Separated polypeptide, kit for detecting EB virus NA1 IgM antibody and application

Abstract

The invention belongs to the field of biological medicines, and relates to a separated polypeptide, a kit for detecting an EB (Epstein-Barr) virus NA1IgM (Immunoglobulin M) antibody and application. Specifically, the separated polypeptide is a truncated fragment of an amino acid sequence as shown in SEQ ID NO: 28, contains sequences from the 408th site to the 498th site of the amino acid sequence as shown in SEQ ID NO: 28, and does not contain sequences from the 1st site to the 90th site of the amino acid sequence as shown in SEQ ID NO: 28. The reagent combination or the kit provided by the invention can effectively diagnose or assist in diagnosing rheumatoid arthritis, is high in sensitivity and strong in specificity, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and relates to an isolated polypeptide, a kit for detecting EB virus NA1 IgM antibody, and uses thereof. Specifically, the isolated polypeptide is a truncated fragment of EB virus nuclear antigen 1 (NA1). The present invention also relates to the preparation and use of an EB virus nuclear antigen 1 (NA1) IgM antibody detection kit. Background Art

[0002] Epstein-Barr virus (EBV) is a widespread human lymphotropic gamma herpes virus that primarily invades B lymphocytes and is a lifelong infection in more than 90% of adults worldwide. [1-3] EBV is associated with a variety of diseases. It is the cause of infectious mononucleosis (IM) and T-cell or natural killer (NK) cell lymphoproliferative diseases, such as chronic active EBV infection (CAEBV) and EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH). It is also associated with a variety of malignancies, such as Burkitt's lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma subtypes, nasopharyngeal carcinoma, gastric cancer, and a variety of autoimmune diseases, such as systemic lupus erythematosus (SLE), multiple sclerosis (MS), Sjögren's syndrome (SS), and rheumatoid arthritis (RA). [1,4-7] .

[0003] Autoimmune diseases (AIDs) are a group of diseases characterized by immune disorders, in which dysfunctional immune activation leads to pathological immune responses of B cells and T cells against host normal cells or organ-specific autoantigens. [8,9] Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, with a global prevalence of 0.46%. It mainly affects synovial joints, manifested as joint pain, swelling, and dysfunction, followed by extra-articular manifestations in organs such as the heart, kidneys, lungs, gastrointestinal tract, and nervous system (including eyes and skin), often causing systemic complications, and in severe cases, disability or death. [7,10-12] At present, the diagnosis of rheumatoid arthritis is mainly assisted by detecting anti-citrullinated protein antibodies (ACPAs) and rheumatoid factor (RF) in the patient's serum. [13,14] However, the sensitivity of both tests is low, at about 60-80%, and because RF is positive in up to 15% of the elderly and 5% of the general population, its specificity is also poor, at only about 60%.[13-15] Since early diagnosis and treatment of rheumatoid arthritis can avoid or significantly slow the progression of joint damage in up to 90% of patients, thereby preventing irreversible disability, it is necessary to develop more accurate diagnostic kits to assist diagnosis.

[12] .

[0004] EBV nuclear antigen 1 (EBNA1) is an important protein of EBV. It consists of 641 amino acids and contains 5 different regions. It plays a variety of important roles in EBV latent infection and is associated with the immortalization of host cells.

[16] EBNA1 is the only EBV protein required to maintain the viral genome, and when B cells are latently infected, EBV only expresses EBNA1 [3,17,18] Compared with healthy people, the antibody titer of EBNA1 IgM in the serum of patients with rheumatoid arthritis was significantly increased, and it had higher diagnostic sensitivity and specificity, with the area under the ROC curve AUC of 0.9954 (95% CI: 0.987-1.004, p < 0.0001) [18,19] .

[0005] Therefore, it is necessary to develop EBNA1IgM antibody detection reagents to assist in the diagnosis of rheumatoid arthritis and better guide the clinical treatment of rheumatoid arthritis. Summary of the invention

[0006] After in-depth research and creative work, the inventors obtained a truncated fragment of EB virus nuclear antigen 1 (NA1). The inventors surprisingly found that the truncated fragment can effectively detect EBNA1 IgM. The present invention also provides a method for preparing a highly sensitive and specific EB virus NA1 IgM antibody detection kit for diagnosis or auxiliary diagnosis of rheumatoid arthritis. The following invention is provided:

[0007] One aspect of the present invention relates to an isolated polypeptide, which is a truncated fragment of the amino acid sequence shown in SEQ ID NO:28, which comprises the sequence of positions 408-498 of the amino acid sequence shown in SEQ ID NO:28, and does not comprise the sequence of positions 1-90 of the amino acid sequence shown in SEQ ID NO:28.

[0008] In some embodiments of the present invention, the isolated polypeptide comprises a first fragment and a second fragment, wherein:

[0009] The first fragment comprises positions 390-408, 377-408, 91-325, 91-377, 91-390 or 91-408 selected from the group consisting of the amino acid sequence of SEQ ID NO: 28, and the second fragment comprises positions 498-641 selected from the group consisting of the amino acid sequence of SEQ ID NO: 28;

[0010] The first fragment is directly connected to the second fragment (for example, via a peptide bond) or connected via a linker;

[0011] Preferably, the first fragment is located at the N-terminus of the isolated polypeptide, and the second fragment is located at the C-terminus of the isolated polypeptide;

[0012] Preferably, the first fragment is located at the C-terminus of the isolated polypeptide, and the second fragment is located at the N-terminus of the isolated polypeptide;

[0013] Preferably, the isolated polypeptide does not contain the sequence of positions 326-389 of the amino acid sequence shown in SEQ ID NO:28, and / or the isolated polypeptide does not contain the sequence of positions 484-571 or 499-571 of the amino acid sequence shown in SEQ ID NO:28;

[0014] Preferably, the linker comprises a sequence shown in SEQ ID NO:37, or multiple tandem sequences shown in SEQ ID NO:37; preferably, the linker comprises a sequence shown in SEQ ID NO:36.

[0015] In some embodiments of the present invention, the isolated polypeptide comprises the sequence of positions 390-498, 390-641, 377-422, 377-498, 377-641 or 408-641 of the amino acid sequence shown in SEQ ID NO:28.

[0016] In some embodiments of the present invention, the isolated polypeptide has an amino acid sequence as shown in any one of SEQ ID NO:30 to SEQ ID NO:35.

[0017] In some embodiments of the present invention, the isolated polypeptide does not contain the sequence of positions 376-470 or 376-407 of the amino acid sequence shown in SEQ ID NO:28, and / or the isolated polypeptide does not contain the sequence of positions 391-410 (or 391-407) and / or 451-470 of the amino acid sequence shown in SEQ ID NO:28.

[0018] The present invention relates to an isolated polypeptide, which is a truncated fragment of the amino acid sequence shown in SEQ ID NO: 28, comprising the sequence of positions 377-422, 377-498, 377-641, 390-498, 390-641 or 408-641 of the amino acid sequence shown in SEQ ID NO: 28, and does not comprise the sequence of positions 1-90 of the amino acid sequence shown in SEQ ID NO: 28.

[0019] In some embodiments of the present invention, the isolated polypeptide, when used to detect the same sample, has a P / N ratio greater than the P / N ratio of the amino acid sequence shown in SEQ ID NO: 28;

[0020] Wherein, the P / N ratio is the ratio of the mean RLU value of the samples of rheumatoid arthritis cases (RA) to the mean RLU value of the samples of healthy subjects (HC);

[0021] Preferably, the P / N ratio is measured by a magnetic particle chemiluminescence platform;

[0022] Preferably, the sample is a serum sample.

[0023] Without being bound by theoretical limitations, P / N is one of the indicators to measure the ability of detection reagents to distinguish between positive samples and negative samples. A higher P / N can reflect to a certain extent that the sensitivity and specificity of the detection method are better.

[0024] In some embodiments of the present invention, the isolated polypeptide is derived from Epstein-Barr virus, such as B95-8 strain or mutant thereof, M81 strain or mutant thereof, or other Epstein-Barr virus or mutant thereof.

[0025] Another aspect of the present invention relates to a fusion polypeptide, which comprises the isolated polypeptide described in any one of the present invention and a protein separation and purification tag, such as a 6His tag.

[0026] Another aspect of the present invention relates to an isolated nucleic acid molecule encoding the isolated polypeptide or the fusion polypeptide of the present invention as described in any one of the present invention.

[0027] Yet another aspect of the present invention relates to a recombinant vector comprising the isolated nucleic acid molecule of the present invention.

