A kit for quantitatively detecting a group of gastric cancer-related serum autoantibodies and its application

Abstract

The present invention discloses a kit for quantitatively detecting serum autoantibodies in gastric cancer and precancerous lesions and its application. The kit includes a solid-phase carrier, a recombinant antigen protein coated on the solid-phase carrier, an enzyme-labeled secondary antibody, a standard product, a chromogenic substrate, a washing solution, and a reaction termination solution. This detection kit can specifically and quantitatively detect the levels of RAE1, PGK1, and NPM1 autoantibodies in serum specimens, and is significantly superior to the currently clinically used serum markers CEA and CA19-9 in terms of sensitivity for early screening and diagnosis of gastric cancer and precancerous lesions.

Description

Technical Field

[0001] The present invention belongs to the field of protein biomarker detection, and particularly relates to a kit for detecting early gastric cancer and precancerous lesions based on the detection of serum autoantibodies. Background Art

[0002] Gastric cancer is one of the most common cancer types in China and the world. According to global cancer data, the incidence of gastric cancer ranks the 5th among malignant tumors, and the average annual number of new gastric cancer cases globally in the past 5 years was 1.806 million. The occurrence process of gastric cancer generally goes from normal gastric mucosa → chronic superficial gastritis → chronic atrophic gastritis without intestinal metaplasia → complete intestinal metaplasia → incomplete intestinal metaplasia → low-grade intraepithelial neoplasia → high-grade intraepithelial neoplasia → invasive gastric cancer. Gastric mucosal atrophy, intestinal metaplasia, and dysplasia / intraepithelial neoplasia are precancerous lesions of the stomach. Early gastric cancer refers to invasive cancer limited to the mucosa or submucosa, regardless of whether there is lymph node metastasis. Early diagnosis is particularly important for reducing the death risk of gastric cancer patients. Currently, endoscopy and endoscopic biopsy are still the gold standard for diagnosing gastric cancer. The combined detection of widely used clinical tumor markers such as CA72-4, carcinoembryonic antigen (CEA), and CA19-9 also has certain value in dynamically observing the occurrence and development of tumors, evaluating clinical efficacy, and predicting patient prognosis. However, the above-mentioned tumor markers are not ideal for diagnosing early gastric cancer or precancerous lesions. Currently, molecules or cells with tumor diagnostic value found in blood include glycoproteins, autoantibodies, microRNAs, circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), exosomes, etc. Autoantibodies (aAbs) against tumor-associated antigens (TAAs) also have certain value in the early detection of cancer. Compared with commonly used clinical tumor markers, autoantibodies can be detected several months or even years before the appearance of clinical symptoms. Moreover, even when the concentration of TAA is very low, after the immunological amplification effect, autoantibodies can reach a relatively high level, which is more conducive to detection.

[0003] In recent years, there have been relevant studies on screening autoantibodies as tumor markers. Four serum autoantibodies (ALDH1B1, UQCRC1, CTAG1, and CENPF) have good diagnostic value and a relatively high positive rate in early colorectal cancer and advanced adenomas (Front Oncol, 2020, 10: 1081). Among a group of liver cancer-related autoantibodies, the AUC of CENPF for diagnosing early HCC is 0.826, and the diagnostic efficiency of ACY1 for liver cirrhosis is the highest, with an AUC of 0.872 (EBioMedicine, 2015, 2: 438 - 446; MolMed Rep, 2016, 14: 4255 - 4262). A systematic review of gastric cancer serum autoantibodies found that the sensitivity of a single autoantibody for diagnosing gastric cancer ranges from 0 to 75%, with a median of 12.35%, while the specificity is generally high, with a median of 99.15% (Int J Cancer, 2015, 136: 2243 - 52). The above studies have demonstrated the great potential of autoantibodies as tumor detection markers. Autoantibodies help improve the sensitivity of detection and the detection rate of gastric cancer. However, there is still a lack of research on autoantibodies in early gastric cancer and precancerous lesions. Summary of the Invention

[0004] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide a new class of autoantibody tumor markers for screening and early diagnosis of gastric cancer and precancerous lesions, and to provide a quantitative detection kit for detecting this marker. The serum autoantibodies detected by this kit are significantly superior to the currently clinically used serum markers CEA and CA19-9 in terms of the sensitivity for diagnosing early gastric cancer and precancerous lesions.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides the use of reagents for detecting autoantibodies of RAE1, PGK1, and NPM1 in the preparation of a kit for early gastric cancer screening and diagnosis and precancerous lesion screening and diagnosis.

