A method for determining anti-NMDAR antibodies using a two-color fluorescent test kit

Abstract

The application discloses a double-color fluorescence detection anti-NMDAR antibody fixing reagent, a method and application thereof, and the fixing reagent comprises a mixture of paraformaldehyde, methanol and acetone, wherein the volume fraction of methanol in the mixture is 10-25%, and the volume fraction of acetone is 75-90%; the fixing effect is better when the fixing is carried out in two steps by using 4% paraformaldehyde in mass-volume concentration and the mixture of 15% methanol and 85% acetone in volume fraction. By using the fixing liquid and the fixing method, the antigenicity of NMDAR is not damaged, obvious green fluorescent cells can be observed in positive samples, red fluorescence is not quenched, and co-localization of red fluorescence and green fluorescence can be observed by using a fluorescence microscope.

Description

[0001] Cross-references to related applications

[0002] This application is a divisional application based on application number 2024114121058, filed on October 11, 2024, entitled "Immobilization Reagent, Method and Application for Dual-Color Fluorescence Detection of Anti-NMDAR Antibody". Technical Field

[0003] This invention belongs to the field of fluorescence detection technology, specifically relating to a method for determining anti-NMDAR antibodies using a dual-color fluorescence detection kit. Background Technology

[0004] N-methyl-D-aspartate receptors (NMDARs) are receptor-protein complexes on nerve cell membranes, belonging to a subfamily of ionotropic glutamate receptors in the brain. They are associated with changes in central nervous system excitability and play a crucial role in mediating brain functions such as learning and memory, in the pathogenesis of chronic neurodegenerative diseases such as Alzheimer's and Parkinson's, and in conditions including traumatic brain injury and acute brain injury such as stroke. The most prominent manifestation of this condition is anti-NMDAR autoantibody-associated encephalitis (ADI). Initially classified as a paraneoplastic syndrome, ADI was a sequela of a cross-reaction between an immune response to a tumor and the brain. Later studies revealed specific anti-NMDAR autoantibodies in samples from patients exhibiting related symptoms. These autoantibodies act on the hippocampus, hence the name anti-NMDAR autoantibody-associated encephalitis. From a pathological perspective, anti-NMDAR antibodies induced by NMDAR penetrate the cerebral cortex to the hippocampus, leading to a sharp decrease in NMDAR levels in the hippocampus. Multiple studies have shown that the infiltration of anti-NMDAR antibodies into the central nervous system and their attack on NMDAR can influence and regulate disease progression. Anti-NMDAR autoantibody-related encephalitis has a relapse risk, but relapse is not necessarily related to a decrease in antibody concentration, suggesting that other physiological factors also play a role in the pathogenesis. Therefore, the detection of anti-NMDAR autoantibodies is helpful in diagnosing parametroma syndrome and other related neurological diseases.

[0005] Cell transfection-based two-color immunofluorescence assay (CBA) is an indirect immunofluorescence assay based on cell transfection. Its working principle is to introduce the antigen gene into mammalian cells, causing the cells to express the target antigen in large quantities. The antibody (primary antibody) in the patient sample binds specifically to the antigen. Then, the primary antibody is bound to the fluorescently labeled secondary antibody, and the result is interpreted based on the fluorescence observed under a microscope.

[0006] In the detection of anti-NMDAR autoantibodies using a cell transfection-based two-color immunofluorescence assay, the CBA reagent fixation principles are: 1. Prevent antigen loss; 2. Permeabilize cells to allow antibody entry; 3. Maintain the antigen in a state where it can bind to the antibody; 4. Maintain normal cell structure. Since different fixatives have different mechanisms of action, and the fixation effect directly affects the final detection results, conventional fixation protocols are not ideal for the detection of anti-NMDAR autoantibodies. Therefore, finding a suitable fixation protocol is essential for the detection of anti-NMDAR autoantibodies. Summary of the Invention

[0007] To address one of the aforementioned problems, this invention provides a fixation reagent, method, and application for dual-color fluorescence detection of anti-NMDAR antibodies.