[0028] Another aspect of the present invention relates to a host cell comprising the isolated nucleic acid molecule of the present invention, or the recombinant vector of the present invention.

[0029] Another aspect of the present invention relates to a load, comprising a solid support and a substance connected or coated with the isolated polypeptide or fusion polypeptide of any one of the present invention;

[0030] Preferably, the solid support is selected from one or more of magnetic microparticles (magnetic beads), agarose beads, polystyrene microparticles, plastic plates, glass plates, nitrocellulose membranes (NC membranes), nylon membranes, polyvinylidene fluoride membranes (PVDF membranes) and semiconductor silicon wafers;

[0031] Preferably, the load is magnetic microparticles coated with the isolated polypeptide described in any one of the present invention or the fusion polypeptide described in the present invention;

[0032] Preferably, the magnetic particles are selected from Magnosphere TM MX100 / Carboxyl、Magnosphere TM MS300 / Carboxyl、Magnosphere TM MS300 / Tosyl and Dynabeads TM One or more of M270 / Carboxyl;

[0033] Preferably, the average particle size of the magnetic particles is 1-3 μm;

[0034] Preferably, the ratio of the separated polypeptide or fusion protein to the magnetic particles is 2-20 μg, such as 2 μg, 5 μg, 10 μg or 20 μg, of the separated polypeptide or fusion protein coated per mg of magnetic particles.

[0035] Another aspect of the present invention relates to a reagent combination, comprising: the loading substance of the present invention, and an anti-human IgM antibody connected with a detectable marker;

[0036] Preferably, the detectable marker is an enzyme, a fluorescent substance, a chemiluminescent substance or a radioactive isotope;

[0037] Preferably, the enzyme is horseradish peroxidase or alkaline phosphatase;

[0038] Preferably, the chemiluminescent substance is an acridine chemiluminescent substance, luminol or its derivatives or an electrochemiluminescent substance;

[0039] Preferably, the acridinium chemiluminescent substance is one or more selected from acridinium esters (such as AE-NHS, DMAE-NHS, Me-DMAE-NHS or NSP-DMAE-NHS), acridinium salts (such as NSP-SA or NSP-SA-NHS), acridinium hydrazide (NSP-SA-ADH), acridinium sulfonamide, 10,10'-dimethyl-9,9'-diacridinium (DMBA), 2,2'-dicarbonyl-9,9'-diacridinium dinitrate (DSBADN), 10,10'-dimethyl-2,2'-disulfonic acid-9,9'-diacridinium (DMDSBA) and 10,10'-dimethyl-9,9'-diacridinium dinitrate (DMBADN, commonly known as luscin);

[0040] Preferably, the luminol or its derivative is selected from luminol and isoluminol;

[0041] Preferably, the electrochemiluminescent substance is Tris(bipyridine)ruthenium(II)chloride hexahydrate.

[0042] In some embodiments of the present invention, the reagent combination, wherein the loading substance of the present invention and the anti-human IgM antibody connected with a detectable marker are not mixed or mixed, preferably not mixed.

[0043] In some embodiments of the present invention, the reagent combination, wherein each reagent exists independently, for example, exists in an independent container or package.

[0044] In some embodiments of the present invention, the reagent combination comprises:

[0045] Magnetic particles coated with the isolated polypeptide or the fusion polypeptide of the present invention as described in any one of the present invention, and an anti-human IgM antibody connected to a detectable marker;

[0046] Preferably, the concentration of the magnetic particles is 2-12 mg / mL or 4-10 mg / mL;

[0047] Preferably, the reagent combination comprises magnetic particles coated with the isolated polypeptide described in any one of the present invention or the fusion polypeptide described in any one of the present invention, and an anti-human IgM antibody connected with an enzyme (such as horseradish peroxidase) or an acridinium chemiluminescent substance (such as acridinium ester, acridinium salt, acridinium hydrazide, acridinium sulfonamide, DMBA, DSBADN, DMDSBA or DMBADN);

[0048] Preferably, the reagent combination further comprises a sample diluent, a negative control and / or a positive control.

[0049] In some embodiments of the present invention, the reagent combination, wherein the negative control refers to a sample that is not expected to produce a positive reaction (negative serum), such as serum known to not contain NA1IgM (ie, the substance to be detected).

[0050] In some embodiments of the present invention, the reagent combination, wherein the positive control refers to a sample known to contain NA1IgM (ie, the substance to be detected), such as serum of a patient infected with EBV virus in which NA1 IgM can be stably detected, or a NA1 IgM humanized antibody.

[0051] Another aspect of the present invention relates to a kit comprising the reagent combination described in any one of the present invention.

[0052] Another aspect of the present invention relates to a combination product, which comprises the kit of the present invention and a commercially available Anti-CCP detection kit, preferably an Anti-CCP detection kit produced by Roche;

[0053] Preferably, the combination product is used for the diagnosis or auxiliary diagnosis of rheumatoid arthritis.

[0054] Another aspect of the present invention relates to use of any of the isolated polypeptides or fusion polypeptides of the present invention in the preparation of a reagent or kit for diagnosing (eg, assisting in the diagnosis of) rheumatoid arthritis or detecting EBNA1 IgM.

[0055] Another aspect of the present invention relates to a method for diagnosing or assisting in the diagnosis of rheumatoid arthritis or a method for detecting EBNA1 IgM, comprising the step of detecting a sample of a subject using the isolated polypeptide, load, reagent combination, kit or combination product of the present invention; preferably, the sample is a serum sample;

[0056] Optionally, the method is combined with other auxiliary diagnostic or confirmatory detection methods for rheumatoid arthritis to diagnose rheumatoid arthritis and / or guide the treatment of rheumatoid arthritis.

[0057] In the present invention, the term "magnetic particles" is composed of a core-shell covering a magnetic layer and a polymer layer, and has a uniform particle size. The end of the polyethylene glycol long chain connected to the surface of the magnetic particles can be connected to an oligoamine group, the long chain can reduce nonspecific adsorption, and the oligoamine end can form a strong covalent bond between the antigen and the solid surface, or the surface of the magnetic particles is covered with a hydrophilic layer or a hydrophobic layer medium, which can make the antigen and the solid surface through non-chemical covalent bonding, such as physical adsorption, ionization bonding, etc.

[0058] In some embodiments of the present invention, magnetic particles having Carboxyl (-COOH) groups on the surface (e.g., magnetic particles M270 Carboxyl), magnetic particles having Tosylactivated (toluenesulfonyl) groups on the surface (e.g., magnetic particles MS300 Tosyl), magnetic particles having amino (-NH2) groups on the surface, and magnetic particles having streptavidin on the surface can all achieve the coupling of the antigen.

[0059] In the present invention, the principle of coupling antigens with magnetic particles is mainly: the functional groups (carboxyl, amino or tosyl, etc.) on the surface of the magnetic particles react chemically with the amino, carboxyl and thiol groups on the antigen, or the streptavidin on the surface of the magnetic particles specifically binds to the biotin labeled on the antigen, or the charged magnetic particle epitopes are linked to the antigen by physical adsorption.

[0060] The commonly used particle size range of magnetic particles is 0.1-10 μm, wherein the preferred particle size range is 0.5-5 μm, and more preferably 1-3 μm.

[0061] In the present invention, if not specifically stated, the term "comprising" may refer to only the object included, or may include one or more other optional elements in addition to the object included. For example, comprising positions 390-641 of the amino acid sequence shown in SEQ ID NO: 28 may include positions 390-641 of the amino acid sequence shown in SEQ ID NO: 28, or may further include other amino acid sequences, for example, may further include amino acid sequences at other positions (except positions 390-641) in SEQ ID NO: 28, and may achieve the effects of the present invention.

[0062] In the present invention, unless otherwise specified, the "first" (eg, the first segment) and the "second" (eg, the second segment) are merely for reference distinction and do not have a typical order meaning.

[0063] Advantageous Effects of the Invention

[0064] The present invention achieves one or more of the following technical effects:

[0065] (1) The isolated polypeptide of the present invention can effectively detect EBNA1IgM and has good specificity and / or affinity;

[0066] (2) The reagent combination or kit can effectively diagnose or assist in the diagnosis of rheumatoid arthritis and has good sensitivity and / or specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1:Evaluation results of the reactivity of different EBNA1 peptides with serum NA1 IgM antibodies (heat map). Note: RA1-RA5 are 5 serum samples from patients clinically diagnosed with rheumatoid arthritis, and HC1-HC3 are 3 serum samples from negative control patients.

