KR20160033580A - Method of detecting a marker for diagnosing rheumatoid arthritis using an immunoassay based on a surface-enhanced raman scattering - Google Patents
Method of detecting a marker for diagnosing rheumatoid arthritis using an immunoassay based on a surface-enhanced raman scattering Download PDFInfo
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/39—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using tunable lasers
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
Abstract
The present invention provides a sample solution which is expected to contain an autoantibody anti-CCP (cyclic citrullinated peptide) antibody; Preparing a magnetic particle on which CCP (cyclic citrullinated peptide) specifically binding to the anti-CCP antibody is immobilized; Preparing a nanoprobe having a secondary antibody and a Raman reporter molecule bound thereto that specifically bind to the anti-CCP antibody; Adding the magnetic particles to the sample solution to cause an immune reaction between the CCP of the magnetic particles and the anti-CCP antibody to form a first immunoconjugate; Adding the nanoprobe to the sample solution in which the first immunocomplex is formed to bind the secondary antibody of the nanoprobe and the anti-CCP antibody on the first immunocomplex to form a sandwich immunoconjugate; Separating said sandwich immunoconjugate using magnetic force; Irradiating the separated sandwich immunoconjugate with laser light; And measuring the surface-enhanced Raman scattering (SERS) signal after the laser light irradiation to confirm the presence of the autoantibody anti-CCP antibody. A marker detection method is provided.
Description
The present invention relates to a method for detecting a rheumatism marker by immunoassay based on Surface-Enhanced Raman Sacattering (hereinafter referred to as 'SERS').
Rheumatoid arthritis is an autoimmune disease caused by chronic inflammation of the lubricated joints. Rheumatoid arthritis is not healing but it is not difficult to control. The sooner you find out and start the treatment, the more effective it is to slow down or stop the process. The most important factor in the treatment of rheumatoid arthritis is how to quickly diagnose and start effective treatment. However, some treatments are toxic and expensive, so you should not proceed with the treatment without a definite diagnosis. A variety of serum biomarkers are being used to speed up the diagnostic process and to speed up the diagnostic process to achieve better disease diagnosis and prognosis. Among them, anti-CCP antibody, which is an autoantibody corresponding to cyclic citrullinated peptide (CCP), is used as a biomarker most suitable for the early diagnosis of rheumatoid arthritis. The advantages of anti-CCP antibodies are detectable in
An enzyme-linked immunosorbent assay (ELISA) is the most widely used test method for rheumatoid arthritis. Various kinds of anti-CCP ELISA test kits capable of quantitative analysis of anti-CCP autoantibodies are available (Non-Patent Document 1) . This experiment is based on the affinity of anti-CCP autoantibodies in serum to CCP adsorbed on microplate surface. Recently, a point-of-care (POC) diagnosis of rheumatoid arthritis using a lateral-flow immunoassay kit using gold nanoparticles is also possible. This is because the gold nanoparticles flow through capillary force with the sample from the patient's serum, causing color change, which makes diagnosis of the disease even with the naked eye. However, both techniques still have sensitivity, specificity and reproducibility as key issues and require more precise and accurate immunoassays.
Recently, immunoassays based on surface-enhanced Raman scattering (SERS) using functional nanotags have become an emerging alternative to sensitive detection in place of ELISA. This technique is attracting attention because it can detect quickly and sensitively (Non-Patent Document 2).
The surface-enhanced Raman scattering (SERS) -based detection method is in the spotlight as a high sensitivity analysis method. The polyclonal antibody (PAb) is immobilized on a solid substrate, and then immune-gold nanoparticles with antigen solution and Raman reporter are added successively. The antigen biomarker can be quantitatively analyzed by measuring the intensity change of the characteristic SERS peak of the reporter molecule. When a reporter molecule is adsorbed to a rough metal surface and exposed to excitation light (laser light), electromagnetic and chemical enhancement occurs at the SERS active site of the reporter molecule known as a " hot junction " . This enhancement effect is expected to solve the problem of low sensitivity which is a disadvantage of conventional Raman and fluorescence detection methods.
The most common form of SERS-based immunoassay is to detect immune complexes in the form of sandwiches immobilized on a solid substrate. However, this method has several disadvantages, requiring several washing steps, and time required accordingly. Detection sensitivity is more important for early diagnosis of RA.
