Autoimmune antibody detection kit based on CBA method

Abstract

The invention provides an autoimmune antibody detection kit based on a CBA method. The autoimmune antibody detection kit comprises an anti-human IgG-Alex594 fluorescent secondary antibody, phosphate buffer saline, a cell chip, a positive quality control product and a negative quality control product. The invention can be stored for a long time, and is fast and convenient to use. The batch effect caused by temporary cell transfection and cell strain construction before each time of use is avoided, and the comparability of a detection result is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to an autoimmune antibody detection kit based on the CBA method. Background Art

[0002] With the increasing demand for autoantibody detection, autoantibody detection technology is also constantly updated and developed. Indirect immunofluorescence technology (IIFT) has become the "gold standard" for autoantibody detection with its unique technical advantages, and is also recognized as the preferred method for autoantibody screening at home and abroad. According to the different detection matrices, IIFT is divided into TBA method with tissue as the matrix and CBA method with cell as the matrix. The CBA method uses target antigen transfected cells as the matrix, which retains the integrity of the antigen spectrum; at the same time, through the target antigen cell transfection technology, the target antigen with a low content can be expressed in large quantities on the cell surface in a natural conformation, greatly improving the sensitivity and specificity of specific antibody detection. However, the traditional CBA method is mostly carried out in laboratories with cell culture conditions. It is difficult for many hospitals to achieve this detection condition, and most of the traditional CBA methods need to reconstruct the plasmid transfected cell model before detection, and then carry out cell culture and detection, which greatly reduces the timeliness of the detection and cannot provide timely and effective guidance for clinical diagnosis and treatment. Summary of the invention

[0003] In view of this, the present invention provides an autoimmune antibody detection kit based on the CBA method, which can be stored for a long time and is quick and convenient to use. It avoids the batch effect caused by temporary cell transfection and construction of cell lines before each use, and improves the comparability of the test results. In addition, the kit can detect multiple indicators at the same time, saving detection time.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides an autoimmune antibody detection kit based on the CBA method, comprising: Anti-human IgG-Alex594 fluorescent secondary antibody, phosphate-buffered saline, cell array, positive control, and negative control.

[0005] Preferably, the cell chip comprises a plurality of microwells for inoculating different cell lines stably expressing the target antigen protein.

[0006] Preferably, the method for preparing the cell line stably expressing the target antigen protein is to insert the desired target antigen into the Lenti-CMV-MCS-3xFLAG-PGK-Puro vector to construct a recombinant plasmid, and after transfection, culture and screening of the recombinant plasmid, obtain a cell line stably expressing the target antigen protein.

[0007] Preferably, the concentration of the cell line stably expressing the target antigen protein inoculated in each microwell is 1x106 cells / well.

[0008] Preferably, the storage temperature of the kit is -80°C.

[0009] Preferably, the method for inoculating the cell line stably expressing the target antigen protein into the cell chip microwells is to first inoculate the cell line stably expressing the target antigen protein into the cell chip microwells, and when the cells in the cell chip microwells grow to 80-90% by adhesion, wash them twice with PBS, add formaldehyde to fix the cells, and after fixation, discard the formaldehyde fixative, wash the cells with PBS to complete the inoculation of the cell line stably expressing the target antigen protein.

[0010] Preferably, the concentration of PBS wash is 0.01M.

[0011] Preferably, when formaldehyde is added to fix the cells, the volume of formaldehyde added each time is 200 μL.

[0012] Preferably, when formaldehyde is added to fix the cells, the fixation temperature is 4° C. and the fixation time is 10 min.

[0013] Compared with the prior art, the present invention has the following beneficial effects: (2) The cell chip of the present invention has multiple micropores, which can be inoculated with one or more cell lines that stably express the target antigen protein to make a single-index cell chip or a multi-detection index cell chip. It can be flexibly matched, that is, the same chip can perform multi-indicator combined detection, thereby improving detection efficiency and meeting different clinical diagnosis and treatment needs.