[0068] Figure 2 :Evaluation results of the reactivity of different EBNA1 peptides with serum NA1IgM antibodies (scatter plot). Note: RA is 5 serum samples from patients clinically diagnosed with rheumatoid arthritis, and HC is 3 serum samples from negative control patients.

[0069] Figure 3 :Evaluation results of the reactivity of different EBNA1 peptides with serum NA1IgG antibodies (heat map). Note: RA1-RA5 are 5 serum samples of patients clinically diagnosed with rheumatoid arthritis, NA1 IgG positive, and N1-N3 are 3 NA1 IgG negative samples.

[0070] Figure 4 :Evaluation results of the reactivity of different EBNA1 peptides with serum NA1IgG antibodies (scatter plot). Note: RA is a collection of 5 serum samples from patients with clinical diagnosis of rheumatoid arthritis, NA1 IgG positive, and N is a collection of 3 NA1 IgG negative samples.

[0071] Figure 5 :Evaluation results of the reactivity of different EBNA1 peptides with serum NA1IgA antibodies (heat map). Note: RA6-RA10 are 5 serum samples of patients clinically diagnosed with rheumatoid arthritis, NA1 IgA positive, and N1-N3 are 3 NA1 IgA negative samples.

[0072] Figure 6 :Evaluation results of the reactivity of different EBNA1 peptides with serum NA1IgA antibodies (scatter plot). Note: RA is 5 serum samples of patients clinically diagnosed with rheumatoid arthritis, NA1 IgA positive, N is 3 NA1 IgA negative samples.

[0073] Figure 7 :Analysis of the diagnostic performance of different recombinant EBNA1 antigens for rheumatoid arthritis (heat map). Note: RA2-RA6 are 5 serum samples from patients clinically diagnosed with rheumatoid arthritis, and HC6-HC8 are 3 serum samples from healthy subjects.

[0074] Figure 8 :Analysis of the diagnostic performance of different recombinant EBNA1 antigens for rheumatoid arthritis (scatter plot). Note: RA is 5 serum samples from patients clinically diagnosed with rheumatoid arthritis, and HC is 3 serum samples from healthy subjects.

[0075] Fig. 9 : Evaluation results of the diagnostic efficacy of 377-498aa, 390-641aa and 1-641aa recombinant NA1 antigens for RA cases on the magnetic particle chemiluminescence platform (ROC curve).

[0076] Fig.10 : Optimization results of magnetic particle model selection for magnetic particle reagent (scatter plot). Note: RA is a sample of rheumatoid arthritis cases, and HC is a sample of healthy people undergoing physical examination.

[0077] Fig.11 :Optimization results of antigen coating amount of magnetic microparticle reagent (scatter plot). Note: RA is a sample of rheumatoid arthritis cases, and HC is a sample of healthy people.

[0078] Fig.12 : Optimization results of magnetic particle coating concentration of magnetic particle reagent (scatter plot). Note: RA is a sample of rheumatoid arthritis cases, and HC is a sample of healthy people.

[0079] Fig.13 :Evaluation of sample dilution multiples during testing (scatter plot). Note: RA is a sample of rheumatoid arthritis cases, and HC is a sample of healthy people undergoing physical examination.

[0080] Fig.14 :Evaluation of sample loading volume during testing (scatter plot). Note: RA is a sample of rheumatoid arthritis cases, and HC is a sample of healthy people undergoing physical examination.

[0081] Fig.15 :Analysis of the detection results of NA1IgM in the Epstein-Barr virus infection positive serum conversion plate SCP-EBV-001 (line graph).

[0082] Fig.16 :Analysis of the detection results of NA1IgM in the Epstein-Barr virus infection positive serum conversion plate SCP-EBV-002 (line graph).

[0083] Fig.17 : Analysis of NA1IgM detection results in EB virus infection positive serum conversion plate ZeptoMetrix 9097 (line graph).

[0084] Fig.18 :The detection results of NA1IgM in samples of different diseases related to EB virus infection (scatter plot). Note: IM is infectious mononucleosis, Crohn is Crohn's disease, SLE is systemic lupus erythematosus, Psoriasis is psoriasis, SS is Sjögren's syndrome, LCA is lung cancer, Lymphoma is lymphoma, GC is gastric cancer, HC is health check, RA is rheumatoid arthritis.

[0085] FIG. 19A to FIG. 19G :Evaluation of the diagnostic value of different Epstein-Barr virus antibodies for rheumatoid arthritis (scatter plot).

[0086] Fig. 20 :The diagnostic efficacy evaluation results of NA1IgM for RA cases on the magnetic particle chemiluminescence platform (ROC curve).

[0087] Fig.21 :Comparative analysis of NA1 IgM antibody test results and Anti-CCP (scatter plot). Note: CCP refers to Anti-CCP, which is translated as anti-cyclic citrullinated peptide antibody, one of the ACPAs; ​​the dotted line in the figure indicates the negative and positive judgment cutoff values ​​of 110314 when the sensitivity and specificity of NA1 IgM reagent are optimal. DETAILED DESCRIPTION

[0088] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0089] Example 1: Synthesis of different polypeptide fragments of EB virus nuclear antigen

[0090] The polypeptide fragments were designed and synthesized according to the full-length amino acid reference sequence (YP_401677, SEQ ID NO: 28) of nuclear antigen 1 (EBNA1) of EBVB95-8 strain retrieved from NCBI (National Center of Biotechnology Information), and a total of 27 polypeptides were obtained, as shown in Table 1.

[0091] Table 1: Synthesis information of EBNA1 peptide from EBV B95-8 strain

[0092]

[0093] EBV B95-8 strain EBNA1 full-length amino acid sequence (YP_401677)

[0094]

[0095] Example 2: Evaluation of the reactivity of different EBNA1 polypeptide fragments with serum NA1 IgM antibodies

[0096] The polypeptides (B1-B27) obtained in Example 1 were re-dissolved with deionized water and diluted to 4 μg / mL with 100 mM carbonate buffer (pH 9.0), and added to a 96-well chemiluminescent microplate at a standard of 100 μL per well. After incubation at 37°C for 2 hours, the plates were washed once with PBST, and 200 μL of blocking solution was added to each well, and incubated at 37°C for 2 hours. After blocking, the blocking solution was discarded, and 50 μL of 5 serum samples of rheumatoid arthritis (RA) cases diluted 1:10 and 50 μL of 3 negative control serum samples diluted 1:10 were added to each of the 8 wells prepared for each polypeptide, and incubated at 37°C for 30 minutes. After the reaction, the plates were washed four times with PBST, and mouse anti-human IgM antibody diluted 1:1000 was added and incubated at 37°C for 30 minutes. After washing four times with PBST, the plates were read using a chemiluminescence microplate reader (Molecular Devices, SpectraMax L). During the plate reading, the excitation solution (Xiamen Wantai Kairui Biotechnology Co., Ltd., Fujian Xia Equipment No. 20160064) and pre-excitation solution (Xiamen Wantai Kairui Biotechnology Co., Ltd., Fujian Xia Equipment No. 20160065) were injected into the instrument in situ, and the sample volume was 100 μL / well.

[0097] The test results are shown in Table 2, and the analysis results are shown in Table 2. Figure 1 and Figure 2 As shown. The results showed that B1-B27 were reactive to rheumatoid arthritis case samples, among which B3, B5, B6, B12, B14, B15, B16, and B17 were relatively reactive (with overall higher RLU values), but B3, B5, B6, B12, B14, B16, and B17 had certain nonspecificity, which was concentrated in the 31aa-485aa region, which was also a relatively reactive region to rheumatoid arthritis case samples. This region contained two Gly / Arg-rich regions, a variable Gly / Ala repeat sequence region, a CK2 binding site, a USP7 binding site, and part of the DNA binding and dimerization domain, and the common feature was the presence of a large number of amino acid repeat sequences. All peptides in the B18-B27, i.e., the 481aa-641aa amino acid region, could well distinguish RA case samples from negative control samples, but the overall reactivity to the samples was lower than that of other peptides, which was manifested as a lower overall detection activity (RLU value). The results showed that different polypeptide fragments in the 1-641aa region were all recognition epitopes of NA1 IgM, some epitopes would have non-specific reactions, and the dominant epitopes or antigenic fragments were 391-410aa and 481aa-641aa.

[0098] Table 2: Evaluation results of the reactivity of different EBNA1 peptides with serum NA1 IgM antibodies (RLU)

[0099]

[0100]

[0101] Note: RA1-RA5 are 5 serum samples from patients clinically diagnosed with rheumatoid arthritis, and HC1-HC3 are 3 negative control cases.