[0007] According to the specific embodiments of the present invention, the reagents for detecting autoantibodies of RAE1, PGK1, and NPM1 are reagents for enzyme-linked immunosorbent assay.

[0008] Furthermore, the reagents for detecting autoantibodies of RAE1, PGK1, and NPM1 are reagents for detecting autoantibodies of RAE1, PGK1, and NPM1 in human serum.

[0009] The key of the present invention lies in that it is determined that the contents of RAE1, PGK1 and NPM1 autoantibodies in human blood are significantly correlated with the risk of gastric cancer. Therefore, the risk of gastric cancer can be judged by detecting the contents of RAE1, PGK1 and NPM1 autoantibodies in human blood.

[0010] The present invention also provides a kit for quantitatively detecting RAE1, PGK1 and NPM1 autoantibodies in serum, which includes a solid-phase carrier, a recombinant antigen protein coated on the solid-phase carrier, an enzyme-labeled secondary antibody, a standard product, a chromogenic substrate, a washing solution and a reaction termination solution.

[0011] According to the specific embodiment of the present invention, the recombinant RAE1 antigen protein is purchased from Origene (the RAE1 protein is a full-length protein, and the protein sequence number is P78406); the recombinant PGK1 antigen protein is a PGK1 fusion protein with His and GST double tags (the PGK1 protein is a protein fragment 321-417 a.a., and the protein sequence number is P00558); the recombinant NPM1 antigen protein is an NPM1 fusion protein with His and GST double tags (the NPM1 protein is a full-length protein, and the protein sequence number is P06748). The PGK1 and NPM1 fusion proteins are obtained by fusing and expressing His and GST tags with PGK1 and NPM1 proteins using DNA in vitro recombination technology. By having His and GST double tags, it is convenient for the expression, detection, tracing and / or purification of the target protein.

[0012] According to the specific embodiment of the present invention, the coating concentration of the recombinant RAE1 antigen protein is 1 μg / ml, the coating concentration of the recombinant PGK1 antigen protein fragment is 2 μg / ml, and the coating concentration of the recombinant NPM1 antigen protein is 4 μg / ml.

[0013] Furthermore, the enzyme-labeled antibody is a commonly used labeled antibody in the art for detection, including but not limited to a horseradish peroxidase-labeled rabbit anti-human IgG antibody.

[0014] In addition, the standard product used in the detection kit of the present invention is a serum specimen containing high levels of RAE1, PGK1 and NPM1 autoantibodies respectively screened by ELISA detection technology.

[0015] The application of the above-mentioned kit for quantitatively detecting RAE1, PGK1 and NPM1 autoantibodies in serum in the preparation of products for early gastric cancer screening and diagnosis, as well as in the preparation of products for screening and diagnosis of precancerous lesions of gastric cancer also belongs to the protection scope of the present invention.

[0016] In this article, "RAE1, PGK1 and NPM1 autoantibodies" refers to "RAE1, PGK1 and NPM1 protein autoantibodies".

[0017] The detection kit of the present invention uses the enzyme-linked immunosorbent assay (ELISA) for determination. The antibody detection reaction plate is a solid-phase carrier coated with recombinant RAE1, PGK1, and NPM1 antigen proteins. For example, an enzyme-labeled plate coated with recombinant RAE1 antigen protein. The serum to be tested can specifically bind to the RAE1 protein in the micro-wells of the enzyme-labeled plate and be adsorbed in the micro-wells. An enzyme-labeled secondary antibody is added for incubation. After thorough washing, a chromogenic substrate is added. After terminating the reaction, the absorbance of each micro-well, that is, the OD value, is measured with an enzyme-labeled instrument. The OD value is directly proportional to the level of RAE1 autoantibody in the serum. The standard curve is plotted through the detection of standard serum samples, and the level of RAE1 antibody in each serum specimen is quantified.