[0008] To achieve the above objectives, the present invention employs the following technical means:

[0009] A first aspect of the present invention provides a fixation reagent for dual-color fluorescence detection of anti-NMDAR antibodies, comprising paraformaldehyde and a mixture of methanol and acetone, wherein the methanol volume fraction is 10-25% and the acetone volume fraction is 75-90%. In a specific embodiment of the present invention, preferably, the fixation reagent is a mixture of paraformaldehyde at a mass-to-volume concentration of 4%, methanol at a volume fraction of 15%, and acetone at a volume fraction of 85%.

[0010] The 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 transfected cell samples with 4% paraformaldehyde for 10 min and washing with PBS;

[0011] (2) Fix the cell samples fixed in step (1) again with a mixture of 10-25% methanol and 75-90% acetone for 5 min. After fixation, wash with PBS.

[0012] 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.

[0013] A third aspect of the present invention provides the application of the immobilization reagent for dual-color fluorescence detection of anti-NMDAR antibodies as described in the first aspect in an anti-NMDAR antibody detection kit.

[0014] A fourth aspect of the present invention provides a two-color fluorescence detection kit for detecting anti-NMDAR antibodies, the two-color fluorescence detection kit containing the immobilization reagent described in the first aspect.

[0015] Furthermore, the detection kit also includes PBS buffer, HEK293T cells, cell culture microplates, blocking solution, and fluorescent secondary antibody, wherein the blocking solution is serum of the species for which the fluorescent secondary antibody is present. 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 a specific overexpression antigen against NMDAR antibody.

[0016] A fifth aspect of the present invention provides a method of using a two-color fluorescence detection kit for detecting anti-NMDAR antibodies, comprising the following steps:

[0017] (1) Take the cell culture microplates in which HEK293T cells with integrated antigen genes adhere, discard the liquid in the wells, add PBS buffer to wash and then discard it;

[0018] (2) Add the sample to be tested into the well of the cell culture microplate, shake gently to mix, and incubate at 37°C;

[0019] (3) After incubation, remove and discard the liquid in the detection well, wash thoroughly with PBS buffer and then discard;

[0020] (4) Add the fluorescent secondary antibody diluted with blocking solution to the detection well, shake gently to mix, and incubate at 37°C in the dark;

[0021] (5) Repeat the cleaning steps in (3), discard the liquid in the well, add PBS buffer to the detection well until it covers the cells, and then observe the green and red fluorescence of the cells under a fluorescence microscope and take pictures.

[0022] (6) Observe whether the two fluorescences co-localize and obtain the test results.

[0023] In some embodiments, the incubation time for the above incubation step is 30-40 minutes, preferably 35 minutes. In some embodiments, the washing step involves removing and discarding the liquid in the test wells after incubation, washing twice with PBS buffer while gently shaking the plate during the washing process, and then placing it on a shaker for a third time for 5 minutes.

[0024] Furthermore, in some implementations, if the test result is positive, and titer verification is required, the sample is diluted proportionally and the experimental process of steps (1) to (6) is repeated.

[0025] In some embodiments of the present invention, 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 undiluted solution is used. In some embodiments of the present invention, the serum sample is diluted 20 times or more with the blocking solution.

[0026] Beneficial effects of the present invention

[0027] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a fixative and fixation method suitable for NMDAR fixation. Using this fixative and fixation method, the antigenicity of NMDAR will not be damaged. In positive samples, obvious green fluorescent cells can be seen, and there is no red fluorescence quenching. Using a fluorescence microscope, co-localization of red fluorescence and green fluorescence can be observed. Attached Figure Description

[0028] Figure 1 The diagram shows the fluorescence detection results of positive and negative samples in the scheme of Embodiment 1 of the present invention;

[0029] Figure 2 The following diagram shows the fluorescence detection results of positive and negative samples in the scheme of Embodiment 2 of the present invention;

[0030] Figure 3 The following diagram shows the fluorescence detection results of positive and negative samples in the scheme of Embodiment 3 of the present invention;

