Method for determining anti-NMDAR antibody by using two-color fluorescence detection kit

Abstract

The invention discloses a fixing reagent and method for double-color fluorescence detection of an anti-NMDAR antibody and application, the fixing reagent comprises paraformaldehyde and a mixture of methanol and acetone, and the mixture comprises 10-25% by volume of methanol, 75-90% by volume of acetone, 10-25% by volume of paraformaldehyde, 10-25% by volume of paraformaldehyde and 10-25% by volume of paraformaldehyde. The fixing effect is better by adopting paraformaldehyde with the mass-volume ratio concentration of 4% and a mixture of methanol with the volume fraction of 15% and acetone with the volume fraction of 85% in two steps. By adopting the fixing liquid and the fixing method, the antigenicity of NMDAR cannot be damaged, obvious green fluorescence cells can be seen in a positive sample, red fluorescence quenching is avoided, and co-localization of red fluorescence and green fluorescence can be observed by using a fluorescence microscope.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a divisional application based on application number 2024114121058, filing date October 11, 2024, and invention name: Fixation reagents, methods and applications for dual-color fluorescence detection of anti-NMDAR antibodies. Technical Field

[0002] The invention belongs to the technical field of fluorescence detection, and in particular relates to a method for measuring anti-NMDAR antibodies using a dual-color fluorescence detection kit. Background Art

[0003] N-methyl-D-aspartate receptor (NMDAR) is a receptor protein complex on the nerve cell membrane. It is a subfamily of ionotropic glutamate receptors in the brain. It is related to changes in the excitability of the central nervous system. It plays a key role in mediating brain functions such as learning and memory, in chronic brain degenerative diseases such as Alzheimer's disease and Parkinson's disease, and in the pathogenesis of conditions including brain trauma and acute brain damage such as stroke. The most important condition is anti-NMDAR autoantibody-associated encephalitis. Anti-NMDAR autoantibody-associated encephalitis was originally classified as a paraneoplastic syndrome, which is a sequelae caused by the cross-reaction of the immune response caused by the tumor with the brain. Later studies found that specific anti-NMDAR autoantibodies were found in samples of patients with related symptoms. These autoantibodies act on the hippocampus of the brain, so the disease was named anti-NMDAR autoantibody-associated encephalitis. From a pathological point of view, the anti-NMDAR antibodies caused by NMDAR penetrate through the cerebral cortex to the hippocampus, resulting in a sharp decrease in NMDAR on the hippocampus. Multiple data show that anti-NMDAR antibodies penetrate into the nerve center and attack NMDAR, which will affect and regulate the progression of the disease. Anti-NMDAR autoantibody-related encephalitis has a risk of recurrence, and its recurrence is not necessarily related to the reduction of antibody concentration. It is inferred that other physiological factors are also involved in the pathogenesis. Therefore, the detection of anti-NMDAR autoantibodies is helpful in the diagnosis of paraneoplastic syndrome and other related brain diseases.

[0004] The cell transfection-based two-color immunofluorescence assay (CBA) is an indirect immunofluorescence assay based on cell transfection. Its working principle is to introduce antigen genes into mammalian cells so that the cells can specifically express the target antigen in large quantities. The antibodies (primary antibodies) in the patient specimens specifically bind to the antigens, and then the fluorescently labeled secondary antibodies are used to bind to the primary antibodies, and the results are interpreted based on the fluorescence observed under a microscope.

[0005] In the detection of anti-NMDAR autoantibodies by two-color immunofluorescence based on cell transfection, the CBA reagent fixation principles are: 1. Prevent antigen loss; 2. Permeabilize cells to allow antibodies to enter; 3. Try to keep the antigen in a state where it can bind to the antibody; 4. Maintain the normal structure of the cell. Since different fixatives have different working principles, and the fixation effect directly affects the final test results, conventional fixation schemes are not ideal for the detection of anti-NMDAR autoantibodies. Therefore, it is very necessary to find a suitable fixation scheme for the detection of anti-NMDAR autoantibodies. Summary of the invention

[0006] In order to solve one of the above problems, the present invention provides a fixation reagent, method and application for dual-color fluorescence detection of anti-NMDAR antibodies.