Therefore, the present inventors continued their research and developed a method for early diagnosis of RA using Raman spectrum and sandwich immunoassay, thereby completing the present invention.
It is an object of the present invention to provide a method for detecting a rheumatism marker by SERS-based immunoassay.
The present invention provides a method for detecting rheumatoid marker by SERS-based immunoassay comprising the following steps:
Preparing a sample liquid which is expected to contain an anti-CCP (cyclic citrullinated peptide) antibody;
Preparing a magnetic particle on which CCP (cyclic citrullinated peptide) specifically binding to the anti-CCP antibody is immobilized;
Preparing a nanoprobe having a secondary antibody and a Raman reporter molecule bound thereto that specifically bind to the anti-CCP antibody;
Adding the magnetic particles to the sample solution to cause an immune reaction between the CCP of the magnetic particles and the anti-CCP antibody to form a first immunoconjugate;
Adding the nanoprobe to the sample solution in which the first immunocomplex is formed to bind the secondary antibody of the nanoprobe and the anti-CCP antibody on the first immunocomplex to form a sandwich immunoconjugate;
Separating said sandwich immunoconjugate using magnetic force;
Irradiating the separated sandwich immunoconjugate with laser light; And
Measuring the surface-enhanced Raman scattering (SERS) signal after the laser light irradiation to confirm the presence of the autoantibody anti-CCP antibody.
The sample fluid is a sample fluid suspected of containing an autoantibody anti-CCP (cyclic citrullinated peptide) antibody, and refers to a sample fluid obtained from a patient generally suspected of having rheumatism. If the sample solution contains an anti-CCP autoantibody, it can be confirmed by the SERS signal.
The sample liquid may be selected from the group consisting of tissue extract, cell lysate, whole blood, plasma, serum, saliva, ocular fluid, cerebrospinal fluid, sweat, urine, milk, It is not.
The Raman reporter molecule may be any of those known in the art including, but not limited to, crystal violet (CV), X-rhodamine-5-isothiocyanate (XRITC), or malachite Green isothiocyanate (MGITC).
The Limit of Detection of the anti-CCP antibody may be less than 25 U / ml. It was difficult to detect anti-CCP antibodies having a concentration of 25 U / ml or less when detecting anti-CCP antibodies using a known ELISA kit. However, detection using the method according to the present invention is possible even when the concentration of the anti-CCP antibody is very low.
The time required for detecting the rheumatism marker may be 30 minutes to 60 minutes. Using the method according to the present invention, rheumatoid markers can be detected early.
The term " sandwich immunoconjugate "refers to an immunoconjugate bound through an antibody-antigen-antibody reaction. The term " sandwich immunoconjugate "Quot; means that the anti-CCP antibody, the CCP and the secondary antibody are sandwiched in the center of the CCP antibody.
Magnetic particles (magnetic beads) and nanoprobes (e.g., hollow gold nanospheres) used in the present invention are well known in the art. Specifically. Korean Patent No. 10-0979727 and Korean Patent Publication No. 10-2012-0017358 disclose a method for producing hollow gold nanoparticles (HGN) and magnetic particles.
In the present invention, a sandwich-type immune complex containing magnetic particles is fixed to a wall of a container (e.g., a microtube) containing a sample solution using a magnet. The SERS signal of the immune complex is then measured. This technique is able to overcome the slow immune response time problems seen in diffusion-limited solid substrates because reactions take place in the liquid phase. As a result, the analysis time is reduced to within one hour. In addition, the SERS signal measures the average signal of the nanoparticles in the liquid phase, resulting in more reproducible results.
The present inventors confirmed the possibility of early diagnosis of rheumatoid arthritis through immunoassay of anti-CCP antibody markers using SERS-based magnetic particles. The inventors of the present invention evaluated the feasibility of the present invention through immunoassay of anti-CCP derived from SABS-based rabbit. To this end, the inventors of the present invention conducted experiments on CBS-immobilized magnetic particles and SERS nanoparticles Were prepared. And measured the SERS signal for their complexes. Next, the present inventors applied this SERS-based analysis method to a 'human-derived anti-CCP standard solution'. This standard solution is commercially available as part of an anti-CCP ELISA kit (BlueGene Biotech, Shanghai, China). In order to confirm the experimental results of clinical samples, the results obtained by the above-described analytical methods and the results obtained by the present inventors using BlueGene's anti-CCP kit were compared. The present invention is the first invention in the field of anti-CCP immunoassay based on SERS and suggests a new direction for the early diagnosis of rheumatoid arthritis.