[0031] Figure 4 The diagram shows the fluorescence detection results of positive and negative samples in the scheme of Embodiment 4 of the present invention;

[0032] Figure 5 The diagram shows the fluorescence detection results of positive and negative samples in the scheme of Embodiment 5 of the present invention;

[0033] Figure 6 The following diagram shows the fluorescence detection results of positive and negative samples in the scheme of Embodiment 6 of the present invention;

[0034] Figure 7 The diagram shows the fluorescence detection results of positive and negative samples in the scheme of Embodiment 7 of the present invention;

[0035] Figure 8 The diagram shows the fluorescence detection results of positive and negative samples in the scheme of Embodiment 8 of the present invention;

[0036] Figure 9 The diagram shows the fluorescence detection results of positive and negative samples in the scheme of Embodiment 9 of the present invention;

[0037] Figure 10 The diagram shows the fluorescence detection results of positive and negative samples in the scheme of Embodiment 10 of the present invention;

[0038] Figure 11 The diagram shows the fluorescence detection results of positive and negative samples in the scheme of Embodiment 11 of the present invention;

[0039] Figure 12The diagram shows the fluorescence detection results of positive and negative samples in the scheme of Embodiment 12 of the present invention;

[0040] Figure 13 The diagram shows the fluorescence detection results of positive and negative samples in the scheme of Embodiment 13 of the present invention;

[0041] Figure 14 The diagram shows the fluorescence detection results of positive and negative samples in the scheme of Embodiment 14 of the present invention;

[0042] In this diagram, red fluorescence represents the antigen, green fluorescence represents the antibody, and overlapping fluorescence indicates co-localization of antigen and antibody fluorescence. Detailed Implementation

[0043] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.

[0044] 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 this invention pertains, and all materials disclosed herein and cited therein are incorporated herein by reference. Many equivalent techniques of specific embodiments of the invention described herein will be recognized or can be understood by ordinary experimentation by those skilled in the art. These equivalents will be included in the claims.

[0045] The technical solution of this application will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the instruments and equipment used in the following embodiments are conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0046] The HEK293T cells with integrated antigen genes and the transfected cells in the cell culture microplates used in the following examples all employed conventional methods.

[0047] Example 1

[0048] A. Fixing method

[0049] 1. Prepare 4% paraformaldehyde solution using PBS;

[0050] 2. Wash the 96-well cell culture microplates with transfected cells twice with PBS;

[0051] 3. Fix with 4% paraformaldehyde for 30 minutes;

[0052] 4. Wash twice with PBS;

[0053] 5. Add 200 μL of PBS as a cell protectant and store under vacuum;

[0054] B. Performance Testing

[0055] (1) Testing principle

[0056] 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). Subsequently, the human secondary antibody bound to the antigen is labeled with goat anti-human fluorescent secondary antibody (green fluorescence). Finally, the co-localization of red fluorescence and green fluorescence is observed using a fluorescence microscope. If co-localization exists, it indicates that the sample contains the antibody; otherwise, the sample does not contain the corresponding antibody.

[0057] (2) Test methods

[0058] Preparation:

[0059] 1. Preparation of PBS buffer: Dilute 10 times the amount of PBS buffer with pure water or distilled water.

[0060] 2. Preparation of blocking solution: Dilute goat serum 10 times with PBS buffer.

[0061] 3. Samples: If it is a serum sample, dilute it at least 20 times with blocking solution; if it is a cerebrospinal fluid sample, use the undiluted solution.

[0062] 4. Fluorescent secondary antibody: Dilute 1500 times with blocking solution.

[0063] Inspection steps:

[0064] 1. Pre-wash: Remove the cell culture microplate and discard the liquid remaining in the wells. Add 200 µL of PBS buffer and wash once, then discard the liquid in the wells again.

[0065] 2. Sample addition: Add 80 μL of diluted serum or cerebrospinal fluid stock solution to the test well. Gently shake to mix, and incubate at 37°C for 35 min.