[0007] In order to achieve the above object, the present invention adopts the following technical means: The first aspect of the present invention provides a fixing reagent for dual-color fluorescence detection of anti-NMDAR antibodies, comprising paraformaldehyde and a mixture of methanol and acetone, wherein the volume fraction of methanol is 10-25%, and the volume fraction of acetone is 75-90%. In a specific embodiment of the present invention, preferably, the fixing reagent is a mixture of paraformaldehyde with a mass volume ratio concentration of 4%, methanol with a volume fraction of 15%, and acetone with a volume fraction of 85%.

[0008] A second aspect of the present invention provides a fixation method for dual-color fluorescence detection of anti-NMDAR antibodies, comprising the following steps: (1) fixing the transfected cell sample with 4% paraformaldehyde for 10 min and washing with PBS; (2) Fix the cell samples fixed in step (1) again with a mixture of 10-25% methanol and 75-90% acetone for 5 min and wash with PBS after fixation.

[0009] In some embodiments of the present invention, the mixture of 10-25% methanol and 75-90% acetone is pre-cooled in a -20°C refrigerator or dry ice before use.

[0010] The third aspect of the present invention provides a use of the fixing reagent for dual-color fluorescence detection of anti-NMDAR antibodies as described in the first aspect in an anti-NMDAR antibody detection kit.

[0011] The fourth aspect of the present invention provides a dual-color fluorescence detection kit for detecting anti-NMDAR antibodies, wherein the dual-color fluorescence detection kit contains the fixing reagent described in the first aspect.

[0012] Further, the detection kit also includes PBS buffer, HEK293T cells, cell culture microplates, blocking solution, fluorescent secondary antibody, and the blocking solution is serum of the species where the fluorescent secondary antibody is located. In some embodiments of the present invention, the blocking solution is goat serum, and the fluorescent secondary antibody is goat anti-human fluorescent secondary antibody. In some embodiments of the present invention, HEK293T cells express specific overexpressed antigens of anti-NMDAR antibodies.

[0013] A fifth aspect of the present invention provides a method for using a dual-color fluorescence detection kit for detecting anti-NMDAR antibodies, comprising the following steps: (1) Take the cell culture microplate with HEK293T cells integrated with the antigen gene, discard the reserved liquid in the well, add PBS buffer to wash and then discard; (2) Add the sample to be tested into the detection well of the cell culture microplate, shake gently to mix, and incubate at 37°C; (3) After incubation, remove and discard the liquid in the test wells, wash thoroughly with PBS buffer and then discard; (4) Add fluorescent secondary antibody diluted in blocking solution to the detection wells, shake gently to mix, and incubate at 37°C in the dark; (5) Repeat the washing steps in (3), discard the liquid in the wells, add PBS buffer to the detection wells until the cells are covered, then observe the green and red fluorescence of the cells under a fluorescence microscope and take pictures; (6) Observe whether the two fluorescences are co-localized and obtain the test results.

[0014] In some embodiments, the incubation time of the incubation step is 30-40 minutes, preferably 35 minutes. In some embodiments, the washing step is to remove and discard the liquid in the detection well after incubation, wash twice with PBS buffer, shake the well plate slightly during the washing process, and place it on a shaker for the third time and wash for 5 minutes.

[0015] Furthermore, in some embodiments, if the test result is positive, if titer verification is required, the sample is diluted according to the ratio and the experimental process of steps (1) to (6) is repeated.

[0016] In some embodiments of the present invention, the sample to be tested needs to be diluted with a blocking solution if it is a serum sample; and the original solution is used if it is a cerebrospinal fluid sample. In some embodiments of the present invention, the serum sample is diluted 20 times or more with the blocking solution.

