MOG-IgG autoantibody detection kit based on living cell CBA method

Abstract

The invention provides an MOG-IgG autoantibody detection kit based on a living cell CBA method, a substrate cell for detecting an MOG-IgG antibody is a living cell strain capable of stably expressing an MOG target antigen, the detection substrate cell GFP-MOG-HEK293 cell strain can stably express the MOG antigen, the living substrate cell can retain the complete spatial conformation of the MOG antigen during detection, and the detection sensitivity is high. The detection sensitivity of the MOG-IgG autoantibody is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a MOG-IgG autoantibody detection kit based on a living cell CBA method. Background Art

[0002] A large number of studies have confirmed that serum MOG-IgG antibodies are biological markers for MOGAD (anti-myelin oligodendrocyte glycoprotein immunoglobulin G antibody-associated disease) and are pathogenic. The current clinical MOG-IgG antibody detection method is the fixed cell CBA method, which differs from the live cell CBA method in the state of the substrate cells. The substrate cells of the fixed cell CBA method need to be fixed before incubation with clinical samples, which will cause the spatial conformation of the antigen to be destroyed, affecting its sensitivity and specificity. Summary of the invention

[0003] In view of this, the present invention provides a MOG-IgG autoantibody detection kit based on the living cell CBA method, which can stably express the MOG antigen and retain the complete spatial conformation of the MOG antigen, thereby improving the sensitivity of MOG-IgG autoantibody detection.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions: The invention provides a MOG-IgG autoantibody detection kit based on a live cell CBA method, comprising an unfixed GFP-MOG-HEK293 live cell line cell sheet.

[0005] Preferably, PBS buffer and fluorescent secondary antibody are also included.

[0006] Preferably, the method for preparing the GFP-MOG-HEK293 cell line comprises the following steps: S1, construction of GFP-MOG recombinant plasmid; S2, transforming and extracting the GFP-MOG recombinant plasmid to obtain a GFP-MOG lentiviral plasmid; S3, culture HEK293 cells; S4. After transfecting the GFP-MOG lentiviral plasmid into HEK293 cells, the cells were cultured and screened to obtain GFP-MOG-HEK293 cell lines.

[0007] Preferably, in step S1, the specific steps of constructing the GFP-MOG recombinant plasmid are: first, EcoRI / BamHI restriction sites are added at both ends of the MOG target gene; the EcoRI / BamHI restriction sites are inserted into the Lenti-CMV-MCS-3xFLAG-PGK-Puro vector; and the MOG target gene is inserted into the vector by a DNA Ligation Kit to obtain a GFP-MOG plasmid.

[0008] Preferably, in step S3, when culturing HEK293 cells, the HEK293 cells are inoculated in a 100 mm cell culture dish containing 10 mL of complete culture medium for culturing.

[0009] Preferably, the complete culture medium comprises DMEM, FBS and P / S, wherein FBS accounts for 10% of the total volume and P / S accounts for 1% of the total volume.

[0010] Preferably, in step S4, the step of transfecting HEK293 cells with the GFP-MOG lentiviral plasmid is: when the growth density of HEK293 cells reaches 60-80%, adding the lentivirus obtained in S2 to a culture dish in which HEK293 cells are cultured, and adding polybrene to perform lentiviral transfection of the cells; and / or, In step S4, after transfection, the steps of culture screening are as follows: 72 hours after infection, puromycin with a final concentration of 2 ug / mL is added, and thereafter, fresh complete medium containing 2 ug / mL puromycin is replaced every 2-3 days for 2 weeks.

[0011] Preferably, the final concentration of polybrene added is 5 μg / mL; and / or, The transfection time is 12-20h.

[0012] Preferably, the method for preparing a cell sheet of an unfixed GFP-MOG-HEK293 live cell line comprises the following steps: successfully screening a GFP-MOG-HEK293 cell line that stably expresses MOG and inoculating it in a 6-well plate to allow it to adhere to the wall and grow to 80-90%.

[0013] Preferably, a glass slide is placed in the 6-well plate, and the cell line grows on the glass slide. The cell seeding density is 1x10 6 cells / mL, and the complete culture medium included DMEM, FBS, and P / S, of which FBS accounted for 10% of the total volume and P / S accounted for 1% of the total volume.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) The GFP-MOG-HEK293 cell line in the kit of the present invention can stably express the MOG antigen and retain the complete spatial conformation of the MOG antigen, thereby improving the sensitivity of MOG-IgG autoantibody detection.