[0066] 3. Washing: After incubation, remove the plate, discard the liquid in the wells, and wash twice with 200 µL PBS buffer. Gently shake the plate during the washing process. For the third wash, place the plate on a shaker and wash for 5 min.

[0067] 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°C in the dark for 35 min.

[0068] 5. Cleaning: Repeat step 3.

[0069] 6. Observation: Discard the liquid in the well, add 100 µL of PBS buffer to the sample well to cover the cells, and then observe the red fluorescence of the cells under a fluorescence microscope and take pictures.

[0070] 7. Observe whether the two fluorescences co-localize to obtain the test result. If a positive result requires titer verification, dilute the sample proportionally and repeat steps 1 to 7.

[0071] (3) Test samples

[0072] Positive sample: Positive reference material for enterprises.

[0073] Negative sample: Negative reference material for enterprises.

[0074] (4) Result Interpretation

[0075] Interpretation criteria

[0076] Negative: No green fluorescent cells were observed in the green fluorescence channel or no overlap of green fluorescent cells with red fluorescence was observed after software synthesis.

[0077] Positive: Green fluorescent cells are visible under the green fluorescent channel, and can be observed to overlap with the red antigen fluorescence after software synthesis.

[0078] The interpretation result is as follows Figure 1 As shown.

[0079] The results showed that no visible green fluorescent cells appeared in the positive samples. Therefore, the 4% paraformaldehyde fixation method cannot be used for NMDAR fixation because 4% paraformaldehyde damages the antigenicity of NMDAR.

[0080] Example 2

[0081] The other steps are the same as in Example 1, except for the fixing method:

[0082] 1. Pre-cool pure acetone in a -20℃ refrigerator or dry ice;

[0083] 2. Wash the 96-well cell culture microplates with transfected cells twice with PBS;

[0084] 3. Fix with pre-cooled acetone for 5 minutes;

[0085] 4. Wash twice with PBS;

[0086] 5. Add 200 μL of PBS as a cell protectant and store under vacuum;

[0087] The interpretation result is as follows Figure 2 As shown.

[0088] The results showed that in 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 although pure acetone does not damage the antigenicity of NMDAR, it will cause the red fluorescence indicating antigen expression to be quenched through some reaction.

[0089] Example 3

[0090] The other steps are the same as in Example 1, except for the fixing method:

[0091] (1) Paraformaldehyde fixation

[0092] 1. Prepare 4% paraformaldehyde solution using PBS;

[0093] 2. Wash the 96-well cell culture microplates with transfected cells twice with PBS;

[0094] 3. Fix with 4% paraformaldehyde for 10 min;

[0095] 4. Wash twice with PBS;

[0096] (2) Fixation with pure methanol

[0097] 1. Pre-cool the pure methanol in a -20℃ refrigerator or dry ice in advance;

[0098] 2. Fix the cell culture microplates fixed in step (1) with pre-cooled pure methanol for 5 minutes;

[0099] 3. Wash twice with PBS;

[0100] 4. Add 200 μL of PBS as a cell protectant and store under vacuum;

[0101] The interpretation result is as follows Figure 3 As shown.

[0102] The results showed that no visible green fluorescent cells appeared in the positive samples, and no red fluorescence was quenched. Therefore, the combination of 4% paraformaldehyde and methanol cannot be used for NMDAR fixation because it damages the antigenicity of NMDAR.

[0103] Example 4

[0104] The other steps are the same as in Example 1, except for the fixing method:

[0105] (1) Paraformaldehyde fixation

[0106] 1. Prepare 4% paraformaldehyde solution using PBS;

[0107] 2. Wash the 96-well cell culture microplates with transfected cells twice with PBS;

[0108] 3. Fix with 4% paraformaldehyde for 10 min;

[0109] 4. Wash twice with PBS;

[0110] (2) Fixation with a mixture of 95% methanol and 5% acetone

[0111] 1. Prepare a mixture of 95% methanol and 5% acetone in advance and pre-cool it in a -20℃ refrigerator or dry ice.

