Anti-CCP antibody was detected with 1 ~ 4 ug / ml conjugate of CCP peptide conjugated with biotin and streptavidin conjugated with phosphor at 37 ° C for 2 hours. The reaction ratio of biotin and streptavidin can be 1: 2 ~ 1: 10 molar ratio. At this time, the marker for detection may be enzymes, gold particles, etc., but not limited to fluorescence.

Anti-human IgM antibodies were used in combination with the fluorescent material (2 ug / ml) and used up to 4 ug / ml. At this time, the marker for detection may be enzymes, gold particles, etc., but not limited to fluorescence.

A total of 50 serum samples were used, including 20 healthy blood samples and 30 rheumatic patient serum samples

In the cartridge in which each inlet was separated, 5 ul of the patient serum was added, and then 100 ul of the solution containing the detection antibody was added dropwise to the solution introduction port, and the amount of fluorescence was quantified after 12 minutes.

Test 1, test 2, and control lines were quantitated and the ratios were determined and the average of the ratio values of negative samples + 2SD was used as the criterion.

As a result of analysis of the positive test results in the same sample, the number of positive test specimens in both anti - CCP antibody test and RF IgM test was 16 samples. And 4 samples in the RF test. Therefore, the number of positive specimens among the 30 specimens was 25 specimens, and the positivity rate was 83.3%. The results of this study suggest that simultaneous testing is more effective in screening patients with RA than in single testing, as a result of a positive diagnosis rate of 70% in the anti-CCP antibody test alone and 66.7% in the RF test alone . In addition, it can be said that it is suitable for judging risk patients according to the diagnosis criteria of rheumatoid arthritis in 2010.

On the other hand, as a result of the analysis of the voice judgment rate, the number of the negative judgment samples for each test was found to be 1 sample in the anti - CCP antibody test and 7 samples in the RF test. In the case of the anti - CCP antibody test and the RF IgM test, The number was 12 samples. Therefore, the number of samples with negative judgment was 12 samples, and the rate of negative judgment was 60.0%. However, the risk level can be determined because the results of the two items are provided simultaneously according to the diagnosis criteria for rheumatoid arthritis in 2010, and when it is used as a screen test in suspected patients, high sensitivity is important.

Therefore, the simultaneous test method of the present invention simultaneously provides positive / negative results of RF and anti-CCP antibody test, thereby providing the results of two items required in the diagnosis guideline of patients with rheumatoid arthritis in 2010 with a high positive detection rate, So that diagnosis and treatment can be promptly performed.

[Table 5]

Claims (7)

Providing a sample from an object requiring diagnosis of rheumatoid arthritis;
Providing said sample with reagents necessary for the detection of anti-CCP antibody and rheumatoid factor;
Loading the mixture into a lateral flow strip comprising the anti-CCP antibody and the rheumatoid factor detection material, resulting in a complex of the anti-CCP antibody and rheumatoid factor in the sample and each detection reagent; Loading the detection antibody that detects the complex, resulting in the formation of a second complex of the complex and the detection antibody; And detecting a signal emitted by the second complex. The method of claim 1, The method according to claim 1,
Wherein said sample is whole blood, serum or plasma. The method according to claim 1,
Wherein said detection is a quantitative or qualitative analysis. The method according to claim 1,
Wherein the amount of the sample is 5 to 20 microns. The method according to claim 1,
Wherein the strip used for the lateral flow analysis comprises a whole blood pad. The method according to claim 1,
Wherein the sample and the reagent necessary for detecting the anti-CCP antibody and the rheumatoid factor are each loaded on the strip. The method according to claim 1,
Wherein said anti-CCP antibody and said rheumatoid factor are all positive, or one of the two is positive, the method further comprises the step of determining that the anti-CCP antibody and the rheumatic factor are high-risk positive.

Images