[0102] Example 3: Comparison of epitopes recognized by EBNA1IgM antibody and EBNA1IgG antibody

[0103] In China, the EB virus infection rate among adults is about 99%, and the positive rate of EB virus nuclear antigen NA1 IgG antibody in serum is greater than 99%. Therefore, it is necessary to fully consider the impact of the widespread existence of EBNA1 IgG on the detection of EBNA1 IgM. The similarities and differences in the epitopes recognized by EBNA1IgM and EBNA1 IgG antibodies are compared through the following examples.

[0104] The polypeptides (B1-B27) obtained in Example 1 were re-dissolved with deionized water and diluted to 4 μg / mL with 100 mM carbonate buffer (pH 9.0), and added to a 96-well chemiluminescent microplate at a standard of 100 μL per well, and 8 wells were prepared for each polypeptide. After incubation at 37°C for 2 hours, wash once with PBST, add 200 μL of blocking solution to each well, and incubate at 37°C for 2 hours. After the blocking is completed, pour out the blocking solution, and add 50 μL of 5 serum samples of rheumatoid arthritis cases diluted at 1:10 (the same samples as those used in Example 2, and EBNA1IgG was confirmed to be positive by commercial product testing) and 50 μL of 3 serum samples diluted at 1:10 confirmed to be negative for EB virus NA1 IgG by commercial product testing, and incubate at 37°C for 30 minutes. After the reaction, the plates were washed four times with PBST, and a 1:1000 diluted acridinium ester (NSP-SA-NHS) labeled mouse anti-human IgG antibody (Wuhan Aokebot Biotechnology Co., Ltd., A0032) was added and incubated at 37°C for 30 minutes. After washing four times with PBST, the plates were read using a chemiluminescence microplate reader (Molecular Devices, SpectraMax L). During the plate reading, the excitation solution and pre-excitation solution were injected in situ by the instrument, and the sample volume was 100 μL / well.

[0105] The test results are shown in Table 3, and the analysis results are shown in Table 3. Figure 3 and Figure 4The results showed that B5, B12-B16 were reactive to EBNA1 IgG antibody-positive samples in rheumatoid arthritis case samples, and the detection of RLU values ​​could distinguish between NA1 IgG antibody-negative and -positive samples. Among them, B5, B12, B13 and B14 had the highest reactivity, indicating that the amino acid sequences 66aa-85aa and 376aa-470aa were the recognition epitopes of NA1 IgG antibodies in serum samples, and the dominant epitopes were 66aa-85aa and 391aa-440aa. Compared with the widely recognized epitopes of the NA1 IgM antibody in the EBNA1 sequence confirmed in Example 2, the recognition epitopes of the two types of antibodies, NA1 IgG and NA1IgM, are not exactly the same, but both have strong reactivity to 66aa-85aa (B5) and 391aa-410aa (B12). The recognition epitope 376aa-470aa of NA1 IgG alone has certain non-specific reactions when used for the detection of NA1 IgM antibodies, which may be because the epitope has only 20 amino acids and there is potential cross-reaction.

[0106] Table 3: Evaluation results of the reactivity of different EBNA1 peptides with serum NA1 IgG antibodies (RLU)

[0107]

[0108]

[0109] Note: RA1-RA5 are 5 serum samples from patients clinically diagnosed with rheumatoid arthritis, NA1 IgG positive, and N1-N3 are 3 NA1 IgG negative samples.

[0110] Example 4: Comparison of epitopes recognized by EBNA1IgM antibody and EBNA1IgA antibody

[0111] The polypeptides (B1-B27) obtained in Example 1 were re-dissolved with deionized water and diluted to 4 μg / mL with 100 mM carbonate buffer (pH 9.0), and added to a 96-well chemiluminescent microplate at a standard of 100 μL per well, and 8 wells were prepared for each polypeptide. After incubation at 37°C for 2 hours, wash once with PBST, add 200 μL of blocking solution to each well, and incubate at 37°C for 2 hours. After the blocking is completed, the blocking solution is poured out, and 50 μL of 5 serum samples of rheumatoid arthritis cases diluted 1:10 (the samples were confirmed to be positive for EBNA1 IgA by commercially available product testing) and 50 μL of 3 serum samples diluted 1:10 by commercially available product testing to confirm that EB virus NA1 IgA is negative were added to each well, and incubated at 37°C for 30 minutes. After the reaction, the plates were washed four times with PBST, and a 1:1000 diluted acridinium ester (NSP-SA-NHS) labeled mouse anti-human IgA antibody (Wuhan Aokebot Biotechnology Co., Ltd., A0052) was added and incubated at 37°C for 30 minutes. After washing four times with PBST, the plates were read using a chemiluminescence microplate reader (Molecular Devices, SpectraMax L). During the plate reading, the excitation solution and pre-excitation solution were injected in situ by the instrument, and the sample volume was 100 μL / well.

[0112] The test results are shown in Table 4, and the analysis results are shown in Table 4. Figure 5 and Figure 6 As shown. The results showed that B3, B5, B12 and B16 had strong reactivity to EBNA1 IgA antibody positive samples in rheumatoid arthritis case samples, indicating that the amino acid sequences 31aa-50aa, 66aa-85aa, 391aa-410aa and 451aa-470aa were the core epitopes recognized by NA1 IgA antibodies in serum samples. Compared with the recognition epitopes of NA1 IgM antibodies confirmed in Example 2, the recognition epitopes of NA1 IgA and NA1 IgM antibodies are not exactly the same.

[0113] Table 4: Evaluation results of the reactivity of different EBNA1 peptides with serum NA1 IgA antibodies (RLU)

[0114]

[0115] Note: RA6-RA10 are 5 serum samples from patients clinically diagnosed with rheumatoid arthritis, NA1 IgA positive, and N1-N3 are 3 NA1 IgA negative samples.

[0116] Example 5: Evaluation of the diagnostic performance of recombinant antigens with different epitope combinations of EBNA1 for rheumatoid arthritis

[0117] Based on the research results of Examples 1-4, an E. coli expression system was designed to express and purify EBNA1 recombinant antigens with different amino acid sequences, and to prepare recombinant EBNA1 antigens with different amino acid sequences. Because the 1-641aa different antigen fragments all have the recognition epitope of NA1 IgM, B3, B5, and B6 are all reactive to NA1 IgM, but there is also non-specificity, and 1-90aa is a region that does not contain Gly / Ala repeating sequences at the N-terminus, and the 1-90aa region is the NA1 IgA recognition epitope region, so 1-90aa is expressed. The 91-325aa region is a highly repeated Gly / Ala region, and recombinant expression was attempted, but failed to express successfully. B9-B10 (326-360aa) is a Gly / Arg repeating sequence, and B11 is a CK2 binding site, so the 326-389aa antigen fragment (containing a Gly / Arg repeating sequence and a CK2 binding site) was selected for expression. B12 is the dominant epitope of NA1 IgM, so the 377-422aa antigen fragment was selected for expression, and the rear end DNA binding fragment was added on this basis, and the 377-498aa fragment was expressed. 481-641aa is the dominant epitope. For further analysis and confirmation, 484-571aa and 408-498aa were expressed. In order to analyze whether the DNA binding site is a dominant epitope, the 484-571aa antigen fragment was expressed. In order to evaluate the non-specific effect of the 1-90aa fragment on the dominant epitope, 1-90aa was expressed in series with 408-498aa and 390-641aa, respectively, and the linker was the GGGGSGGGGS (SEQ ID NO: 36) amino acid sequence, i.e., 2 flexible linkers (G4S) (SEQ ID NO: 37). The full-length recombinant antigen of 1-641aa was transiently expressed in 293F cells due to its large molecular weight. The purity of the 10 recombinant antigens was greater than 90% as detected by SDS-PAGE electrophoresis. The information of the expressed EBNA1 antigen is shown in Table 5.

[0118] Table 5: Information on recombinantly expressed antigenic epitopes or antigenic fragments

[0119]

[0120]

[0121] The 10 recombinantly expressed antigens were diluted to 4 μg / mL with 100 mM carbonate buffer (pH 9.0) and added to a 96-well chemiluminescent microplate at a standard of 100 μL per well. 8 wells were prepared for each recombinantly expressed antigen. After incubation at 37°C for 2 hours, the plates were washed once with PBST, 200 μL of blocking solution was added to each well, and the plates were incubated at 37°C for 2 hours. After blocking, the blocking solution was discarded, and 50 μL of 5 serum samples of rheumatoid arthritis cases diluted 1:10 and 50 μL of 3 serum samples of healthy people diluted 1:10 were added to each of the 8 wells prepared for each recombinant antigen, and the plates were incubated at 37°C for 30 minutes. After the reaction, the plates were washed four times with PBST, and a 1:1000 diluted acridinium ester (NSP-SA-NHS) labeled mouse anti-human IgM antibody (Wuhan Aokebot Biotechnology Co., Ltd., A0043) was added and incubated at 37°C for 30 minutes. After washing four times with PBST, the plates were read using a chemiluminescence microplate reader (Molecular Devices, SpectraMax L). During the plate reading, the excitation solution and pre-excitation solution were injected in situ by the instrument, and the sample volume was 100 μL / well.