[0018] The beneficial effects of the present invention are as follows:

[0019] The inventors have screened out antigen proteins with relatively high levels in the sera of gastric cancer and precancerous lesion patients through serum proteomics analysis and mass spectrometry identification. By purchasing or recombinating these antigen proteins and verifying the levels of corresponding serum autoantibodies through ELISA, serum autoantibodies capable of detecting early gastric cancer and precancerous lesions have been screened out. It is found that the mRNA export factor (RAE1) autoantibody has the highest differential detection value. The AUC of RAE1 autoantibody for differentiating early gastric cancer from healthy controls is 0.745, and the sensitivity and specificity are 94.9% and 47.1% respectively; the AUC for differentiating precancerous lesions of gastric cancer is 0.710, and the sensitivity and specificity are 84.3% and 50.4% respectively; the positive rates of RAE1 autoantibody for detecting early gastric cancer and precancerous lesions are 41.9% and 45.2% respectively, while the positive rates of CEA for detecting early gastric cancer and precancerous lesions are 6.5% and 9.7% respectively. The differential detection values of PGK1 and NPM1 autoantibodies are second only to RAE1. It is worth noting that when combining the three autoantibodies of RAE1, PGK1, and NPM1, the AUCs for diagnosing early gastric cancer and precancerous lesions are 0.769 and 0.760 respectively. By further combining age and gender, the AUCs can be significantly increased to 0.857 and 0.787, and the diagnostic efficacy is higher than that of single-index detection. Therefore, the sensitivity of autoantibodies such as RAE1 for diagnosing early gastric cancer and precancerous lesions is significantly higher than that of the commonly used index CEA at present, indicating that RAE1, PGK1, and NPM1 autoantibodies and their combined detection with age and gender have potential value in the early diagnosis of gastric cancer. Description of the Drawings

[0020] Figure 1Detection of autoantibodies in sera of gastric cancer patients and healthy individuals. (a) Coomassie Brilliant Blue-stained protein gel of gastric cancer; (b) Immunoblot using sera from gastric cancer (GC) patients as primary antibodies; (c) Immunoblot using sera from healthy controls (HC) as primary antibodies. The circles indicate immunoreactive spots recognized by gastric cancer sera. PGK1 and NPM1 are from protein spot 1, and RAE1 is from protein spot 2.

[0021] Figure 2 SDS-PAGE of recombinant protein purification. 1. Marker; 2. Uninduced Escherichia coli; 3. IPTG-induced Escherichia coli; 4. Precipitate of sonicated bacterial lysate; 5. Supernatant of sonicated bacterial lysate; 6. Purification by HIS column; 7. Purification by His and GST columns.

[0022] Figure 3 Positive rates of CEA, CA19-9, and autoantibodies RAE1, PGK1, NPM1 in diagnosing gastric cancer and precancerous lesions.

[0023] Figure 4 Scatter plot of autoantibodies RAE1, PGK1, NPM1 in detecting gastric cancer and precancerous lesions.

[0024] Figure 5 ROC curves of autoantibodies RAE1, PGK1, NPM1 and combined diagnosis of gastric cancer and precancerous lesions.

[0025] Figure 6 Western blot verification of RAE1 autoantibody levels in different sera. a-c. Serum samples with high, medium, and low RAE1 autoantibody levels shown by ELISA results respectively; +. RAE1 antibody (ABclonal); M. Marker. Detailed implementation manners

[0026] To illustrate the present invention more clearly, the following further describes the present invention in conjunction with preferred embodiments. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0027] Example 1 Serum proteomics analysis and mass spectrometry identification of gastric cancer-related autoantibodies

[0028] I. Materials

[0029] Three human gastric cancer cell lines, AGS, HGC-27, and SNU-1, were all obtained from the Department of Gastroenterology, Beijing Friendship Hospital; the 2-D protein quantification kit, 2-D purification kit, IPG buffer, IPG strip, and PVDF membrane were purchased from GE; the ECL luminescent solution (MILLIPORE); HRP-rabbit anti-human IgG (Sigma); the isoelectric focusing instrument (Ettan IPGphor3 GE Healthcare); the electrophoresis instrument (BIO-RAD POWER PAC3000); the S.N-75S / 01450 ultraviolet gel imager (BIO-RAD); the mass spectrometer Nano-LC-Q-TOF-MS / MS (Sciex).

[0030] II. Extraction and Purification of Total Cellular Proteins

[0031] All cells were digested and centrifuged. Five volumes of lysis buffer were added to the precipitate, and it was frozen and thawed three times in liquid nitrogen. RNase and DNase were added to digest nucleic acids, and it was incubated on ice for 30 min. Then, it was centrifuged at 4°C and 14,000 rpm for 40 min, and the supernatant was collected. The proteins were quantified and purified using the 2-D protein quantification kit and 2-D purification kit, respectively.