[0017] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a fixative solution and a fixation method suitable for NMDAR fixation, and the use of the fixative solution and the fixation method will not damage the antigenicity of NMDAR. In positive samples, obvious green fluorescent cells can be seen without red fluorescence quenching, and co-localization of red fluorescence and green fluorescence can be observed using a fluorescence microscope. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A graph showing the fluorescence detection results of positive samples and negative samples in the scheme of Example 1 of the present invention is shown; Figure 2 A graph showing the fluorescence detection results of positive samples and negative samples in the scheme of Example 2 of the present invention; Figure 3 A graph showing the fluorescence detection results of positive samples and negative samples in the solution of Example 3 of the present invention is shown; Figure 4 A graph showing the fluorescence detection results of positive samples and negative samples in the scheme of Example 4 of the present invention; Figure 5 A graph showing the fluorescence detection results of positive samples and negative samples in the scheme of Example 5 of the present invention; Figure 6 A graph showing the fluorescence detection results of positive samples and negative samples in the scheme of Example 6 of the present invention; Figure 7 A graph showing the fluorescence detection results of positive samples and negative samples in Example 7 of the present invention; Figure 8 A graph showing the fluorescence detection results of positive samples and negative samples in the scheme of Example 8 of the present invention is shown; Fig. 9 A graph showing the fluorescence detection results of positive samples and negative samples in the scheme of Example 9 of the present invention; Fig.10 A graph showing the fluorescence detection results of positive samples and negative samples in the scheme of Example 10 of the present invention; Fig.11 A graph showing the fluorescence detection results of positive samples and negative samples in the scheme of Example 11 of the present invention; Fig.12 A graph showing the fluorescence detection results of positive samples and negative samples in the scheme of Example 12 of the present invention; Fig.13 A graph showing the fluorescence detection results of positive samples and negative samples in the scheme of Example 13 of the present invention; Fig.14 A graph showing the fluorescence detection results of positive samples and negative samples in the scheme of Example 14 of the present invention; Among them, red fluorescence is antigen, green fluorescence is antibody, and overlapping fluorescence is co-localization of antigen fluorescence and antibody fluorescence. DETAILED DESCRIPTION

[0019] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0021] The technical solution of the present application is further described in detail below in conjunction with specific implementation methods. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The instruments and equipment used in the following embodiments are all conventional laboratory instruments and equipment unless otherwise specified; the test materials used in the following embodiments are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0022] The HEK293T cells integrated with antigen genes and transfected cells involved in the following examples are cultured in cell culture microplates using conventional methods.

[0023] Example 1 A. Fixation Method 1. Prepare 4% paraformaldehyde in PBS; 2. Wash the 96-well cell culture microplate with transfected cells twice with PBS; 3. Fix with 4% paraformaldehyde for 30 minutes; 4. Wash twice with PBS; 5. Add 200 μL PBS as a cell protectant and store in a vacuum; B. Performance Test (1) Inspection principle The detection principle of this embodiment is indirect immunofluorescence. The antibody corresponding to NMDAR in the sample forms an antigen-antibody complex with the specific overexpressed antigen expressed on HEK293T cells in the reagent. This antigen carries mCherry fluorescent protein (red fluorescence). Then, goat anti-human fluorescent secondary antibody (green fluorescence) is used to label the human secondary antibody bound to the antigen. Finally, a fluorescence microscope is used to observe whether the red fluorescence and the green fluorescence are co-localized. If there is co-localization, it means that the sample contains the antibody, otherwise the sample does not contain the corresponding antibody.

[0024] (2) Inspection method Preparation: 1. Preparation of PBS buffer: dilute 10 times PBS buffer with pure water or distilled water.

[0025] 2. Preparation of blocking solution: dilute goat serum 10 times with PBS buffer.

[0026] 3. Sample: If it is a serum sample: dilute it at least 20 times with blocking solution; if it is a cerebrospinal fluid sample, use the original solution.