[0122] The test results are shown in Table 6, and the analysis results are shown in Table 6. Figure 7 and Figure 8 shown.

[0123] The results show:

[0124] 1-90aa can detect some RA case samples, but there is a nonspecific reaction;

[0125] The epitope 326-389aa (containing the Gly / Arg repeat sequence and the CK2 binding site) had low reactivity and could not distinguish case samples from healthy human serum samples;

[0126] 377-422aa and 408-498aa can distinguish RA case samples from healthy human serum samples, and 408-498aa has better distinguishing effect, but there is a certain degree of missed detection. However, there is detection complementarity between the two epitopes. The antigen effect of 377-498aa epitope after the two epitopes are combined is better than 377-422aa and 408-498aa, indicating that 377-498aa epitope is the dominant epitope of NA1 IgM.

[0127] The 1-90aa epitope and the 408-498aa epitope are expressed in tandem, which can solve the problem of missed detection of a single epitope, but the overall reactivity is weak and there is a risk of false positives;

[0128] 484-571aa is the epitope in the DNA binding site region, with weak overall reactivity and the presence of false positives;

[0129] The recombinantly expressed 390-641aa epitope showed the strongest reactivity and better RA case differentiation, and the reactivity became weaker after tandem expression of the 1-90aa epitope;

[0130] The recombinantly expressed 1-641aa full-length antigen can also distinguish RA case samples from healthy subjects, but has a certain reactivity to healthy subjects samples ( Figure 7 and Figure 8 ), there is a risk of false positive.

[0131] Table 6: Analysis of diagnostic performance of different recombinant EBNA1 antigens for rheumatoid arthritis (RLU)

[0132]

[0133] Note: RA2-RA6 are 5 serum samples from patients with clinical diagnosis of rheumatoid arthritis, and HC6-HC8 are 3 serum samples from healthy subjects. The P / N ratio is the ratio of the mean RLU value of the rheumatoid arthritis case (RA) sample test to the mean RLU value of the healthy subject (HC) sample test.

[0134] Antigenic fragment 1-90 aa:

[0135]

[0136] Antigenic fragment 390-641 aa:

[0137]

[0138] 390-498 aa:

[0139]

[0140] 377-498 aa:

[0141]

[0142] 377-641 aa:

[0143]

[0144]

[0145] 408-641aa:

[0146]

[0147] Antigenic fragments 390-483 and 572-641aa:

[0148]

[0149] Example 6: Comparison of the auxiliary diagnostic efficacy of the recombinant antigen containing the dominant epitope NA1 and the full-length recombinant antigen for RA cases

[0150] The recombinantly expressed 377-498aa recombinant NA1 antigen, 390-641aa recombinant NA1 antigen and 1-641aa recombinant NA1 antigen were coated with magnetic microparticles Magnosphere TM MS300 / Tosyl, the amount of antigen added per milligram of magnetic particles is 20 μg, the concentration of magnetic particles is 4 mg / mL, the coating buffer is 50 mM borate buffer (pH 9.5), after coating at 37°C for 18 hours, the magnetic particles are collected and the supernatant is removed, and the same volume of 10% BSA is added to block at 37°C for 2 hours. After blocking, the magnetic particles are collected and washed 4 times with PBST, and then resuspended and mixed with pH 7.4 phosphate buffer to prepare reagent 1.

[0151] Acridinium ester (NSP-SA-NHS) labeled mouse anti-human IgM antibody (Wuhan Aokebot Biotechnology Co., Ltd., A0043) diluted 1:1000 was used as reagent 2.

[0152] The prepared reagents were used to test 40 samples from healthy people and 48 samples from cases clinically diagnosed with rheumatoid arthritis on a fully automatic chemiluminescence immunoassay produced by Xiamen Youmai Medical Instrument Co., Ltd. The detection method was as follows: 20 μL of the sample diluted 10 times was taken, 50 μL of reagent 1 was added, mixed and incubated at 37°C for 15 minutes, magnetic collection and washing with PBST twice, 50 μL of reagent 2 was added, mixed and incubated at 37°C for 10 minutes, magnetic collection and washing four times, 100 μL of excitation solution and pre-excitation solution were added, and the light signal was detected. The results are shown in Table 7.

[0153] The test results were analyzed by ROC curve, and the results are shown in Table 8 and Fig. 9 The results showed that the area under the ROC curve of NA1 antigen at 377-498aa epitope was 0.8438 (95% CI: 0.7608-0.9267), the area under the ROC curve of NA1 antigen at 390-641aa epitope was 0.8895 (95% CI: 0.8184-0.9605), and the area under the ROC curve of NA1 antigen at 1-641aa epitope was 0.8362 (95% CI: 0.7459-0.9265). The AUC of NA1 antigen at 390-641aa epitope was the highest, with the best diagnostic efficacy. The 499-641aa epitope was also an extremely important part for the recognition of NA1 IgM antibodies.

[0154] Table 7: Results of the diagnostic efficacy evaluation of 377-498aa, 390-641aa and 1-641aa recombinant NA1 antigens for RA cases on the magnetic microparticle chemiluminescence platform

[0155]

[0156]

[0157]

[0158]

[0159] Note: RA1-RA48 are 48 serum samples from patients clinically diagnosed with rheumatoid arthritis, and TJ1-TJ40 are 36 serum samples from healthy subjects.

[0160] Table 8: Evaluation results of the diagnostic efficacy of 390-641aa recombinant NA1 antigen and 1-641aa recombinant NA1 antigen for RA cases on the magnetic microparticle chemiluminescence platform

[0161] Recombinant Antigen AUC (95% CI) 377-498aa 0.8438 (95% CI: 0.7608-0.9267) 390-641aa 0.8895 (95% CI: 0.8184-0.9605) 1-641aa 0.8362 (95% CI: 0.7459-0.9265)

[0162] Example 7: Process Optimization of EB Virus Nuclear Antigen NA1IgM Antibody Detection Kit (Magnetic Particle Chemiluminescence Method)

[0163] 1. Selection of Antigen-Coated Magnetic Microparticles

[0164] The 390-641aa recombinant NA1 antigen was coated on Magnosphere TM MX100 / Carboxyl (JSR Life Sciences), Magnosphere TM MS300 / Carboxyl(JSR Life Sciences)、Magnosphere TM MS300 / Tosyl(JSR Life Sciences) and Dynabeads TM M270 / Carboxyl (Life Technologies Corporation), and the coated magnetic particles were diluted to 0.8 mg (calculated according to the mass of the pure magnetic particles) / mL as reagent 1, and combined with acridinium ester-labeled mouse anti-human IgM antibody reagent 2 to detect 10 rheumatoid arthritis case samples and 10 healthy physical examination serum samples. The test results are shown in Table 9 and Fig.10 shown.

[0165] The results showed that different types of magnetic particles can be used for coating NA1 antigen to prepare NA1 IgM antibody detection reagent. The coating pH ranged from 5.5 to 9.6, and the particle size of the magnetic particles ranged from 1 μm to 3 μm. TMMS300 / Tosyl has the best effect and the best P / N value (the ratio of the mean RLU value of case samples to the mean RLU value of healthy population samples). It can well distinguish case samples from healthy population samples and has the highest diagnostic sensitivity.