Using the disease marker detection method according to the present invention, rheumatism can be diagnosed early.
1 is a schematic diagram for detecting an anti-CCP marker based on SERS according to the present invention. (1) shows the preparation of CCP-immobilized magnetic particles; (2) indicates that the anti-CCP autoantibody marker is selectively bound to CCP and captured by magnetic particles; (3) adding a nanoprobe conjugated with a secondary antibody to form a sandwich denture immune complex; And (4) indicates separation of the sandwich immune complex and measurement of SERS.
FIG. 2 (a) is a schematic diagram showing that CCP is immobilized on magnetic particles, and FIG. 2 (b) is a graph showing SERS intensity variation according to CCP concentration.
FIG. 3 (a) shows a process for producing nanoprobes (hollow gold nanoparticles: HGN) according to the present invention. MGITC was used as a Raman reporter molecule. (b) is a transmission electron microscope (TEM) image of HGNs. (c) shows the dynamic light scattering (DLS) distribution of HGNs (black bars) and SERS nanoprobes (red bars). (d) shows the UV / Vis absorption spectrum of HGNs (black line) and SERS nanoprobe (red line).
(A) is a blank, BSA, RF, SERS spectrum of the anti--CCP, and mixtures thereof, and (b) of Figure 4 is 1617cm - a Raman intensity for the corresponding at 1.
Figure 5 is a TEM image of a sandwich immune complex of 12 different rabbit anti-CCP ranging from 10-400 ng / mL.
Figure 6 (a) is a SERS spectrum of rabbit anti -CCP autoantibody, (b) it is 1617cm - is the Raman intensity, the corresponding a, a function of concentration from 1 to (b) shows the change in SERS intensity in the low concentration range.
FIG. 7 shows the results of detection of human anti-CCP antibodies at 12 different concentrations according to the immunoassay (a) according to the present invention and the method (b) using a commercially available ELISA kit. The graph inserted inside shows the analysis result of low concentration (0-25 UmL -1 ).
FIG. 8 shows the result of comparing anti-CCP detection results using SERS and ELISA.
It is to be understood that both the foregoing general description and the following detailed description of the present invention are exemplary and explanatory and are intended to be exemplary and explanatory only and are not restrictive of the invention, It is obvious to those skilled in the art that such variations and modifications fall within the scope of the appended claims.
Example 1: Experimental method
1-1: anti- CCP Autoantibody-containing Of the sample liquid Ready
Rabbit anti-CCP autoantibody was purchased from Bioss, product number bs-1053R. Concentration of this product was prepared by diluting 100 μg / mL solution at high concentration. C, D, E, F (0, 25, 50, 100, 250, 500) contained in the product number E01C0708 Human CCP ELISA kit manufactured by BlueGene was used for human anti- The remaining low concentration solutions were prepared by dilution with the included dilution buffer.
1-2: CCP end Combined Preparation of magnetic particles
Magnetic particles with CCP (Peptron Inc., Daejeon, South Korea) were prepared by the following method: 500 μL of 300 nM biotinylated CCP (biotin-HQCHQEST-Cit-GRSRGRCGRSGS-COOH, Cys3 and Cys16 Mu] l of 0.5 mg / mL streptavidin-bound magnetic beads (1 [mu] m in size, Dynabeads® MyOne ™, 2.6 kDa, Peptron Inc., Daejeon, South Korea). Invitrogen, Eugene, OR, USA) with streptavidin-biotin. The mixture was allowed to react for 2 hours at room temperature and then unreacted streptavidin was blocked with 500 μL of 300 nM biotin. Unreacted reagents were washed twice with PBS buffer solution.
The CCP antibody used in the present invention was synthesized according to a published sequence (biotin-HQCHQEST-Cit-GRSRGRCGRSGS-COOH, addition of a sulfur bond between Cys3 and Cys16) through a peptone company.
1-3: Metal nano Of the probe Ready
Secondary antibody conjugation that binds to the anti-CCP antibody attached to the SERS probe HGNs (Hollow gold nanoparticles) was prepared according to the method described in known literature (Lee, S. et al., Biosens. J. Phys. Chem. B 2005, 109 (1987), < RTI ID = 0.0 > , 11083-11087). The secondary antibody used in this example is an anti-human IgG antibody (manufacturer: abcam, product number: ab2410).