[0032] III. Treatment of Purified Samples and Passive Hydration

[0033] The purified samples were shaken and centrifuged at 4°C and 13,000 rpm for 8 min. The supernatant was discarded. After centrifuging for 8 min without adding samples, the supernatant was completely aspirated, leaving the precipitate, which was dried until it became semi-transparent. 124 μL of hydration solution (10 mL of hydration solution: 4.2 g of urea, 1.524 g of thiourea, 0.1 g of DTT, 0.4 g of chaps, colored with bromophenol blue) was melted at room temperature, 1 μL of IPG buffer was added, and then it was added to the protein sample and incubated at room temperature for 30 min. Then, it was centrifuged at 18°C and 14,000 rpm for 40 min. The IPG strip was equilibrated at room temperature. After centrifugation, the protein was loaded onto a 7-cm, pH 3-10 IPG strip and passively hydrated in an IPG BOX for 16 h.

[0034] IV. Two-Dimensional Gel Electrophoresis and Immunoblotting

[0035] Three hydrated gel strips were subjected to isoelectric focusing (IEF), followed by gel electrophoresis (SDS-PAGE). Two of them were then transferred to PVDF membranes for blocking, and the other gel was stained with Coomassie Brilliant Blue. Then, 7 gastric cancer sera were randomly selected and mixed as the primary antibody for the gastric cancer group, and 7 healthy control sera were randomly selected and mixed as the primary antibody for the control group. The mixed sera were diluted at 1:100 and incubated overnight at 4°C. After washing 3 times with TBST containing 0.1% Tween 20, a rabbit anti-human IgG conjugated with horseradish peroxidase (HRP) (diluted at 1:5000) was used as the secondary antibody and incubated for 1 hour at room temperature. After washing 3 times in the same manner, it was developed with an ECL kit and exposed using an imager. Protein spots that reacted positively with gastric cancer patient sera and negatively with normal healthy human sera in the immunoblotting reaction were identified and excised on parallel gels for mass spectrometry identification. The results are as Figure 1 , PGK1 and NPM1 were derived from protein spot 1, and RAE1 was derived from protein spot 2.

[0036] Example 2 Preparation of Recombinant PGK1 and NPM1 Antigen Proteins

[0037] I. Materials

[0038] The protein marker (10 - 180KD) was a product of Thermo Fisher; Escherichia coli BL21(DE3) was preserved in our laboratory; isopropyl β-D-thiogalactoside (IPTG) was a product of Amresco; plasmid construction and DNA sequence determination were completed by Bomed Company. Other reagents were all domestic analytical pure.

[0039] II. Construction and Identification of Prokaryotic Expression Vectors for Human PGK1 and NPM1

[0040] The PGK1 protein fragment 321 - 417 a.a. of the present invention was recombinantly expressed, with the protein sequence number P00558, a GST tag added to its N-terminus, and a His tag added to its C-terminus. The molecular weight was 38KD. The full-length NPM1 protein of the present invention was recombinantly expressed, with the protein sequence number P06748, a GST tag added to its N-terminus, and a His tag added to its C-terminus. The molecular weight was 59KD. Genes were synthesized by chemical synthesis method, with the vector being pET-6p vector. Recombinant plasmids were constructed and sequenced and identified (completed by Bomed Company).

[0041] III. Induced Expression of GST-PGK1-His and GST-NPM1-His Fusion Proteins in Escherichia coli BL21(DE3) Strains

[0042] The correctly sequenced recombinant expression plasmid was transformed into competent Escherichia coli BL21(DE3) cells. A single colony was picked and placed into liquid LB medium (containing Amp), and cultured with shaking at 37°C for 5 - 6 h. A small amount of the bacterial solution was taken as the control group without IPTG induction, and cultured with shaking at 16°C for 12 h. IPTG was added to the remaining bacterial solution to a final concentration of 0.5 mmol / L, and cultured with shaking at 16°C for 12 h. The bacterial solution was collected into a 1.5 ml centrifuge tube, centrifuged at 12000 r / min for 10 min, and the supernatant was discarded to retain the precipitate. The precipitate was resuspended with distilled water, added with loading buffer, boiled for 10 min, and subjected to 10% SDS-PAGE electrophoresis. Coomassie Brilliant Blue staining was used to observe the expression of the fusion protein. The results are shown in the appendix Figure 2 。