[0112] 2. Fix the cell culture microplates fixed in step (1) with a pre-cooled mixture of 95% methanol and 5% acetone for 5 minutes;

[0113] 3. Wash twice with PBS;

[0114] 4. Add 200 μL of PBS as a cell protectant and store under vacuum;

[0115] The interpretation result is as follows Figure 4 As shown.

[0116] The results showed that no visible green fluorescent cells appeared in the positive samples, and no red fluorescence was quenched. Therefore, the combination of 4% paraformaldehyde with 95% methanol and 5% acetone cannot be used for NMDAR fixation because it damages the antigenicity of NMDAR.

[0117] Example 5

[0118] The other steps are the same as in Example 1, except for the fixing method:

[0119] (1) Paraformaldehyde fixation

[0120] 1. Prepare 4% paraformaldehyde solution using PBS;

[0121] 2. Wash the 96-well cell culture microplates with transfected cells twice with PBS;

[0122] 3. Fix with 4% paraformaldehyde for 10 min;

[0123] 4. Wash twice with PBS;

[0124] (2) Fixation with a mixture of 85% methanol and 15% acetone

[0125] 1. Prepare a mixture of 85% methanol and 15% acetone in advance and pre-cool it in a -20℃ refrigerator or dry ice.

[0126] 2. Fix the cell culture microplates fixed in step (1) with a pre-cooled mixture of 85% methanol and 15% acetone for 5 minutes;

[0127] 3. Wash twice with PBS;

[0128] 4. Add 200 μL of PBS as a cell protectant and store under vacuum;

[0129] The interpretation result is as follows Figure 5 As shown.

[0130] The results showed that no visible green fluorescent cells appeared in the positive samples, and no red fluorescence was quenched. Therefore, the combination of 4% paraformaldehyde with 85% methanol and 15% acetone cannot be used for NMDAR fixation because it damages the antigenicity of NMDAR.

[0131] Example 6

[0132] The other steps are the same as in Example 1, except for the fixing method:

[0133] (1) Paraformaldehyde fixation

[0134] 1. Prepare 4% paraformaldehyde solution using PBS;

[0135] 2. Wash the 96-well cell culture microplates with transfected cells twice with PBS;

[0136] 3. Fix with 4% paraformaldehyde for 10 min;

[0137] 4. Wash twice with PBS;

[0138] (2) Fixation with a mixture of 75% methanol and 25% acetone

[0139] 1. Prepare a mixture of 75% methanol and 25% acetone in advance and pre-cool it in a -20℃ refrigerator or dry ice.

[0140] 2. Fix the cell culture microplates fixed in step (1) with a pre-cooled mixture of 75% methanol and 25% acetone for 5 minutes;

[0141] 3. Wash twice with PBS;

[0142] 4. Add 200 μL of PBS as a cell protectant and store under vacuum;

[0143] The interpretation result is as follows Figure 6 As shown.

[0144] The results showed that no visible green fluorescent cells appeared in the positive samples, and no red fluorescence was quenched. Therefore, the combination of 4% paraformaldehyde with 75% methanol and 25% acetone cannot be used for NMDAR fixation because it damages the antigenicity of NMDAR.

[0145] Example 7

[0146] The other steps are the same as in Example 1, except for the fixing method:

[0147] (1) Paraformaldehyde fixation

[0148] 1. Prepare 4% paraformaldehyde solution using PBS;

[0149] 2. Wash the 96-well cell culture microplates with transfected cells twice with PBS;

[0150] 3. Fix with 4% paraformaldehyde for 10 min;

[0151] 4. Wash twice with PBS;

[0152] (2) Fixation with a mixture of 65% methanol and 35% acetone

[0153] 1. Prepare a mixture of 65% methanol and 35% acetone in advance and pre-cool it in a -20℃ refrigerator or dry ice.

[0154] 2. Fix the cell culture microplates fixed in step (1) with a pre-cooled mixture of 65% methanol and 35% acetone for 5 minutes;

[0155] 3. Wash twice with PBS;

[0156] 4. Add 200 μL of PBS as a cell protectant and store under vacuum;

[0157] The interpretation result is as follows Figure 7 As shown.