Briefly, cobalt nanoparticles were synthesized by reducing CoCl 2 with NaBH 4 under
Conjugation of the Raman reporter molecule to the HGNs of the secondary antibody was performed as described in the known literature (Chon, H et al., Anal. Chem. 2009, 81, 3029-3034). The Raman reporter molecule, MGITC (malachite green isothiocyanate, INVIROGEN), was adsorbed on the surface of HGNs. Specifically, 1 [mu] L of 50 nM MGIT was added to 1.0 mL of 0.7 nM HGN colloid. The surface of HGNs was treated with 10 μL of 30 μM poly (ethylene glycol) 2-mercaptoethyl ether acetic acid (PEG linker, Aldrich) for 2 hours at room temperature Was modified. Unreacted reagents were then removed by centrifugation at 6000 rpm for 10 minutes. Next, 1.0 mL PBS buffer (pH 7.4) was added to disperse the HGNs. Carboxy groups on the surface of HGNs reacted with 10 μL of 0.1 μM rabbit anti-IgG (or human anti-IgG), a secondary antibody, for 10 minutes at room temperature. Unreacted succinimidyl groups were then inactivated using 10 μL of 0.01 M ethanolamine for 3 hours. Unreacted reagents were removed by centrifugation at 5,000 rpm for 10 min and the solution was washed twice with PBS buffer.
1-4: SERS Signal measurement method
A
Example 2: SERS Autoimmune Antibody by Immunoassay - CCP detection
Figure 1 shows the overall flow of quantitative analysis of anti-CCP autoantibody markers via SERS-based immunoassay. First, biotin-immobilized CCP binds to magnetic particles to which streptavidin is immobilized through biotin-streptavidin interaction (FIG. 2 (a)). In order to find the optimum amount of CCP that can bind to magnetic particles in this part, five different concentrations of biotin-immobilized CCP were incubated at 100 ng / mL anti-CCP and secondary antibody immobilized at 40 to 400 nM intervals The optimum concentration of CCP was determined to be 300 nM (Fig. 2 (b)) based on the obtained SERS measurement results.
Secondly, the magnetic particles were added to the sample solution containing the anti-CCP antibody. RF (rheumatoid factor) and BSA (bovine serum albumin) were used as negative controls, which were used to evaluate the selectivity of anti-CCP antibodies. After the anti-CCP autoantibody selectively binds to the surface of the magnetic particle, a sandwich-type immunoconjugate is formed by the SERS nanoprobe immobilized with the secondary antibody. In this section, hollow type gold nanoparticles were used as SERS nanoparticles, enabling a very uniform level of immunoassay. In the case of hollow gold nanoparticles, it is characterized by a hollow structure. This means that the nanoparticles are highly homogeneous and reproducible by local electromagnetic fields that remain inside a number of pinholes. Therefore, these nanoparticles are used as highly reproducible detection tools for the quantitative analysis of immune-specific biomarkers. Where MGITC was used as a Raman labeling molecule.
FIG. 3 shows a step-by-step process for producing a nanoparticle immobilized with a secondary antibody. These nanoparticles were analyzed by transmission electron microscopy, DLS and UV / VIS spectroscopy. Finally, the sandwich-type anti-CCP immunoconjugate is separated from the solution by a strong attraction between the magnetic particles and the magnet, thereby separating the filtrate from the immune complex. In this procedure, two washing steps were performed using a specific buffer solution. The magnet was removed for SERS measurement and the immune complex was redispersed on the buffer solution.
Example 3: SERS Selectivity of an immune-based system
To evaluate the selectivity of the SERS-based immunoassay system, the SERS spectra shown in FIG. 4 (a) were measured in a mixture of anti-CCP, RF and BSA based on the same concentration of rabbit. As expected, the strongly enhanced Raman signal was measured only in anti-CCP autoantibodies and mixtures thereof, whereas in the presence of only RF or BSA without anti-CCP autoantibodies in solution, the Raman signal was not measured. As shown in Figure 4 (b) 1617cm - relative to the Raman signal that appears at one shows that the CCP is a combination of only -CCP wherein the magnetic particles are fixed autoantibodies, such SERS-based analysis is also used for selective assessment .