[0027] 4. Fluorescent secondary antibody: dilute 1500 times with blocking solution.

[0028] Inspection steps: 1. Pre-wash: Take out the cell culture microplate and discard the remaining liquid in the well. Add 200 µL PBS buffer to wash once and discard the liquid in the well again.

[0029] 2. Sample addition: Add 80ul diluted serum or cerebrospinal fluid to the test well. Shake gently to mix, and incubate at 37℃ for 35min.

[0030] 3. Washing: After incubation, remove the wells, discard the liquid in the test wells, and add 200 µL PBS buffer to wash twice. Slightly shake the wells during the washing process. Place the wells on a shaker for the third time and wash for 5 min.

[0031] 4. Add fluorescent secondary antibody: discard the liquid in the well, add 80 µL of diluted fluorescent secondary antibody to the detection well, shake gently to mix, and incubate at 37℃ in the dark for 35 minutes.

[0032] 5. Cleaning: Repeat step 3.

[0033] 6. Observation: Discard the liquid in the wells, add 100 µL of PBS buffer to the sample wells to cover the cells, then observe the red fluorescence of the cells under a fluorescence microscope and take photos.

[0034] 7. Observe whether the two fluorescences are co-localized to obtain the test results. If the positive result needs to be titered, dilute the sample according to the proportion and repeat the experimental process from steps 1 to 7.

[0035] (3) Test samples Positive sample: Enterprise positive reference product.

[0036] Negative sample: enterprise negative reference.

[0037] (4) Interpretation of results Interpretation criteria Negative: No green fluorescent cells were observed in the green fluorescent channel or no overlap between green fluorescent cells and red fluorescence was observed after software synthesis.

[0038] Positive: Green fluorescent cells can be seen in the green fluorescence channel, and overlap with the red antigen fluorescence can be observed through software synthesis.

[0039] The results are as follows Figure 1 shown.

[0040] The results showed that no green fluorescent cells were visible in the positive samples, so the 4% paraformaldehyde fixation method could not be used for NMDAR fixation because 4% paraformaldehyde damaged the antigenicity of NMDAR.

[0041] Example 2 The other steps are the same as those in Example 1, except for the fixing method: 1. Pre-cool pure acetone in a -20℃ refrigerator or dry ice; 2. Wash the 96-well cell culture microplate with transfected cells twice with PBS; 3. Fix with pre-cooled acetone for 5 minutes; 4. Wash twice with PBS; 5. Add 200 μL PBS as a cell protectant and store in a vacuum; The results are as follows Figure 2 shown.

[0042] The results showed that in the positive samples, obvious green fluorescent cells were visible, but the red fluorescence was quenched. Therefore, the pure acetone fixation method cannot be used for NMDAR fixation because pure acetone does not damage the antigenicity of NMDAR, but will cause the red fluorescence indicating antigen expression to be quenched through some reaction.

[0043] Example 3 The other steps are the same as those in Example 1, except for the fixing method: (1) Paraformaldehyde fixation 1. Prepare 4% paraformaldehyde in PBS; 2. Wash the 96-well cell culture microplate with transfected cells twice with PBS; 3. Fix with 4% paraformaldehyde for 10 min; 4. Wash twice with PBS; (2) Pure methanol fixation 1. Pre-cool pure methanol in a -20℃ refrigerator or dry ice; 2. Fix the cell culture microplate fixed in step (1) with pre-cooled pure methanol for 5 minutes; 3. Wash twice with PBS; 4. Add 200 μL PBS as a cell protectant and store in a vacuum; The results are as follows Figure 3 shown.

[0044] The results showed that in the positive samples, no visible green fluorescent cells appeared and no red fluorescence was quenched, so the combination of 4% paraformaldehyde and methanol could not be used for NMDAR fixation because it damaged the antigenicity of NMDAR.