[0158] The results showed that no visible green fluorescent cells appeared in the positive samples, and no red fluorescence was quenched. Therefore, the combination of 4% paraformaldehyde with 65% methanol and 35% acetone cannot be used for NMDAR fixation because it damages the antigenicity of NMDAR.

[0159] Example 8

[0160] The other steps are the same as in Example 1, except for the fixing method:

[0161] (1) Paraformaldehyde fixation

[0162] 1. Prepare 4% paraformaldehyde solution using PBS;

[0163] 2. Wash the 96-well cell culture microplates with transfected cells twice with PBS;

[0164] 3. Fix with 4% paraformaldehyde for 10 min;

[0165] 4. Wash twice with PBS;

[0166] (2) Fixation with a mixture of 55% methanol and 45% acetone

[0167] 1. Prepare a mixture of 55% methanol and 45% acetone in advance and pre-cool it in a -20℃ refrigerator or dry ice.

[0168] 2. Fix the cell culture microplates fixed in step (1) with a pre-cooled mixture of 55% methanol and 45% acetone for 5 minutes;

[0169] 3. Wash twice with PBS;

[0170] 4. Add 200 μL of PBS as a cell protectant and store under vacuum;

[0171] The interpretation result is as follows Figure 8 As shown.

[0172] The results showed that no visible green fluorescent cells appeared in the positive samples, and no red fluorescence was quenched. Therefore, the combination of 4% paraformaldehyde with 55% methanol and 45% acetone cannot be used for NMDAR fixation because it damages the antigenicity of NMDAR.

[0173] Example 9

[0174] The other steps are the same as in Example 1, except for the fixing method:

[0175] (1) Paraformaldehyde fixation

[0176] 1. Prepare 4% paraformaldehyde solution using PBS;

[0177] 2. Wash the 96-well cell culture microplates with transfected cells twice with PBS;

[0178] 3. Fix with 4% paraformaldehyde for 10 min;

[0179] 4. Wash twice with PBS;

[0180] (2) Fixation with a mixture of 45% methanol and 55% acetone

[0181] 1. Prepare a mixture of 45% methanol and 55% acetone in advance and pre-cool it in a -20℃ refrigerator or dry ice.

[0182] 2. Fix the cell culture microplates fixed in step (1) with a pre-cooled mixture of 45% methanol and 55% acetone for 5 minutes;

[0183] 3. Wash twice with PBS;

[0184] 4. Add 200 μL of PBS as a cell protectant and store under vacuum;

[0185] The interpretation result is as follows Figure 9 As shown.

[0186] The results showed that no visible green fluorescent cells appeared in the positive samples, and no red fluorescence was quenched. Therefore, the combination of 4% paraformaldehyde with 45% methanol and 55% acetone cannot be used for NMDAR fixation because it damages the antigenicity of NMDAR.

[0187] Example 10

[0188] The other steps are the same as in Example 1, except for the fixing method:

[0189] (1) Paraformaldehyde fixation

[0190] 1. Prepare 4% paraformaldehyde solution using PBS;

[0191] 2. Wash the 96-well cell culture microplates with transfected cells twice with PBS;

[0192] 3. Fix with 4% paraformaldehyde for 10 min;

[0193] 4. Wash twice with PBS;

[0194] (2) Fixation with a mixture of 35% methanol and 65% acetone

[0195] 1. Prepare a mixture of 35% methanol and 65% acetone in advance and pre-cool it in a -20℃ refrigerator or dry ice.

[0196] 2. Fix the cell culture microplates fixed in step (1) with a pre-cooled mixture of 35% methanol and 65% acetone for 5 minutes;

[0197] 3. Wash twice with PBS;

[0198] 4. Add 200 μL of PBS as a cell protectant and store under vacuum;

[0199] The interpretation result is as follows Figure 10 As shown.

[0200] The results showed that no visible green fluorescent cells appeared in the positive samples, and no red fluorescence was quenched. Therefore, the combination of 4% paraformaldehyde with 35% methanol and 65% acetone cannot be used for NMDAR fixation because it damages the antigenicity of NMDAR.