FIG. 5 is a transmission electron microscope measurement result showing a sandwich immunoconjugate when anti-CCP in the range of 10 to 400 ng / mL is introduced. In the presence of anti-CCP autoantibodies, SERS nanoparticles adhere to the surface of the magnetic particles through interaction between anti-IgG immobilized on the hollow gold nanoparticles and anti-CCP immobilized on the surface of the magnetic particles. From these results, it can be seen that, when there is a larger amount of anti-CCP, a larger amount of nanoparticles will be bonded to the surface of the magnetic particle. This transmission electron microscopy image provides strong evidence that a higher amount of sandwich immunoconjugate will form as the concentration of anti-CCP increases.
Example 4: Raman signal intensity according to antibody concentration
Figure 6 according to the concentration of rabbit anti -CCP antibody (a) SERS spectra and (b) 1617cm - shows the relative intensity of Raman signal in the first. The SERS spectrum was measured within the same concentration range as that of the TEM instrument. Here, 1617cm as rabbit anti -CCP increasing the concentration of antibody were increased intensity of the SERS signal of the first. As can be seen in Figure 6 (b), when the concentration of this antibody reached 50 ng / ml, the formation of the immune complexes was almost complete. It is also noteworthy that the intensity of the SERS signal changes even at a low concentration range of 0-100 pg / mL. In other words, it means that highly accurate quantitative evaluation of rabbit anti-CCP antibody can be performed using SERS-based immunoassay. The detection limit for rabbit anti-CCP antibodies is expected to be around 13 pg / mL.
Comparative Example : ELISA Comparison with Analytical Methods
To evaluate the possibility of applying the SERS-based assay described above to actual clinical trials, we compared it with the commercially available anti-CCP ELISA assay kit (BlueGene Biotech). To compare these two, human anti-CCP antibody samples with 12 different concentrations were prepared. The concentrations of the anti-CCP solutions (0, 25, 50, 100, 250 and 500 U / mL) contained in the kit were the standard reagents contained in the kit. . ELISAs were performed for all of these concentrations using the BlueGene kit and the SERS-based assay. Figure 7 compares the results of performing anti-CCP ELISA using these two methods. The method of SERS-based, 1617cm for a quantitative assessment - the SERS signal was observed in the first.
Overall, as can be seen in FIGS. 7 (a) and 7 (b), the results obtained with the SERS assay were similar to those obtained with the ELISA assay at various concentration ranges. In the low concentration range (0-25 U / mL), more consistent results were obtained using the SERS-based assay than ELISA kits. In other words, using the SERS-based immunoassay method means that a more sensitive quantitative evaluation of human anti-CCP antibodies can be performed. Therefore, this method can be a new method for early diagnosis of rheumatoid arthritis.
Test Example : Clinical Trial
As shown in FIG. 7, the SERS analysis results were more accurate than the ELISA as a result of comparing the two measurement methods, the SERS method and the ELISA, with a low concentration of anti-CCP (anti-CCP) standard of 0 to 25 U / mL. Based on this, the possibility of using early diagnosis of RA in SERS method will be verified by using actual patient blood. For this, anti-CCP levels of patient blood samples (n = 43) identified as anti-CCP negative were confirmed by SERS analysis. The blood of the patients with the negative anti - CCP selected was first checked by ELISA, and the results were compared with the SERS analysis. The values determined by both methods are shown in Table 1. Differences between SERS and ELISA were assessed by Passing-Boblok analysis. As a result, there was a significant difference between the anti-CCP negative group results performed with the two assays, as in Fig. 8a. This is a very different result from the results of the anti-CCP positive group of 25 U / mL or more, which showed little difference between the two methods. The slope and slope of the 95% confidence interval range of the analytical results were -6.35 ~ 2.52 U / mL and 0.0004 ~ 1.07 U / mL, respectively. This is because the range of the confidence interval is large and the correlation coefficient P = 0.01 is very small. Bland-Altman plot analysis was performed to evaluate the agreement between the two methods. The 95% agreement upper / lower limit between the two methods was as wide as -24.9 ~ 16.4 U / mL. In other words, the correlation was very low in the anti-CCP negative group (n = 43). Were also significantly different from those with high correlation in the anti-CCP positive group (n = 31). To analyze the accuracy of the SERS and ELISA methods, the gold standard method should be used to obtain the true value of the anti-CCP concentration of the blood sample. Among the existing assay methods, There is no way to measure the true value of a sample. Therefore, the accuracy of each method was evaluated by comparing the standard deviation of the two methods instead of the standard method and the comparative method. As shown in FIGS. 8D and 8E, it was confirmed that the standard deviation of the SERS method was much narrower than the ELISA. This confirms that the SERS assay shows a more consistent result than the ELISA in the 25 U / mL low concentration range samples.