[0014] (3) The kit of the present invention can be applied to the detection of autoantibodies for various autoimmune diseases according to different detection indicators, providing stable products and technical support for autoantibody detection. At the same time, the kit has a fluorescently labeled secondary antibody to provide antibody information, and the cell transfected antigen is also fluorescently labeled. The results are located and interpreted by dual fluorescence, effectively eliminating false positive results caused by serum matrix, improving detection specificity, and thus improving the accuracy of clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a schematic diagram of the cell chip provided in Example 1 of the present invention; Figure 2 A comparison chart of cell growth after the cell chip provided by the embodiment of the present invention and the traditional well plate are inoculated with the target antigen cell line; Figure 3 This is a fluorescence image of the clinical sample provided in Example 3 of the present invention, which is positive for antibodies when detected using the kit of the present invention; Figure 4This is a fluorescence image showing the absence of antibody positivity in the clinical sample provided in Example 3 of the present invention when detected using the kit of the present invention. DETAILED DESCRIPTION

[0016] 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.

[0017] Example 1: Preparation of an autoimmune antibody detection kit based on the CBA method Taking the combined detection of NMDAR, LGI1, CASPR2, GAD65, GABABR, and mGluR5 autoantibodies as an example, a kit is prepared, including the following steps: ① Obtain the above gene IDs through the GenBank website and successfully construct recombinant plasmids of different target antigens; ②HKE293 cells cultured in DMEM+10%FBS+1%P / S complete medium were transfected with GFP-NMDAR recombinant plasmid, GFP-LGI1 recombinant plasmid, GFP-CASPR2 recombinant plasmid, GFP-GAD65 recombinant plasmid, GFP-GABABR recombinant plasmid, and GFP-mGluR5 recombinant plasmid to obtain cell lines stably expressing target antigen proteins; ③ The obtained stable cell lines were divided into 1x10 6 cells / well were inoculated in the microwells of the cell chip, and different cell strains were inoculated in each well, with one replicate well for each cell strain; ④ Add 1 mL of freezing solution (90% FBS + 10% DMSO) to each of the remaining cell lines, mix well and transfer into cryopreservation tubes; place in a cell program cooling box, in a -80°C refrigerator, and transfer to a liquid nitrogen tank for storage overnight to prepare for continuous output of stable cell chip products.

[0018] ⑤ When the cells in the microwells of the cell chip have grown to 80-90% adherence, discard the culture medium, wash twice with 0.01M PBS, then add 200μL formaldehyde to each well and fix the cells at 4℃ for 10min.

[0019] ⑥ After discarding the formaldehyde fixative, wash the cells twice with 0.01M PBS, and then dry them naturally to obtain a cell chip containing NMDAR, LGI1, CASPR2, GAD65, GABABR, and mGluR5 target antigens, which can be used for the above autoimmune encephalitis antibody CBA method combined detection. The prepared cell chip can be stored at -80℃ for more than 12 months and at -20℃ for more than 1 month.

[0020] ⑦ The autoimmune antibody detection kit based on the CBA method includes the prepared cell chip products, positive quality control products, negative quality control products, sample diluent, 10x washing solution (phosphate buffered saline), anti-human IgG-Alex594 fluorescent secondary antibody, and the assembled kit can be stored at -80℃ for more than 12 months and at -20℃ for more than 1 month. Figure 1 As shown in FIG. 1 , a glass chip with a special surface modification is shown. The cell growth of the cell chip used in the present invention is compared with that of the traditional well plate after inoculation of the target antigen cell line. Figure 2 As shown, the special material of the cell chip enhances the cell's adhesion and improves the cell growth state.

[0021] Example 2 Application of the autoantibody detection kit based on the CBA method ① The kit components obtained in Example 1 were rewarmed to room temperature (about 24° C.); ② Clinical serum or cerebrospinal fluid samples to be tested are diluted with 0.01M PBS buffer, with the initial dilution ratio of serum being 1:10 and that of cerebrospinal fluid being 1:1; subsequent dilution ratios are 1:10, 1:32, 1:100, 1:320, and 1:1000.

[0022] ③ Take 30 μL of the diluted sample to be tested and add it to the corresponding microwells of the cell chip, and incubate it with the cell lines in the microwells of the cell chip. The incubation conditions are: 37°C, 45 minutes.