[0015] (2) The live cell CBA method for detecting MOG-IgG provided by the present invention can obtain a GFP-MOG-HEK293 cell line that can be cultured stably for a long time, enabling instant detection of serum samples and significantly shortening the detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph showing the electrophoresis results of MOG protein expression after the recombinant plasmid provided in Example 1 of the present invention was transferred into HEK293 cells; Figure 2 This is a diagram showing the screening results of the GFP-MOG-HEK293 stable cell line provided in Example 1 of the present invention; Figure 3 This is a diagram of the GFP-MOG-HEK293 cell state before incubation of the clinical sample provided in Example 2 of the present invention; Figure 4 This is a flow chart of the live cell CBA method for detecting human serum MOG-IgG provided in Example 3 of the present invention; Figure 5 This is a graph showing the results of detecting human serum MOG-IgG using the live cell CBA method provided in Example 3 of the present invention.

[0017] in, Figure 3 3A and 3B are bright field (10x) images of the middle field of view and the peripheral field of view, respectively, and 3C and 3D are fluorescence (10x) images of the middle field of view and the peripheral field of view, respectively. Figure 5 Among them, 5A is a negative result and 5B is a positive result. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0019] Example 1 Preparation of MOG-IgG autoantibody detection kit based on live cell CBA method The preparation method comprises the following steps: Step 1: Construction of GFP-MOG recombinant plasmid The MOG target gene with EcoRI / BamHI restriction sites was inserted into the Lenti-CMV-MCS-3xFLAG-PGK-Puro vector to obtain a recombinant full-length GFP-MOG plasmid. Plasmid transformation and extraction were then performed to obtain the GFP-MOG lentiviral plasmid. The gene ID of MOG was obtained from the GenBank website, which was NM_002433.5.

[0020] Step 2: Construction of GFP-MOG-293 cell line HEK293 cell culture: HEK293 cells were inoculated in a 100 mm cell culture dish containing 10 mL of complete medium. The complete medium was prepared with DMEM + 10% FBS + 1% P / S; the number of cells inoculated was about 2 x 10 6 cells / mL; the incubator was maintained at a constant temperature of 37°C, 5% CO2, and humidity of 45-65%.

[0021] Construction of GFP-MOG-HEK293 stable cell line: Lentivirus infection: When the growth density of HEK293 cells reaches 60-80%, add the lentivirus obtained in step 1 to the above cell culture dish, and add polybrene to perform cell lentivirus transfection, where the final concentration of polybrene in the cell sample is 5ug / mL. After 12-20h of infection, discard the culture medium in the culture dish and add 10mL of fresh complete culture medium to continue culturing.

[0022] Screening of stable cell lines: 72 hours after infection, add puromycin at a final concentration of 2ug / mL. After that, replace the culture medium with fresh complete medium containing 2ug / mL puromycin every 2-3 days. After about 2 weeks, screen the stable cell lines and perform gel electrophoresis on the cell lines. Figure 1 As shown, and fluorescent photos were taken as Figure 2 shown.

[0023] Cryopreservation of GFP-MOG-HEK293 stable cell line: The above-obtained adherent GFP-MOG-HEK293 stable cell line was digested into single cells with 0.05% trypsin. Some cells were re-seeded in a 6-well plate with a glass slide at the bottom of the well to prepare GFP-MOG-HEK293 live cell sheets. The remaining cells were cryopreserved.

[0024] Among them, the cell freezing method is: add 1 mL of freezing solution (50% FBS + 40% DMEM + 10% DMSO) to the single cell suspension after trypsin digestion, and transfer the mixture of freezing solution and cells into a cryopreservation tube; place it in a -80℃ refrigerator, and transfer it to a liquid nitrogen tank for storage after overnight.

[0025] Step 3: Preparation of cell sheets of unfixed GFP-MOG-293 live cell lines The GFP-MOG-HEK293 cell line was inoculated on a glass slide and cultured with complete culture medium in an incubator at 37°C, 5% CO2, and 45-65% humidity; when the cells on the glass slide in the 6-well plate grew to 80-90% adherent, an unfixed cell sheet was obtained, which can be used for subsequent MOG-IgG antibody detection.

[0026] Example 2 Detection process of MOG-IgG antibody in human serum by live cell detection The cell sheet inoculated with unfixed GFP-MOG-HEK293 living cells obtained in Example 1 was washed twice with 0.01 M PBS.

[0027] The serum samples to be tested were diluted with 0.01 M PBS buffer at dilution ratios of 1:10, 1:32, 1:100, 1:320, and 1:1000.

[0028] Then, serum samples with different dilution ratios were directly added to the cell sheet in the above 6-well plate and incubated with the GFP-MOG-HEK293 living cells on the cell sheet. The incubation conditions were: 37° C., 1 h.

[0029] After incubation, the liquid was discarded and the cells were washed twice with 0.01 M PBS.

[0030] The fluorescent secondary antibody was diluted with 0.01M PBS at a ratio of 1:100, and the diluted fluorescent secondary antibody was added to the above 6-well plate, and the fluorescent secondary antibody was incubated with GFP-MOG-HEK293 cells at 37°C for 1 hour.

[0031] After the incubation, the liquid was discarded and 0.01 M PBS was added again for washing twice.