[0166] Table 9: Optimization results of magnetic particle model selection for magnetic particle reagents

[0167] Magnetic particle model MX100 Carboxyl M270 Carboxyl MS300 Carboxyl MS300 Tosyl Magnetic particle size 1μm 2.7μm 3μm 3μm Coating buffer MES pH 5.5 MES pH 5.5 MES pH 5.5 CB pH9.6 Sample No. RLU RLU RLU RLU HC1 73176 59014 100196 48911 HC2 125719 106433 212969 98247 HC3 31533 35559 61388 22340 HC4 30331 47438 82725 23329 HC5 61714 63406 100810 44380 HC6 30292 26747 44922 29734 HC7 18842 32119 47522 14344 HC8 24397 36694 83406 16741 HC9 78821 106971 302042 142137 HC10 30988 35586 40048 26580 RA1 792354 906900 2305435 1163629 RA2 1718257 1262474 2716884 2605174 RA3 2348186 1884469 3343944 3427483 RA4 3072651 3190191 4056129 4258757 RA5 1012175 1098968 2208342 1714380 RA6 1658040 1236970 2753087 2512951 RA7 643616 1038156 2157870 1841917 RA8 457998 715573 1646901 1088512 RA9 1465231 1009256 2276698 2141641 RA10 1271586 1231871 2370218 2409673 P 1444009 1357483 2583551 2316412 N 50581 54997 107603 46674 P / N 28.55 24.68 24.01 49.63

[0168] Note: MES is 2-Morpholinoethanesulphonic acid; CB is carbonate buffer; P / N is the ratio of the mean RLU values ​​of rheumatoid arthritis case samples (RA1-RA10) and healthy people samples (HC1-HC10).

[0169] 2. Assessment of Antigen Coating Amount

[0170] Magnosphere coated at different coating ratios TM MS300 / Tosyl, the antigen coating amount per mg of magnetic particles was 2μg, 5μg, 10μg and 20μg respectively, and the effect of antigen coating amount on the detection performance of the reagent was evaluated. The results are shown in Table 10 and Fig.11 The results showed that the P / N difference was not large for the antigen coating amount of 2μg-20μg, and only the RLU value was different. The antigen coating amount can be selected from 2μg to 20μg.

[0171] Table 10: Evaluation results of different magnetic particle antigen coating amounts

[0172] Raw material coating ratio 2μg 5μg 10 μg 20 μg Sample No. RLU RLU RLU RLU HC1 36359 47667 48991 54399 HC2 75294 83695 90296 95723 HC3 16423 21297 19853 25008 HC4 18826 23986 20699 23202 HC5 20124 25270 42048 61055 HC6 21902 27266 28690 30236 HC7 9452 10901 13392 14855 HC8 11438 15306 16798 20367 HC9 105629 127154 127158 116104 HC10 18737 22474 24771 24208 RA1 853255 1221209 1166703 1320637 RA2 1553440 2137491 2356188 2394036 RA3 3168967 3464680 3572210 3571353 RA4 4084552 4310023 4254683 4383635 RA5 1095841 1472813 1554507 1478839 RA6 2108344 2122266 2391674 2226631 RA7 829557 1274827 1680150 1649630 RA8 799455 960621 1123517 1214215 RA9 825234 1580892 2002872 2224938 RA10 2013766 2038920 2256467 2385221 P 1733241 2058374 2235897 2284914 N 33418 40502 43270 46516 P / N 51.86 50.82 51.67 49.12

[0173] 3. Evaluation of Magnetic Particle Coating Concentration

[0174] The amount of antigen coated per mg of magnetic particles is 10 μg. TM MS300 / Tosyl, the concentration of magnetic particles during coating was 4mg / mL, 10mg / mL and 20mg / mL respectively, and the effect of magnetic particle concentration on performance during coating was evaluated. The results are shown in Table 11 and Fig.12 The results showed that the P / N value was the highest when the magnetic particle coating concentration was 4 mg / mL and 10 mg / mL, indicating that the reagent performance was excellent. When the magnetic particle coating concentration was increased to 20 mg / mL, the P / N value decreased, because the RLU value of some rheumatoid arthritis case samples decreased.

[0175] Table 11: Effect of magnetic particle coating concentration on performance

[0176]

[0177]

[0178] Example 8: Optimization evaluation of reaction system

[0179] 1. Sample dilution multiple evaluation

[0180] The 390-641aa recombinant NA1 antigen was coated on the Magnosphere at a rate of 10 μg protein per mg magnetic particle. TM MS300 / Tosyl, the coated magnetic particles were diluted to 0.8 mg / mL as reagent 1, and combined with acridinium ester labeled anti-human IgM antibody reagent 2 to detect 10 rheumatoid arthritis case samples and 10 healthy physical examination serum samples. The samples were diluted 10 times and 100 times respectively during the test, and the original samples were used as controls. The sample volume was 20 μL. The test results are shown in Table 12 and Fig.13 When the sample dilution factor was 10 times, the discrimination between the test results of RA case samples and the test results of healthy people samples was the best, with a P / N value of 44.19.

[0181] Table 12: Evaluation of sample dilution factors during testing

[0182]

[0183]

[0184] Note: P / N is the ratio of the mean RLU values ​​of rheumatoid arthritis case samples (RA21-RA30) and healthy checkup population samples (HC23-HC32).

[0185] 2. Sample loading volume evaluation

[0186] The samples diluted 10 times were tested, and the sample loading volume was set to 20 μL and 50 μL respectively. The test results are shown in Table 13 and Fig.14 As shown, the optimal sample loading volume after 10-fold dilution is 20 μL. As the sample loading volume increases, the nonspecific reaction increases.

[0187] Table 13: Sample loading volume evaluation

[0188] Sample loading volume 20μL 50μL Sample No. RLU RLU RA21 5213581 7255290 RA22 8792511 8301135 RA23 1420318 1687786 RA24 8109660 9172011 RA25 8705483 8697754 RA26 7813710 8761696 RA27 3299831 7694747 RA28 6589294 8997563 RA29 8544188 8967957 RA30 1332190 3718088 HC23 183169 1292392 HC24 85053 579758 HC25 63805 122199 HC26 49975 139008 HC27 95331 1867806 HC28 215889 329795 HC29 250373 587767 HC30 145389 899730 HC31 139993 394839 HC32 124834 315906 P 5982077 7325403 N 135381 652920 P / N 44.19 11.22

[0189] Example 9: Relationship between NA1IgM antibody and acute EB virus infection The 390-641aa recombinant NA1 antigen was coated on the Magnosphere at a rate of 10 μg protein per mg magnetic particle. TMMS300 / Tosyl, the coated magnetic particles were diluted to 0.8 mg / mL as reagent 1, and combined with acridinium ester labeled anti-human IgM antibody reagent 2. The sample dilution multiple was 10 times, and the sample volume was 20 μL. The Epstein-Barr virus infection positive conversion disks SCP-EBV-001 and SCP-EBV-002 from BIOMEX and the Epstein-Barr virus infection positive conversion disk 9097 from ZeptoMetrix were tested on the fully automatic chemiluminescence immunoassay produced by Xiamen Youmaike Medical Instrument Co., Ltd. The results are as follows Figures 15 to 17 The test results are shown in Tables 14 to 16. The test results of three sets of EB virus infection positive conversion discs showed that within the longest 210 days after EB virus infection, the NA1 IgM test results were all negative, and the antibody could not be detected, indicating that during the acute infection period of EB virus, the EB virus nuclear antigen NA1 did not stimulate the immune system to produce EBNA1 IgM antibodies, or the concentration of the produced antibodies was very low and failed to reach the detection capacity of the reagent. This example strongly supports the specificity of EBNA1 IgM in the auxiliary diagnosis of rheumatoid arthritis, and the specificity will not be interfered by the widespread EB virus infection.

[0190] Table 14: Detection results of NA1 IgM in Epstein-Barr virus infection positive serum conversion plate SCP-EBV-001

[0191]

[0192]

[0193] Note:

[0194] The test results indicated by the underlined numbers in the table are positive test results, and the data test results in the table are considered positive when they are greater than or equal to 1.00;

[0195] InnoDx refers to Wantai Kairui, which here refers to the EBNA1IgM detection reagent developed by Kairui (i.e., the magnetic beads coated with 390-641aa recombinant NA1 antigens described in this embodiment). This reagent is used for detection of NA1 IgM in the table;

[0196] Diasorin Liaison is a product launched by Diasorin Diagnostics, usually referring to the company's fully automatic chemiluminescence immunoassay system. Here, NA1 IgG / VCA IgG / VCA IgM in the indication table are detected using Diasorin reagent.

[0197] Table 15: Detection results of NA1IgM in Epstein-Barr virus infection positive serum conversion plate SCP-EBV-002

[0198]

[0199]

[0200] Note:

[0201] The test results indicated by the underlined numbers in the table are positive test results, and the data test results in the table are considered positive when they are greater than or equal to 1.00;

[0202] InnoDx refers to Wantai Kairui, which here refers to the EBNA1IgM detection reagent developed by Kairui (i.e., the magnetic beads coated with 390-641aa recombinant NA1 antigens described in this embodiment). This reagent is used for detection of NA1 IgM in the table;

[0203] Diasorin Liaison is a product launched by Diasorin Diagnostics, usually referring to the company's fully automatic chemiluminescence immunoassay system. Here, NA1 IgG / VCA IgG / VCA IgM in the indication table are detected using Diasorin reagent.