[0023] ④ After incubation, discard the liquid and wash twice with 0.01M PBS; then place the cell chip in a 37°C incubator to dry for 1-2 minutes for later use.

[0024] ⑤ Place the dried cell chip in a wet box, add 30 μL of the prepared fluorescent secondary antibody working solution into the microwell, and incubate in a 37°C incubator for 45 minutes. The fluorescent secondary antibody is diluted with 0.01M PBS at a ratio of 1:100.

[0025] ⑥After incubation, discard the liquid and add 0.01M PBS to wash twice.

[0026] ⑦ After slightly wiping off excess liquid from the cell chip, immediately place it under a fluorescence microscope to observe the results and collect images.

[0027] Example 3 Interpretation of microscopic examination results under a fluorescence microscope The results of the cell chip treated in Example 2 were interpreted under a microscope: ① Observe green fluorescence and red fluorescence at wavelengths EX BP 545 / 25 and EX BP 470 / 40 respectively. Green fluorescence represents GFP-target antigen protein, and red fluorescence represents antibody fluorescence in the sample to be tested. Merge 2 sets of fluorescence images to determine whether there is fluorescence co-localization, so as to determine whether the sample contains the antibody to be tested. ② If green fluorescence and red fluorescence are observed in cells under a microscope at the same time, and the green fluorescence and red fluorescence are co-localized on the cells, it is a positive result; ③If only green fluorescence but no red fluorescence is observed in the cells under a microscope, it is a negative result.

[0028] In this embodiment 2, cell chip microscopy was performed, wherein: Figure 3 In the microwells of the chip inoculated with GFP-NMDAR-HEK293 cell line, co-localization of green fluorescence and red fluorescence was observed for a cerebrospinal fluid sample. No red fluorescence was observed in the microwells of other target antigen cell lines, indicating that anti-NMDAR antibodies existed in the cerebrospinal fluid to be tested.

[0029] Figure 4 A negative result means that no red fluorescence is observed in any well of the cell chip for the sample to be tested, only antigen fluorescence.

[0030] The multiple indicators of the present invention can be flexibly combined and jointly detected, which improves the detection efficiency and contributes to efficient and accurate clinical diagnosis.

[0031] The specific raw materials not described in the present invention are all existing materials and can be directly purchased from the market.

[0032] 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. An autoimmune antibody detection kit based on the CBA method, characterized in that: include: Anti-human IgG-Alex594 fluorescent secondary antibody; Phosphate-buffered saline; Cell chips; Positive quality control; as well as, Negative control.

2. The kit according to claim 1, characterized in that The cell chip comprises a plurality of micropores for inoculating different cell lines stably expressing target antigen proteins.

3. The kit according to claim 2, characterized in that The preparation method of the cell line stably expressing the target antigen protein is to insert the required target antigen into the Lenti-CMV-MCS-3xFLAG-PGK-Puro vector to construct a recombinant plasmid, and obtain the cell line stably expressing the target antigen protein after transfection, culture and screening of the recombinant plasmid.

4. The kit according to claim 3, characterized in that The concentration of the cell line stably expressing the target antigen protein inoculated in each microwell is 1x10 6 cells / well.

5. The kit according to claim 1, characterized in that The storage temperature of the kit is -80°C.

6. The kit according to claim 2, characterized in that The method for inoculating the cell line stably expressing the target antigen protein into the microwells of the cell chip is as follows: first, the cell line stably expressing the target antigen protein is inoculated into the microwells of the cell chip, and when the cells in the microwells of the cell chip grow to 80-90% by adhesion, the cells are washed twice with PBS, and then formaldehyde is added to fix the cells. After fixation, the formaldehyde fixative is discarded, and the cells are washed with PBS to complete the inoculation of the cell line stably expressing the target antigen protein.

7. The kit according to claim 6, characterized in that The concentration of PBS for washing was 0.01M.

8. The kit according to claim 6, characterized in that When adding formaldehyde to fix the cells, the volume of formaldehyde added each time was 200 μL.

9. The kit according to claim 8, characterized in that When formaldehyde is added to fix the cells, the fixation temperature is 4°C and the fixation time is 10 minutes.

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