[0045] Example 4 The other steps are the same as those in Example 1, except for the fixing method: (1) Paraformaldehyde fixation 1. Prepare 4% paraformaldehyde in PBS; 2. Wash the 96-well cell culture microplate with transfected cells twice with PBS; 3. Fix with 4% paraformaldehyde for 10 min; 4. Wash twice with PBS; (2) Fixation with a mixture of 95% methanol and 5% acetone 1. Prepare a mixture of 95% methanol and 5% acetone in advance and pre-cool it in a -20℃ refrigerator or dry ice; 2. Fix the cell culture microplate fixed in step (1) with a pre-cooled mixture of 95% methanol and 5% acetone for 5 minutes; 3. Wash twice with PBS; 4. Add 200 μL PBS as a cell protectant and store in a vacuum; The results are as follows Figure 4 shown.

[0046] The results showed that in the positive samples, no visible green fluorescent cells appeared and no red fluorescence was quenched, so the combination of 4% paraformaldehyde and a mixture of 95% methanol and 5% acetone could not be used for NMDAR fixation because it damaged the antigenicity of NMDAR.

[0047] Example 5 The other steps are the same as those in Example 1, except for the fixing method: (1) Paraformaldehyde fixation 1. Prepare 4% paraformaldehyde in PBS; 2. Wash the 96-well cell culture microplate with transfected cells twice with PBS; 3. Fix with 4% paraformaldehyde for 10 min; 4. Wash twice with PBS; (2) Fixation with a mixture of 85% methanol and 15% acetone 1. Prepare a mixture of 85% methanol and 15% acetone in advance and precool it in a -20℃ refrigerator or dry ice; 2. Fix the cell culture microplate fixed in step (1) with a pre-cooled mixture of 85% methanol and 15% acetone for 5 minutes; 3. Wash twice with PBS; 4. Add 200 μL PBS as a cell protectant and store in a vacuum; The results are as follows Figure 5 shown.

[0048] The results showed that in the positive samples, no visible green fluorescent cells appeared and no red fluorescence was quenched. Therefore, the combination of 4% paraformaldehyde and a mixture of 85% methanol and 15% acetone could not be used for NMDAR fixation because it damaged the antigenicity of NMDAR.

[0049] Example 6 The other steps are the same as those in Example 1, except for the fixing method: (1) Paraformaldehyde fixation 1. Prepare 4% paraformaldehyde in PBS; 2. Wash the 96-well cell culture microplate with transfected cells twice with PBS; 3. Fix with 4% paraformaldehyde for 10 min; 4. Wash twice with PBS; (2) Fixation with a mixture of 75% methanol and 25% acetone 1. Prepare a mixture of 75% methanol and 25% acetone in advance and pre-cool it in a -20℃ refrigerator or dry ice; 2. Fix the cell culture microplate fixed in step (1) with a pre-cooled mixture of 75% methanol and 25% acetone for 5 minutes; 3. Wash twice with PBS; 4. Add 200 μL PBS as a cell protectant and store in a vacuum; The results are as follows Figure 6 shown.

[0050] The results showed that in the positive samples, no visible green fluorescent cells appeared and no red fluorescence was quenched, so the combination of 4% paraformaldehyde and a mixture of 75% methanol and 25% acetone could not be used for NMDAR fixation because it damaged the antigenicity of NMDAR.

[0051] Example 7 The other steps are the same as those in Example 1, except for the fixing method: (1) Paraformaldehyde fixation 1. Prepare 4% paraformaldehyde in PBS; 2. Wash the 96-well cell culture microplate with transfected cells twice with PBS; 3. Fix with 4% paraformaldehyde for 10 min; 4. Wash twice with PBS; (2) Fixation with a mixture of 65% methanol and 35% acetone 1. Prepare a mixture of 65% methanol and 35% acetone in advance and pre-cool it in a -20℃ refrigerator or dry ice; 2. Fix the cell culture microplate fixed in step (1) with a pre-cooled mixture of 65% methanol and 35% acetone for 5 minutes; 3. Wash twice with PBS; 4. Add 200 μL PBS as a cell protectant and store in a vacuum; The results are as follows Figure 7 shown.