[0204] Table 16: Detection results of NA1IgM in ZeptoMetrix 9097, a serum conversion plate for EBV infection

[0205]

[0206]

[0207] Note:

[0208] The underlined numbers in the table indicate that the test results are positive results. The data test results in the table are considered positive when they are greater than or equal to 1.00;

[0209] InnoDx refers to Wantai Kairui, which here refers to the EBNA1IgM detection reagent developed by Kairui (i.e., the magnetic beads coated with 390-641aa recombinant NA1 antigens described in this embodiment). This reagent is used for detection of NA1 IgM in the table;

[0210] Diasorin Liaison is a product launched by Diasorin Diagnostics, usually referring to the company's fully automatic chemiluminescence immunoassay system. Here, NA1 IgG / VCA IgG / VCA IgM in the indication table are detected using Diasorin reagent.

[0211] Example 10: Evaluation of the detection rate of NA1IgM in different EB virus infection-related diseases

[0212] The 390-641aa recombinant NA1 antigen was coated on the Magnosphere at a rate of 10 μg protein per mg magnetic particle. TMMS300 / Tosyl, the coated magnetic particles were diluted to 0.8 mg / mL as reagent 1, and combined with acridinium ester labeled anti-human IgM antibody reagent 2. The sample dilution multiple was 10 times, and the sample volume was 20 μL. The samples of EB virus infection-related disease cases (including infectious mononucleosis, lung cancer, lymphoma, gastric cancer and autoimmune diseases such as Crohn's disease, systemic lupus erythematosus, Sjögren's syndrome, psoriasis and rheumatoid arthritis) and healthy people were tested on the fully automatic chemiluminescence immunoassay produced by Xiamen Youmaike Medical Instrument Co., Ltd. The test results are analyzed as follows Fig.18 As shown in Table 17, in the rheumatoid arthritis case samples, the detection results of NA1 IgM showed a trend of being significantly higher than those of other disease groups, and there were significant differences with the detection results of the healthy physical examination population. This embodiment shows that by detecting NA1 IgM, an important reference basis can be provided for the diagnosis of rheumatoid arthritis, and clinicians can be assisted to more accurately determine whether a patient suffers from rheumatoid arthritis, especially in the differential diagnosis of other EB virus infection-related diseases, the detection result of NA1 IgM may become one of the key indicators.

[0213] Table 17: ROC curve analysis of NA1IgM test results in samples of different diseases related to EB virus infection

[0214] AUC Std.Error 95% CI p Value HC vs.IM 0.794 0.05138 0.7095-0.8786 <0.0001 HC vs. Crohn 0.5854 0.07782 0.4574-0.7134 0.3013 HC vs. SLE 0.5915 0.06221 0.4891-0.6938 0.1493 HC vs. Psoriasis 0.5536 0.1022 0.3854-0.7217 0.654 HC vs.SS 0.6317 0.0578 0.5366-0.7267 0.0262 HC vs. LCA 0.639 0.06235 0.5364-0.7415 0.0294 HC vs. lymphoma 0.5375 0.08353 0.4001-0.6749 0.6273 HC vs. GC 0.5268 0.0612 0.4262-0.6275 0.6423 HC vs.RA 0.9900 0.0069 0.9765-1.000 <0.0001

[0215] Example 11: Evaluation of the diagnostic value of different EB virus antibodies for rheumatoid arthritis

[0216] 100 rheumatoid arthritis case samples and 100 healthy physical examination serum samples were tested using the NA1IgM detection reagent prepared in Example 10 and the NA1 IgA, NA1 IgG, VCA IgA, VCA IgM, VCA IgG and EA IgG detection reagents produced by Xiamen Wantai Kairui Biotechnology Co., Ltd., respectively, and the test results were plotted as scatter plots as shown in FIG. FIG. 19A to FIG. 19G The results of ROC curve analysis are shown in Table 18. Compared with other EB virus antibody markers, NA1 IgM has the best diagnostic efficacy for rheumatoid arthritis cases.

[0217] Table 18: ROC curve analysis results of the diagnostic value of different EB virus antibodies for rheumatoid arthritis

[0218]

[0219]

[0220] Draw a ROC curve for the test results of NA1IgM, such as Fig. 20As shown, when the RLU value of 110314 is used as the negative and positive judgment boundary, the detection sensitivity and specificity of the reagent are optimal. As shown in Table 19, the detection sensitivity of NA1 IgM is 0.9200 (95% CI: 0.8500-0.9589), and the specificity is 0.9697 (95% CI: 0.9147-0.9917).

[0221] Table 19: Detection sensitivity and specificity of NA1IgM

[0222] Evaluation indicators result 95% CI Sensitivity 0.9200 0.8500 to 0.9589 Specificity 0.9697 0.9147 to 0.9917

[0223] Example 12: Comparison of NA1IgM and Anti-CCP, a marker of rheumatoid arthritis

[0224] The NA1IgM antibody detection kit prepared in Example 10 was used to detect 69 rheumatoid arthritis case samples with a detection background of the Anti-CCP detection kit produced by Roche, and the test results were analyzed by scatter plot. The results are as follows: Fig.21 As shown, the results show that the NA1IgM antibody detection kit is complementary to the Anti-CCP reagent, and can detect 6 rheumatoid arthritis case samples that Anti-CCP failed to detect. This detection complementarity will promote a more comprehensive diagnosis of rheumatoid arthritis, effectively reduce the possibility of missed diagnosis and misdiagnosis, and the relevant combination strategy deserves further research and promotion and application. In addition, the sensitivity of the kit of the present invention is 92.75%, which is better than the sensitivity of Anti-CCP (85.51%).

[0225]

References

[0226] [1]Jog NR, James J A.Epstein Barr Virus and Autoimmune Responses inSystemic Lupus Erythematosus[J].Frontiers inImmunology,2021,11:623944.DOI:10.3389 / fimmu.2020.623944.

[0227] [2]Holck DA, Karen D, Gunnar H.Epstein-Barr Virus in SystemicAutoimmune Diseases[J].Clinical&DevelopmentalImmunology,2013,2013:535738.DOI:10.1155 / 2013 / 535738.

[0228] [3]Soldan S S,Lieberman P M.Epstein-Barr virus and multiplesclerosis[J].Nature reviews.Microbiology,2023.

[0229] [4]Fujiwara S,Takei M.pstein-Barr virus and autoimmunediseases[J].Clinical and Experimental Neuroimmunology,2015,6:38-48.DOI:10.1111 / cen3.12263.

[0230] [5]Epstein-Barr Virus and Cytomegalovirus in AutoimmuneDiseases:AreThey Truly Notorious?A Preliminary Report[J].Blackwell Publishing Inc,2007,1108(none):567-577.DOI:10.1196 / annals.1422.059.

[0231] [6]Rigopoulou E I,Smyk D S,Matthews C E,et al.Epstein-BarrVirus as aTrigger of Autoimmune Liver Diseases[J].Advances inVirology,2012,2012(5):987471.DOI:10.1155 / 2012 / 987471.

[0232] [7]Zeng L,Yang K,Yu G,et al.Advances in research onimmunocyte ironmetabolism,ferroptosis,and their regulatory roles inautoimmune andautoinflammatory diseases[J].Cell Death&Disease,2024,15(7).DOI:10.1038 / s41419-024-06807-2.

[0233] [8]Zhang L.A common mechanism links Epstein-Barr virusinfections andautoimmune diseases.J Med Virol.2023Jan;95(1):e28363.doi:10.1002 / jmv.28363.

[0234] [9]Pisetsky D.Pathogenesis of autoimmune disease[J].NatureReviews.Nephrology,2023:1-16.DOI:10.1038 / s41581-023-00720-1.

[0235]

[10] Pratesi F,Tommasi C,Anzilotti C,et al.Deiminated Epstein-Barrvirus nuclear antigen 1 is a target of anti-citrullinated proteinantibodiesin rheumatoid arthritis[J].Arthritis&Rheumatism,2006,54(3).DOI:10.1002 / art.21629.

[0236]

[11] Increased Epstein-Barr Virus DNA Load and AntibodiesAgainst EBNA1and EA in Sardinian Patients with RheumatoidArthritis[J].Viral Immunology,2015,28(7):385.DOI:10.1089 / vim.2015.0035.