[0043] IV. Large-scale expression and purification of GST-PGK1-His and GST-NPM1-His fusion proteins

[0044] A single colony of BL21(DE3) containing the recombinant plasmid was picked and placed into 5 - 10 ml of liquid LB medium (containing Amp), and cultured with shaking at 37°C overnight. The next day, the above bacterial solution was added to 800 ml of liquid LB medium (containing Amp) at a ratio of 1%, cultured with shaking at 37°C for 4 - 5 h, IPTG was added to a final concentration of 0.5 mmol / L, and cultured with shaking at 16°C for 12 h. The bacterial cells were collected by the same method as above. 1 g of bacterial cells was dissolved in 20 ml of lysis buffer (pH 7 - 8), and ultrasonically lysed on ice bath for 10 min, centrifuged at 12000 r / min at 4°C for 1 h, and the supernatant and precipitate were collected. The soluble recombinant protein was purified by Ni-IDA-Sefinose pre-packed column and / or GST gravity pre-packed column, and the purity was detected by electrophoresis after purification. The results are shown in the appendix Figure 2 。

[0045] Example 3 Preparation of enzyme-linked immunosorbent assay (ELISA) plates and standards

[0046] I. Materials

[0047] The 96-well ELISA plates were purchased from Corning, USA; the recombinant PGK1 and NPM1 antigen proteins were prepared according to the method of Example 1; the recombinant RAE1 antigen protein was purchased from Origene; newborn bovine serum (NBS) was purchased from Gibco, USA; ELISA coating buffer, TMB chromogenic solution and termination solution were all purchased from Beijing Solarbio Science & Technology Co., Ltd.; the washing buffer was purchased from BOSTER; HRP-labeled rabbit anti-human IgG was purchased from Sigma, USA; other reagents were all of domestic analytical purity.

[0048] II. Determination of the coating concentration of ELISA plates and the concentration of secondary antibody

[0049] 1. Coating of ELISA plates

[0050] The checkerboard method was used to determine the optimal coating concentration of the enzyme-linked immunosorbent assay (ELISA) plate. The RAE1, PGK1, and NPM1 proteins were diluted to different concentrations with ELISA coating buffer for coating. 100 μl of the above proteins were added to each reaction well, and 100 μl of the coating buffer without protein was added to the control well. The plate was placed in a refrigerator at 4 °C overnight. Then, the liquid was discarded. 250 μl of wash buffer (added with 0.05% Tween 20) was added to each reaction well. After gently shaking the ELISA plate on the laboratory bench, the wash buffer was discarded, and the ELISA plate was patted dry on absorbent paper.

[0051] 2. Blocking of the ELISA plate

[0052] 100 μl of blocking solution (10% NBS + 0.5% sucrose, PBS) was added to each reaction well of the above ELISA plate. After incubating at 37 °C for 2 hours, the blocking solution was discarded, and then the plate was washed 1 - 2 times in the same way.

[0053] 3. Detection of positive and negative serum samples on the ELISA plate

[0054] The positive (P) and negative (N) sera were diluted 1:100 with sample diluent (10% NBS, PBS). 50 μl was added to each well. The ELISA plate was placed on a microplate shaker at 500 / min and incubated for 1 h. After discarding the liquid, the plate was washed 3 times. Then, 50 μl of HRP-labeled rabbit anti-human IgG at different dilution multiples (1:4000, 1:5000, 1:8000, 1:16000) was added to each reaction well in each row. After incubating on the shaker for 30 min, the plate was washed 3 times. The TMB chromogenic solution (equal volume of solution A + solution B) was prepared, and 100 μl of the chromogenic solution was added to each reaction well. The reaction was carried out for 15 min under light-proof conditions, and then 50 μl of stop solution was added to each well to terminate the reaction. The OD values of each reaction well were measured at 450 nm using an ELISA reader, and the results are shown in Table 1 below.

[0055] Table 1 OD values of reaction wells and control wells with different coating concentrations and different secondary antibody dilution degrees on the ELISA plate detected by the checkerboard method

[0056]

[0057] 4. Determination of the optimal coating concentration

[0058] Based on the above experiment, when the P / N value was around 10, the coating concentration of the RAE1 antigen protein was preliminarily determined to be 1 μg / ml, the coating concentration of the PGK1 antigen protein fragment was 2 μg / ml, and the coating concentration of the NPM1 antigen protein was 4 μg / ml.