[0052] The results showed that in the positive samples, no visible green fluorescent cells appeared and no red fluorescence was quenched, so the combination of 4% paraformaldehyde and a mixture of 65% methanol and 35% acetone could not be used for NMDAR fixation because it damaged the antigenicity of NMDAR.

[0053] Example 8 The other steps are the same as those in Example 1, except for the fixing method: (1) Paraformaldehyde fixation 1. Prepare 4% paraformaldehyde in PBS; 2. Wash the 96-well cell culture microplate with transfected cells twice with PBS; 3. Fix with 4% paraformaldehyde for 10 min; 4. Wash twice with PBS; (2) Fixation with a mixture of 55% methanol and 45% acetone 1. Prepare a mixture of 55% methanol and 45% acetone in advance and pre-cool it in a -20℃ refrigerator or dry ice; 2. Fix the cell culture microplate fixed in step (1) with a pre-cooled mixture of 55% methanol and 45% acetone for 5 minutes; 3. Wash twice with PBS; 4. Add 200 μL PBS as a cell protectant and store in a vacuum; The results are as follows Figure 8 shown.

[0054] The results showed that in the positive samples, no visible green fluorescent cells appeared and no red fluorescence was quenched. Therefore, the combination of 4% paraformaldehyde and a mixture of 55% methanol and 45% acetone could not be used for NMDAR fixation because it damaged the antigenicity of NMDAR.

[0055] Example 9 The other steps are the same as those in Example 1, except for the fixing method: (1) Paraformaldehyde fixation 1. Prepare 4% paraformaldehyde in PBS; 2. Wash the 96-well cell culture microplate with transfected cells twice with PBS; 3. Fix with 4% paraformaldehyde for 10 min; 4. Wash twice with PBS; (2) Fixation with a mixture of 45% methanol and 55% acetone 1. Prepare a mixture of 45% methanol and 55% acetone in advance and precool it in a -20℃ refrigerator or dry ice; 2. Fix the cell culture microplate fixed in step (1) with a pre-cooled mixture of 45% methanol and 55% acetone for 5 minutes; 3. Wash twice with PBS; 4. Add 200 μL PBS as a cell protectant and store in a vacuum; The results are as follows Fig. 9 shown.

[0056] The results showed that in the positive samples, no visible green fluorescent cells appeared and no red fluorescence was quenched, so the combination of 4% paraformaldehyde and a mixture of 45% methanol and 55% acetone could not be used for NMDAR fixation because it damaged the antigenicity of NMDAR.

[0057] Example 10 The other steps are the same as those in Example 1, except for the fixing method: (1) Paraformaldehyde fixation 1. Prepare 4% paraformaldehyde in PBS; 2. Wash the 96-well cell culture microplate with transfected cells twice with PBS; 3. Fix with 4% paraformaldehyde for 10 min; 4. Wash twice with PBS; (2) Fixation with a mixture of 35% methanol and 65% acetone 1. Prepare a mixture of 35% methanol and 65% acetone in advance and pre-cool it in a -20℃ refrigerator or dry ice; 2. Fix the cell culture microplate fixed in step (1) with a pre-cooled mixture of 35% methanol and 65% acetone for 5 minutes; 3. Wash twice with PBS; 4. Add 200 μL PBS as a cell protectant and store in a vacuum; The results are as follows Fig.10 shown.

[0058] The results showed that in the positive samples, no visible green fluorescent cells appeared and no red fluorescence was quenched. Therefore, the combination of 4% paraformaldehyde and a mixture of 35% methanol and 65% acetone could not be used for NMDAR fixation because it damaged the antigenicity of NMDAR.