[0032] In a fume hood, the cells on the cell sheet in the 6-well plate were fixed with acetone-mixed fixative for 10 min, and then the liquid was discarded and the cells were blown dry.

[0033] Finally, the cell sheet was washed twice with 0.01M PBS to remove the excess fixative, and the cell sheet was taken out from the 6-well plate and placed under a fluorescence microscope with a 20x objective lens for microscopic examination. The results were analyzed, such as Figure 3 shown.

[0034] Example 3 Validation of human serum MOG-IgG antibody detection by living cells Serum samples of 119 suspected MOGAD patients were tested for serum MOG-IgG antibodies according to the steps in Example 2. Figure 4 As shown, the results are Figure 5 The conventional fixed cell CBA method was also used to detect serum MOG-IgG antibodies in the same batch of samples. The test results are shown in Table 1 below: Table 1

[0035] Specifically: the live cell CBA technology detected 19 cases of serum MOG-IgG antibody positive and 100 cases of negative; the fixed cell CBA method detected 22 cases of serum MOG-IgG antibody positive and 97 cases of negative.

[0036] Finally, 14 patients were diagnosed as MOGAD based on clinical phenotypes, including 14 true positives (serum MOG-IgG antibody positive + clinical diagnosis of MOGAD) and 5 false positives (serum MOG-IgG antibody positive + clinical diagnosis excluding MOGAD) by live cell CBA method; 11 true positives and 11 false positives by fixed cell CBA method.

[0037] After statistical analysis, the sensitivity of the live cell CBA method of the present invention for detecting MOG-IgG antibodies was 100%, and the specificity was 95%; the sensitivity of the conventional fixed cell CBA method for detecting MOG-IgG antibodies was 85% and the specificity was 90%.

[0038] Compared with the fixed cell CBA method, the live cell CBA method has higher sensitivity and specificity when detecting MOG-IgG antibodies, which is helpful for accurate diagnosis of the disease.

[0039] The specific raw materials not described in the present invention are all existing materials and can be directly purchased from the market.

[0040] The above are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A MOG-IgG autoantibody detection kit based on live cell CBA method MOG-IgG autoantibody detection kit, characterized in that, Includes unfixed GFP-MOG-HEK293 live cell sheet.

2. The kit according to claim 1, characterized in that It also includes PBS buffer and fluorescent secondary antibody.

3. The kit according to claim 1, characterized in that The preparation method of the GFP-MOG-HEK293 cell line comprises the following steps: S1, construction of GFP-MOG recombinant plasmid; S2, transforming and extracting the GFP-MOG recombinant plasmid to obtain a GFP-MOG lentiviral plasmid; S3, culture HEK293 cells; S4. After transfecting the GFP-MOG lentiviral plasmid into HEK293 cells, the cells were cultured and screened to obtain GFP-MOG-HEK293 cell lines.

4. The kit according to claim 3, characterized in that In step S1, the specific steps of constructing the GFP-MOG recombinant plasmid are: first, EcoRI / BamHI restriction sites are added at both ends of the MOG target gene; the EcoRI / BamHI restriction sites are inserted into the Lenti-CMV-MCS-3xFLAG-PGK-Puro vector; the MOG target gene is inserted into the vector using a DNA Ligation Kit to obtain a GFP-MOG plasmid.

5. The kit according to claim 3, characterized in that In step S3, when culturing HEK293 cells, the HEK293 cells are seeded in a 100 mm cell culture dish containing 10 mL of complete culture medium for culturing.

6. The kit according to claim 5, characterized in that Complete culture medium includes DMEM, FBS and P / S, wherein FBS accounts for 10% of the total volume and P / S accounts for 1% of the total volume.

7. The kit according to claim 3, characterized in that In step S4, the step of transfecting HEK293 cells with the GFP-MOG lentiviral plasmid is as follows: when the growth density of HEK293 cells reaches 60-80%, the lentivirus obtained in S2 is added to a culture dish in which HEK293 cells are cultured, and polybrene is added to perform lentiviral transfection of the cells; and / or, In step S4, after transfection, the steps of culture screening are as follows: 72 hours after infection, puromycin with a final concentration of 2 ug / mL is added, and thereafter, fresh complete medium containing 2 ug / mL puromycin is replaced every 2-3 days for 2 weeks.

8. The kit according to claim 7, characterized in that The final concentration of polybrene added is 5ug / mL; and / or, The transfection time is 12-20h.

9. The kit according to claim 3, characterized in that The steps of the method for preparing a cell sheet of an unfixed GFP-MOG-HEK293 living cell line are as follows: a successfully selected GFP-MOG-HEK293 cell line that stably expresses MOG is inoculated in a 6-well plate and allowed to adhere to the wall and grow to 80-90%.

10. The kit according to claim 9, characterized in that A glass slide is placed in a 6-well plate, and the cell lines grow adherently on the glass slide.

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