[0059] III. Preparation of standards and establishment of a standard curve

[0060] The standard samples were serum specimens containing high levels of RAE1, PGK1, and NPM1 autoantibodies respectively, which were screened by ELISA detection technology. The OD values measured for standard serum samples at different dilutions under the above coating concentrations were used to plot the standard curve.

[0061] Example 4 Diagnostic value of RAE1, PGK1, and NPM1 autoantibodies in gastric cancer and precancerous lesions and verification of RAE1 autoantibody level

[0062] Three autoantibodies were detected in 364 samples, including 122 healthy sample sera, 51 precancerous lesions, 78 early gastric cancers, and 113 advanced gastric cancer sera. The diagnostic value was evaluated using the ROC curve and AUC value.

[0063] The results showed that compared with healthy controls, the levels of RAE1, PGK1, and NPM1 autoantibodies in the sera of patients with precancerous lesions, early gastric cancer, and advanced gastric cancer were significantly increased ( Figure 3 ). The AUCs of RAE1 autoantibody for diagnosing early gastric cancer and precancerous lesions were 0.745 and 0.710 respectively, and the positive rates were 41.9% and 45.2%; the AUCs of PGK1 autoantibody for diagnosing early gastric cancer and precancerous lesions were 0.648 and 0.709 respectively, and the positive rates were 41.9% and 38.7%; the AUCs of NPM1 autoantibody for diagnosing early gastric cancer and precancerous lesions were 0.611 and 0.664 respectively, and the positive rates were 29% and 25.8%. The positive rates of CEA for diagnosing early gastric cancer and precancerous lesions were 6.5% and 9.7% respectively, and those of CA19-9 were even lower, being 1.6% and 0% respectively ( Figure 4 ). When diagnosing with a single autoantibody, the value of RAE1 was the best. When combining the three autoantibodies of RAE1, PGK1, and NPM1, the AUCs for diagnosing early gastric cancer and precancerous lesions were 0.769 and 0.760 respectively. When further combining age and gender, the AUCs could be significantly increased to 0.857 and 0.787, and the diagnostic efficiency was higher than that of single-index detection ( Figure 5 ). The levels of RAE1 autoantibody in different sera were randomly verified by Western blot ( Figure 6 ), and the results showed a consistent trend with the relative levels detected by ELISA.

[0064] Obviously, the above-mentioned embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation manners here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. Use of a reagent for detecting autoantibodies in the preparation of a kit for screening and diagnosing early gastric cancer and precancerous lesions of gastric cancer; The reagent for detecting autoantibodies is a reagent for detecting RAE1, PGK1 and NPM1 autoantibodies in human serum.

2. The use according to claim 1, wherein, The reagent for detecting RAE1, PGK1 and NPM1 autoantibodies in human serum is a reagent for enzyme-linked immunosorbent assay.

3. Use of a kit for quantitative detection of serum autoantibodies in the preparation of a product for screening and diagnosing early gastric cancer and / or precancerous lesions of gastric cancer, wherein, The kit is used to detect the contents of RAE1, PGK1 and NPM1 autoantibodies in human serum; The kit includes a solid-phase carrier and a recombinant antigen protein coated on the solid-phase carrier; the recombinant antigen protein is a recombinant RAE1 antigen protein with a His tag and / or a GST tag, a recombinant PGK1 antigen protein fragment, and a recombinant NPM1 antigen protein.

4. The use according to claim 3, wherein, The kit also includes an enzyme-labeled secondary antibody, a standard, a chromogenic substrate, a washing solution, and a reaction termination solution.

5. The use according to claim 3, wherein, The coating concentration of the recombinant RAE1 antigen protein is 1 μg / ml, the coating concentration of the recombinant PGK1 antigen protein fragment is 2 μg / ml, and the coating concentration of the recombinant NPM1 antigen protein is 4 μg / ml.

6. The use according to claim 4, wherein, The enzyme-labeled secondary antibody is a horseradish peroxidase-labeled rabbit anti-human IgG antibody.

7. The use according to claim 4, wherein, The standard is a serum specimen containing high levels of RAE1, PGK1 and NPM1 autoantibodies respectively screened by ELISA detection technology.

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