[0201] Example 11

[0202] The other steps are the same as in Example 1, except for the fixing method:

[0203] (1) Paraformaldehyde fixation

[0204] 1. Prepare 4% paraformaldehyde solution using PBS;

[0205] 2. Wash the 96-well cell culture microplates with transfected cells twice with PBS;

[0206] 3. Fix with 4% paraformaldehyde for 10 min;

[0207] 4. Wash twice with PBS;

[0208] (2) Fixation with a mixture of 25% methanol and 75% acetone

[0209] 1. Prepare a mixture of 25% methanol and 75% acetone in advance and pre-cool it in a -20℃ refrigerator or dry ice.

[0210] 2. Fix the cell culture microplates fixed in step (1) with a pre-cooled mixture of 25% methanol and 75% acetone for 5 minutes;

[0211] 3. Wash twice with PBS;

[0212] 4. Add 200 μL of PBS as a cell protectant and store under vacuum;

[0213] The interpretation result is as follows Figure 11 As shown.

[0214] The results showed that weak green fluorescent cells were visible in the positive samples, and there was no red fluorescence quenching. Therefore, the combination of 4% paraformaldehyde with 25% methanol and 75% acetone can be used for NMDAR fixation, but it is not the optimal choice.

[0215] Example 12

[0216] The other steps are the same as in Example 1, except for the fixing method:

[0217] (1) Paraformaldehyde fixation

[0218] 1. Prepare 4% paraformaldehyde solution using PBS;

[0219] 2. Wash the 96-well cell culture microplates with transfected cells twice with PBS;

[0220] 3. Fix with 4% paraformaldehyde for 10 min;

[0221] 4. Wash twice with PBS;

[0222] (2) Fixation with a mixture of 15% methanol and 85% acetone

[0223] 1. Prepare a mixture of 15% methanol and 85% acetone in advance and pre-cool it in a -20℃ refrigerator or dry ice.

[0224] 2. Fix the cell culture microplates fixed in step (1) with a pre-cooled mixture of 15% methanol and 85% acetone for 5 minutes;

[0225] 3. Wash twice with PBS;

[0226] 4. Add 200 μL of PBS as a cell protectant and store under vacuum;

[0227] The interpretation result is as follows Figure 12 As shown.

[0228] 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 with 15% methanol and 85% acetone can be used for NMDAR fixation and is a better choice.

[0229] Example 13

[0230] The other steps are the same as in Example 1, except for the fixing method:

[0231] (1) Paraformaldehyde fixation

[0232] 1. Prepare 4% paraformaldehyde solution using PBS;

[0233] 2. Wash the 96-well cell culture microplates with transfected cells twice with PBS;

[0234] 3. Fix with 4% paraformaldehyde for 10 min;

[0235] 4. Wash twice with PBS;

[0236] (2) Fixation with a mixture of 10% methanol and 90% acetone

[0237] 1. Prepare a mixture of 10% methanol and 90% acetone in advance and pre-cool it in a -20℃ refrigerator or dry ice.

[0238] 2. Fix the cell culture microplates fixed in step (1) with a pre-cooled mixture of 10% methanol and 90% acetone for 5 minutes;

[0239] 3. Wash twice with PBS;

[0240] 4. Add 200 μL of PBS as a cell protectant and store under vacuum;

[0241] The interpretation result is as follows Figure 13 As shown.

[0242] The results showed that weak green fluorescent cells were visible in the positive samples, and there was no red fluorescence quenching. Therefore, the combination of 4% paraformaldehyde with 10% methanol and 90% acetone can be used for NMDAR fixation, but it is not the optimal choice.

[0243] Example 14

[0244] The other steps are the same as in Example 1, except for the fixing method:

[0245] (1) Paraformaldehyde fixation

[0246] 1. Prepare 4% paraformaldehyde solution using PBS;

[0247] 2. Wash the 96-well cell culture microplates with transfected cells twice with PBS;

[0248] 3. Fix with 4% paraformaldehyde for 10 min;

[0249] 4. Wash twice with PBS;

[0250] (2) Fixation with a mixture of 5% methanol and 95% acetone

[0251] 1. Prepare a mixture of 5% methanol and 95% acetone in advance and pre-cool it in a -20℃ refrigerator or dry ice.