[0059] Embodiment 11 The other steps are the same as those in Example 1, except for the fixing method: (1) Paraformaldehyde fixation 1. Prepare 4% paraformaldehyde in PBS; 2. Wash the 96-well cell culture microplate with transfected cells twice with PBS; 3. Fix with 4% paraformaldehyde for 10 min; 4. Wash twice with PBS; (2) Fixation with a mixture of 25% methanol and 75% acetone 1. Prepare a mixture of 25% methanol and 75% acetone in advance and pre-cool it in a -20℃ refrigerator or dry ice; 2. Fix the cell culture microplate fixed in step (1) with a pre-cooled mixture of 25% methanol and 75% acetone for 5 minutes; 3. Wash twice with PBS; 4. Add 200 μL PBS as a cell protectant and store in a vacuum; The results are as follows Fig.11 shown.

[0060] The results showed that in the positive samples, weak green fluorescent cells were visible, and there was no quenching of red fluorescence. Therefore, the combination of 4% paraformaldehyde, 25% methanol and 75% acetone mixture can be used for NMDAR fixation, but it is not the best choice.

[0061] Example 12 The other steps are the same as those in Example 1, except for the fixing method: (1) Paraformaldehyde fixation 1. Prepare 4% paraformaldehyde in PBS; 2. Wash the 96-well cell culture microplate with transfected cells twice with PBS; 3. Fix with 4% paraformaldehyde for 10 min; 4. Wash twice with PBS; (2) Fixation with a mixture of 15% methanol and 85% acetone 1. Prepare a mixture of 15% methanol and 85% acetone in advance and pre-cool it in a -20℃ refrigerator or dry ice; 2. Fix the cell culture microplate fixed in step (1) with a pre-cooled mixture of 15% methanol and 85% acetone for 5 minutes; 3. Wash twice with PBS; 4. Add 200 μL PBS as a cell protectant and store in a vacuum; The results are as follows Fig.12 shown.

[0062] The results showed that in the positive samples, obvious green fluorescent cells were visible, and there was no red fluorescence quenching. Therefore, the combination of 4% paraformaldehyde, 15% methanol and 85% acetone mixture can be used for NMDAR fixation and is a better choice.

[0063] Embodiment 13 The other steps are the same as those in Example 1, except for the fixing method: (1) Paraformaldehyde fixation 1. Prepare 4% paraformaldehyde in PBS; 2. Wash the 96-well cell culture microplate with transfected cells twice with PBS; 3. Fix with 4% paraformaldehyde for 10 min; 4. Wash twice with PBS; (2) Fixation with a mixture of 10% methanol and 90% acetone 1. Prepare a mixture of 10% methanol and 90% acetone in advance and pre-cool it in a -20℃ refrigerator or dry ice; 2. Fix the cell culture microplate fixed in step (1) with a pre-cooled mixture of 10% methanol and 90% acetone for 5 minutes; 3. Wash twice with PBS; 4. Add 200 μL PBS as a cell protectant and store in a vacuum; The results are as follows Fig.13 shown.

[0064] The results showed that in the positive samples, weak green fluorescent cells were visible, and there was no quenching of red fluorescence. Therefore, the combination of 4% paraformaldehyde, 10% methanol and 90% acetone mixture can be used for NMDAR fixation, but it is not the best choice.

[0065] Embodiment 14 The other steps are the same as those in Example 1, except for the fixing method: (1) Paraformaldehyde fixation 1. Prepare 4% paraformaldehyde in PBS; 2. Wash the 96-well cell culture microplate with transfected cells twice with PBS; 3. Fix with 4% paraformaldehyde for 10 min; 4. Wash twice with PBS; (2) Fixation with a mixture of 5% methanol and 95% acetone 1. Prepare a mixture of 5% methanol and 95% acetone in advance and pre-cool it in a -20℃ refrigerator or dry ice; 2. Fix the cell culture microplate fixed in step (1) with a pre-cooled mixture of 5% methanol and 95% acetone for 5 minutes; 3. Wash twice with PBS; 4. Add 200 μL PBS as a cell protectant and store in a vacuum; The results are as follows Fig.14 shown.