[0237]

[12] Kaushik P,Kaushik R.Diagnosis and Management ofRheumatoidArthritis[J].American Journal of Medicine,2008,121(6):e7-e7.DOI:10.1016 / j.amjmed.2007.11.026.

[0238]

[13] Cush J J.Rheumatoid Arthritis Early Diagnosis andTreatment[J].heMedical Clinics of North America,2021(2):105.

[0239]

[14] Brown P,Pratt AG,Hyrich KL.Therapeutic advances inrheumatoidarthritis.BMJ[J].2024 Jan 17;384:e070856.doi:10.1136 / bmj-2022-070856.

[0240]

[15] Jeffrey,A,Sparks.Rheumatoid Arthritis.[J].Annals ofInternalMedicine,2019.DOI:10.7326 / AITC201901010.

[0241]

[16] Christian Münz.Epstein Barr Virus Volume 2:One HerpesVirus:ManyDiseases[J].Current Topics in Microbiology andImmunology,2015,391.DOI:10.1007 / 978-3-319-22834-1.

[0242]

[17] Svendsen A J,Westergaard M C W,Draborg A H,et al.Altered AntibodyResponse to Epstein-Barr Virus in Patients WithRheumatoid Arthritis andHealthy Subjects Predisposed to the Disease.A Twin Study[J].Frontiers inimmunology,12:650713[2024-09-13].DOI:10.3389 / fimmu.2021.650713.

[0243]

[18] Wulff M, Westergaard, Holck A, et al. Isotypes of Epstein-Barr virusantibodies in rheumatoid arthritis: association with rheumatoid factors and citrulline-dependent antibodies. [J]. BioMedresearch international, 2015. DOI: 10.1155 / 2015 / 472174.

[0244]

[19] Trier NH,Draborg AH,Sternbk L,et al.EBNA1 IgM-BasedDiscrimination Between Rheumatoid Arthritis Patients,SystemicLupusErythematosus Patients and Healthy Controls[J].Antibodies,2019,8(2):35-.DOI:10.3390 / antib8020035.

[0245] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art. According to all the teachings disclosed, various modifications and replacements can be made to those details, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. An isolated polypeptide, which is a truncated fragment of the amino acid sequence shown in SEQ ID NO: 28, which comprises the sequence of positions 408-498 of the amino acid sequence shown in SEQ ID NO: 28, and does not comprise the sequence of positions 1-90 of the amino acid sequence shown in SEQ ID NO:

28.

2. The isolated polypeptide according to claim 1, comprising a first fragment and a second fragment, wherein: The first fragment comprises positions 390-408, 377-408, 91-325, 91-377, 91-390 or 91-408 selected from the group consisting of the amino acid sequence of SEQ ID NO: 28, and the second fragment comprises positions 498-641 selected from the group consisting of the amino acid sequence of SEQ ID NO: 28; The first segment is directly connected to the second segment or connected via a linker; Preferably, the first fragment is located at the N-terminus of the isolated polypeptide, and the second fragment is located at the C-terminus of the isolated polypeptide; Preferably, the first fragment is located at the C-terminus of the isolated polypeptide, and the second fragment is located at the N-terminus of the isolated polypeptide; Preferably, the isolated polypeptide does not contain the sequence at positions 326-389 of the amino acid sequence shown in SEQ ID NO: 28, and / or the isolated polypeptide does not contain the sequence at positions 499-571 of the amino acid sequence shown in SEQ ID NO: 28; Preferably, the linker comprises a sequence shown in SEQ ID NO:37, or multiple tandem sequences shown in SEQ ID NO:37; preferably, the linker comprises a sequence shown in SEQ ID NO:

36.

3. The isolated polypeptide according to any one of claims 1 to 2, comprising the sequence of positions 390-641, 390-498, 377-422, 377-498, 377-641 or 408-641 of the amino acid sequence shown in SEQ ID NO:

28.

4. The isolated polypeptide according to any one of claims 1 to 4, wherein the amino acid sequence is shown in any one of SEQ ID NO: 30 to SEQ ID NO:

35.

5. The isolated polypeptide according to any one of claims 1 to 4, when used to detect the same sample, has a P / N ratio greater than the P / N ratio of the amino acid sequence shown in SEQ ID NO: 28; in, The P / N ratio is the ratio of the mean RLU value of rheumatoid arthritis case samples to the mean RLU value of healthy person samples; Preferably, the P / N ratio is measured by a magnetic particle chemiluminescence platform; preferably, the sample is a serum sample.

6. A fusion polypeptide comprising the isolated polypeptide according to any one of claims 1 to 5, and a protein separation and purification tag.

7. An isolated nucleic acid molecule encoding the isolated polypeptide of any one of claims 1 to 5 or the fusion polypeptide of claim 6.

8. A recombinant vector comprising the isolated nucleic acid molecule of claim 7.

9. A host cell comprising the isolated nucleic acid molecule of claim 7, or the recombinant vector of claim 8.

10. A load, comprising a solid support and a substance connected or coated with the isolated polypeptide according to any one of claims 1 to 5 or the fusion polypeptide according to claim 6; Preferably, the solid support is selected from one or more of magnetic microparticles, agarose beads, polystyrene microparticles, plastic plates, glass plates, nitrocellulose membranes, nylon membranes, polyvinylidene fluoride membranes and semiconductor silicon wafers; Preferably, the load is the isolated polypeptide according to any one of claims 1 to 5 or the magnetic microparticles coated with the fusion polypeptide according to claim 6; Preferably, the magnetic particles are selected from Magnosphere TM MX100 / Carboxyl、Magnosphere TM MS300 / Carboxyl、Magnosphere TM MS300 / Tosyl and Dynabeads TM One or more of M270 / Carboxyl; Preferably, the average particle size of the magnetic particles is 1 μm-3 μm; Preferably, the ratio of the separated polypeptide or fusion protein to the magnetic particles is 2-20 μg, such as 2 μg, 5 μg, 10 μg or 20 μg, of the separated polypeptide or fusion protein coated per mg of magnetic particles.

11. Reagent combination, comprising: The load substance of claim 10, and an anti-human IgM antibody linked to a detectable marker; Preferably, the detectable marker is an enzyme, a fluorescent substance, a chemiluminescent substance or a radioactive isotope; Preferably, the enzyme is horseradish peroxidase or alkaline phosphatase; Preferably, the chemiluminescent substance is an acridine chemiluminescent substance, luminol or its derivatives or an electrochemiluminescent substance; Preferably, the acridinium chemiluminescent substance is one or more selected from acridinium esters (such as AE-NHS, DMAE-NHS, Me-DMAE-NHS or NSP-DMAE-NHS), acridinium salts (such as NSP-SA or NSP-SA-NHS), acridinium hydrazides, acridinium sulfonamides, 10,10'-dimethyl-9,9'-diacridinium, 2,2'-dicarbonyl-9,9'-diacridinium dinitrate, 10,10'-dimethyl-2,2'-disulfonic acid-9,9'-diacridinium and 10,10'-dimethyl-9,9'-diacridinium dinitrate; Preferably, the luminol or its derivative is selected from luminol and isoluminol; Preferably, the electrochemiluminescent substance is ruthenium terpyridine.

12. The reagent combination according to claim 11, comprising: Magnetic microparticles coated with the isolated polypeptide according to any one of claims 1 to 5 or the fusion polypeptide according to claim 6, and an anti-human IgM antibody linked to a detectable marker; Preferably, the concentration of magnetic particles is 2-12 mg / mL; Preferably, the reagent combination comprises magnetic particles coated with the isolated polypeptide according to any one of claims 1 to 5 or the fusion polypeptide according to claim 6, and an anti-human IgM antibody linked to an enzyme (e.g., horseradish peroxidase) or an acridinium chemiluminescent substance (e.g., acridinium ester, acridinium salt, acridinium hydrazide, acridinium sulfonamide, DMBA, DSBADN, DMDSBA or DMBADN); Preferably, the reagent combination further comprises a sample diluent, a negative control and / or a positive control.

13. A kit comprising the reagent combination according to any one of claims 11 to 12.

14. A combination product comprising the kit according to claim 13 and a commercially available Anti-CCP detection kit such as the Anti-CCP detection kit produced by Roche; Preferably, the combination product is used for the diagnosis or auxiliary diagnosis of rheumatoid arthritis.

15. Use of the isolated polypeptide according to any one of claims 1 to 5 or the fusion polypeptide according to claim 6 in the preparation of a reagent or kit for diagnosing or assisting in the diagnosis of rheumatoid arthritis or detecting EBNA1 IgM.

Images