[0252] 2. Fix the cell culture microplates fixed in step (1) with a pre-cooled mixture of 5% methanol and 95% acetone for 5 minutes;

[0253] 3. Wash twice with PBS;

[0254] 4. Add 200 μL of PBS as a cell protectant and store under vacuum;

[0255] The interpretation result is as follows Figure 14 As shown.

[0256] The results showed that no visible green fluorescent cells appeared in the positive samples, and no red fluorescence was quenched. Therefore, the combination of 4% paraformaldehyde with 5% methanol and 95% acetone cannot be used for NMDAR fixation because it damages the antigenicity of NMDAR.

[0257] In summary, in the detection of anti-NMDAR antibodies using a two-color immunofluorescence assay based on cell transfection, the fixation reagent is a mixture of paraformaldehyde, methanol, and acetone, with a methanol volume fraction of 10-25% and an acetone volume fraction of 75-90%. Preferably, the fixation reagent is a mixture of 4% paraformaldehyde, 15% methanol, and 85% acetone. The fixation step is performed in two steps as follows: (1) the transfected cell samples are fixed with 4% paraformaldehyde for 10 min and washed with PBS; (2) the cell samples fixed in step (1) are fixed again with a mixture of 10-25% methanol and 75-90% acetone for 5 min, and then washed with PBS. Using this fixation solution and method will not damage the antigenicity of NMDAR. In positive samples, obvious green fluorescent cells are visible, and there is no red fluorescence quenching. Co-localization of red and green fluorescence can be observed using a fluorescence microscope.

[0258] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by this application.

Claims

1. The application of a fixation reagent for dual-color fluorescence detection of anti-NMDAR antibodies in an anti-NMDAR antibody detection kit, characterized in that: The fixatives consist of a mixture of paraformaldehyde at a mass-to-volume ratio of 4% and methanol and acetone at a volume fraction of 15% and 85%, respectively.

2. The application according to claim 1, characterized in that: The fixation method of the fixation reagent according to claim 1 includes the following steps: (1) Cell samples were fixed with 4% paraformaldehyde for 10 min and washed with PBS; (2) The cell samples fixed in step (1) were fixed again with a mixture of 15% methanol and 85% acetone for 5 min. After fixation, the samples were washed with PBS.

3. The application according to any one of claims 1-2, characterized in that: Before use, the mixture of 15% methanol and 85% acetone should be pre-cooled in a -20°C refrigerator or dry ice.

4. The application according to claim 1, characterized in that: The kit also includes PBS buffer, HEK293T cells, cell culture microplates, blocking solution, and fluorescent secondary antibody, wherein the blocking solution is serum of the species for which the fluorescent secondary antibody is present.

5. The application according to claim 4, characterized in that: The detection of anti-NMDAR antibodies using the kit includes the following steps: (1) Take the cell culture microplates in which HEK293T cells with integrated antigen genes adhere, discard the liquid in the wells, add PBS buffer to wash and then discard it; (2) Add the sample to be tested into the 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 detection well, wash thoroughly with PBS buffer and then discard; (4) Add the fluorescent secondary antibody diluted with blocking solution to the detection well, shake gently to mix, and incubate at 37°C in the dark; (5) Repeat the cleaning steps in (3), discard the liquid in the well, add PBS buffer to the detection well until it covers the cells, and then observe the green and red fluorescence of the cells under a fluorescence microscope and take pictures. (6) Observe whether the two fluorescences co-localize and obtain the test results.

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

7. The application according to claim 5, characterized in that: If the test result is positive, and if it is necessary to verify the titer of the positive sample, then the sample is diluted according to the ratio and the experimental process of steps (1) to (6) is repeated.

8. The application according to claim 5, 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 undiluted solution should be used.

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