[0066] The results showed that in the positive samples, no visible green fluorescent cells appeared and no red fluorescence was quenched, so the combination of 4% paraformaldehyde, 5% methanol and 95% acetone mixture could not be used for NMDAR fixation because it damaged the antigenicity of NMDAR.

[0067] In summary, in the dual-color immunofluorescence method based on cell transfection to detect anti-NMDAR antibodies, the fixing reagent is a mixture of paraformaldehyde and methanol and acetone, and the volume fraction of methanol in the mixture is 10-25%, and the volume fraction of acetone is 75-90%. Preferably, the fixing reagent is a mixture of paraformaldehyde with a mass volume ratio of 4%, methanol with a volume fraction of 15%, and acetone with a volume fraction of 85%. And the fixing step is carried out in two steps, as follows: (1) the transfected cell sample is fixed with 4% paraformaldehyde for 10 min and washed with PBS; (2) the cell sample fixed in step (1) is fixed again with a mixture of 10-25% methanol and 75-90% acetone for 5 min, and washed with PBS after fixation. The use of this fixing solution and fixing method will not damage the antigenicity of NMDAR. In the positive sample, obvious green fluorescent cells can be seen, and there is no red fluorescence quenching. The co-localization of red fluorescence and green fluorescence can be observed using a fluorescence microscope.

[0068] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined in the present application.

Claims

1. A method for measuring anti-NMDAR antibodies using a dual-color fluorescence detection kit, characterized in that: The method for determining anti-NMDAR antibodies is for non-diagnostic and non-therapeutic purposes and comprises the following steps: (1) HEK293T cells with integrated antigen genes are added to a cell culture microplate. After adherence culture, a fixation reagent is added to fix the cells. PBS buffer is added to the cell culture microplate for pre-washing and then discarded; (2) Add the sample to be tested into the detection well of the cell culture microplate, shake gently to mix, and incubate at 37°C; (3) After incubation, remove and discard the liquid in the test wells, wash thoroughly with PBS buffer and then discard; (4) Add fluorescent secondary antibody diluted in blocking solution to the detection wells, shake gently to mix, and incubate at 37°C in the dark; (5) Repeat the washing steps in (3), discard the liquid in the wells, add PBS buffer to the detection wells until the cells are covered, then observe the green and red fluorescence of the cells under a fluorescence microscope and take pictures; (6) Observe whether the two fluorescences are co-localized and obtain the test results; The fixing agent used for cell fixation in step (1) comprises a mixture of paraformaldehyde with a mass volume ratio of 4%, methanol and acetone, wherein the volume fraction of methanol is 10-25% and the volume fraction of acetone is 75-90%.

2. The method according to claim 1, characterized in that: The fixing agent is a mixture of 4% paraformaldehyde in a mass volume ratio, 15% methanol and 85% acetone in a volume fraction.

3. The method according to claim 1, characterized in that: The cell fixation comprises the following steps: (1) After adherent culture, the cell samples were fixed with 4% paraformaldehyde for 10 min and washed with PBS; (2) Fix the cell samples fixed in step (1) again with a mixture of 10-25% methanol and 75-90% acetone for 5 min and wash with PBS after fixation.

4. The method according to claim 3, characterized in that: The mixture of 10-25% methanol and 75-90% acetone was precooled in a -20°C refrigerator or dry ice before use.

5. The method according to claim 1, characterized in that: The incubation time in step (2) is 30-40 minutes.

6. The method according to claim 1, characterized in that: If the test result is positive, if the titer of the positive sample needs to be verified, the sample is diluted according to the ratio and the experimental process of steps (1) to (6) is repeated.

7. The method according to claim 1, characterized in that: If the sample to be tested is a serum sample, it needs to be diluted with a blocking solution; if it is a cerebrospinal fluid sample, the original solution is used.

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