[Table 1]
Quantitative analysis of anti-CCP negative group (n = 43,> 25 U / mL) performed by ELISA and SERS
For early diagnosis of rheumatoid arthritis, the present inventors have developed a novel SERS-based immunoassay which can detect anti-CCP antibodies. Here, magnetic particles immobilized with CCP were used as a substrate capable of capturing anti-CCP antibodies, and hollow gold nanoparticles bound with a secondary antibody were used as markers capable of emitting the SERS signal. The detection limit for rabbit anti-CCP is expected to be about 13 pg / mL. In addition, the detection of human anti-CCP antibodies in a low concentration range demonstrated that SERS-based immunoassay can detect more sensitively than a commonly used ELISA kit. This analysis demonstrates that this technique can be a new way to diagnose rheumatoid arthritis early.
Claims (5)
Preparing a magnetic particle on which CCP (cyclic citrullinated peptide) specifically binding to the anti-CCP antibody is immobilized;
Preparing a nanoprobe having a secondary antibody and a Raman reporter molecule bound thereto that specifically bind to the anti-CCP antibody;
Adding the magnetic particles to the sample solution to cause an immune reaction between the CCP of the magnetic particles and the anti-CCP antibody to form a first immunoconjugate;
Adding the nanoprobe to the sample solution in which the first immunocomplex is formed to bind the secondary antibody of the nanoprobe and the anti-CCP antibody on the first immunocomplex to form a sandwich immunoconjugate;
Separating said sandwich immunoconjugate using magnetic force;
Irradiating the separated sandwich immunoconjugate with laser light; And
Measuring the surface-enhanced Raman scattering (SERS) signal after the laser light irradiation to confirm the presence of the autoantibody anti-CCP antibody;
A method for detecting rheumatism markers by SERS-based immunoassay.
Wherein the sample liquid is selected from the group consisting of tissue extract, cell lysate, whole blood, plasma, serum, saliva, ocular fluid, cerebrospinal fluid, sweat, urine, milk, multiple fluid, synovial fluid and peritoneal fluid.
Wherein the Raman reporter molecule is crystal violet (CV), X-rhodamine-5-isothiocyanate (XRITC) or malachite green isothiocyanate (MGITC).
Wherein the limit of detection of the anti-CCP antibody is 25 U / ml.
Wherein the time required for detecting the rheumatoid marker is 30-60 minutes.
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| KR20180047853A (en) * | 2016-11-01 | 2018-05-10 | 주식회사 더웨이브톡 | Virus detecting device with chaotic sensor and virus detecting method using the same |
| KR20190059089A (en) * | 2017-11-22 | 2019-05-30 | 바디텍메드(주) | Method for diagnosing rheumatoid arthritis based on lateral flow assay using anti-CCP antibody and Rheumatoid Factor |
| KR102381031B1 (en) * | 2021-05-31 | 2022-03-30 | 가천대학교 산학협력단 | Electrochemical Biosensing Platform for Detection of Rheumatoid Arthritis Biomarker |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20180047853A (en) * | 2016-11-01 | 2018-05-10 | 주식회사 더웨이브톡 | Virus detecting device with chaotic sensor and virus detecting method using the same |
| KR20190059089A (en) * | 2017-11-22 | 2019-05-30 | 바디텍메드(주) | Method for diagnosing rheumatoid arthritis based on lateral flow assay using anti-CCP antibody and Rheumatoid Factor |
| WO2019103324A1 (en) * | 2017-11-22 | 2019-05-31 | 바디텍메드(주) | Lateral flow immunoassay-based rheumatoid arthritis diagnosis method using anti-ccp antibody and rheumatoid factor |
| KR102381031B1 (en) * | 2021-05-31 | 2022-03-30 | 가천대학교 산학협력단 | Electrochemical Biosensing Platform for Detection of Rheumatoid Arthritis Biomarker |
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