A kit for detecting AChR blocking antibodies, a detection method and application

Abstract

The application provides a kit for detecting AChR blocking antibodies, a detection method and application, and belongs to the technical field of medical detection. The application provides a compound for detecting AChR blocking antibodies instead of alpha-BGT. Compared with alpha-BGT, the compound is non-toxic, cheap, easy to obtain, and can still detect AChR blocking antibodies after high-temperature treatment. The application also provides a method for detecting AChR blocking antibodies by using the compound. The compound does not need to be labeled with radioactive iodine or other substances, thereby reducing the cost, avoiding the influence of radioactive substances on the environment and the harm to experimenters, and being green and environmentally friendly. The compound can also be incubated with a serum sample at the same time, thereby shortening the detection time and improving the detection efficiency.

Description

[0001] This application is a divisional application of application number 202211280761.8 filed on October 19, 2022, entitled "A kit, detection method and application for detecting AChR blocking antibodies". Technical Field

[0002] This invention belongs to the field of medical testing technology, and in particular relates to a kit, detection method and application for detecting AChR blocking antibodies. Background Technology

[0003] Myasthenia gravis (MG) is an acquired autoimmune disease mediated by autoantibodies, characterized by weakness of some or all skeletal muscles, easy fatigue, and symptoms that worsen with activity and improve with rest. Acetylcholine receptor (AChR) antibodies are the most prevalent pathogenic antibodies in MG. According to previous literature, AChR antibodies can be classified into three types based on their different effector mechanisms: binding, blocking, and modulating. AChR binding antibodies can bind to the receptor and simultaneously activate the complement system, forming a membrane attack complex at the postsynaptic membrane of the neuromuscular junction (NMJ), leading to severe endplate membrane damage and subsequent destruction of the receptor and receptor-related protein structures. Modulating antibodies, through cross-linking, can internalize the bound receptor and accelerate its degradation, resulting in the loss of AChR expression. Blocking antibodies can directly bind to the acetylcholine binding site on the receptor, blocking the binding of acetylcholine and causing functional blockage of the acetylcholine binding site, leading to poor muscle contraction.

[0004] In myocardial infarction (MG) patients, AChR antibodies are detectable in the serum of approximately 50%–60% of ocular MG and 85%–90% of systemic MG. Studies have shown that double positivity for both AChR binding and blocking antibodies is primarily observed in systemic MG, with patients exhibiting double positivity having a worse prognosis and an increased risk of thymoma. However, in ocular MG and mild systemic MG patients, single positivity for either blocking or binding antibodies is more common. Furthermore, patients with only blocking antibodies are predominantly ocular MG. Therefore, detecting blocking AChR antibodies is of significant practical value in the auxiliary diagnosis of ocular and systemic MG, especially in patients with negative binding AChR antibodies. Children and adolescents account for up to 50% of MG patients, predominantly ocular MG, and rarely convert to systemic MG. Overall, the prevalence of ocular MG is high; therefore, detecting AChR blocking antibodies is crucial for the diagnosis and prognosis of MG patients.

[0005] Alpha-bungarotoxin (α-BGT) is a peptide extracted from the venom of the krait (Bungarus fasciatus). Because α-BGT can bind highly selectively to the α-subunit of AChR, it is widely used for the detection of AChR antibodies. Currently, the most widely used serological detection method for AChR blocking antibodies is radioimmunoprecipitation (RIPA). This involves first binding the AChR blocking antibody in the sample to AChR, then adding 125I radiolabeled α-bungarotoxin (125I-α-BGT) to bind to the remaining binding sites on the receptor. The receptor precipitates with Sepharose-ConA. After centrifugation, the precipitate contains 125I-α-BGT. The content of 125I-α-BGT is determined by measuring the radioactivity of the precipitate, and it is inversely proportional to the concentration of AChR-blocking antibody in the original sample.

[0006] However, existing technologies still have the following defects and shortcomings:

[0007] 1) α-BGT is a toxin with a certain degree of toxicity and is not easily obtained;

[0008] 2) When RIPA detects AChR blocking antibodies, it is necessary not only to use α-BGT, but also to label with the radioactive isotope 125I. The manufacture and processing of radioactive iodine are expensive, have a greater impact on the environment, and are also harmful to laboratory personnel. In addition, relevant training and safety monitoring are required for laboratory personnel who come into contact with radioactive materials.

[0009] 3) For RIPA detection of AChR blocking antibodies, the AChR blocking antibodies in the sample must first bind to AChR. This process takes about 2 hours, and sometimes even longer, requiring overnight. Then, 125I-α-BGT is added to bind to the remaining binding sites on AChR. The whole experimental process is quite time-consuming. Summary of the Invention

[0010] In view of this, the purpose of the present invention is to provide a kit, detection method and application for detecting AChR blocking antibodies. The detection of AChR blocking antibodies using acetylcholine bromide or carbachol is safer, and the above compounds are lower in cost and easier to obtain.

[0011] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0012] This invention provides the application of a compound in the preparation of a reagent for detecting AChR blocking antibodies, said compound comprising acetylcholine bromide or carbachol.

[0013] Preferably, the concentration of the acetylcholine bromide solution is 0.2 mg / mL to 125 mg / mL; and the concentration of the carbacholine solution is 0.01 mg / mL to 100 mg / mL.

[0014] This invention provides a kit for detecting AChR blocking antibodies, the kit comprising a working solution, a blocking reagent, an AChR cell smear, and a pcDNA3.1 cell smear, wherein the blocking reagent is a solution of the aforementioned compound.

[0015] Preferably, the working solution is PBST or PBS, more preferably PBST.

[0016] The blocking reagent is a solution of the above-mentioned compound, and the compound solution (such as acetylcholine chloride solution, acetylcholine bromide solution, carbacholine solution, or cis-sulfatracurium solution, etc.) is prepared from the working solution.

[0017] Preferably, the kit further includes a washing solution, which is a phosphate buffer.

[0018] This invention provides a method for detecting AChR blocking antibodies based on the CBA method, comprising the following steps:

[0019] Serum diluted with the above working solution was used as the primary antibody for the non-blocking group, and serum diluted with the above blocking reagent was used as the primary antibody for the blocking group. Both were reacted with cell smears in the sample application area. After washing, the fluorescently labeled secondary antibody was incubated with the treated cell smears. The changes in fluorescence signals between the blocking group and the non-blocking group were compared to determine whether the AChR antibody in the serum was a blocking type.

[0020] Preferably, the reaction time is 2 to 3 hours.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The present invention provides a compound that can replace α-BGT for detecting AChR blocking antibodies. Compared with α-BGT, the compound is non-toxic, inexpensive, readily available, and can still detect AChR blocking antibodies after high-temperature treatment.

[0023] (2) The present invention also provides a method for detecting AChR blocking antibodies. The compound described in the present invention does not require radioactive iodine or other substances for labeling, which reduces costs and avoids the impact of radioactive substances on the environment and the harm to experimental personnel, making it green and environmentally friendly.

[0024] (3) The present invention provides a method for detecting AChR blocking antibodies. The compound described in the present invention can be incubated with serum samples simultaneously, which shortens the detection time and improves the detection efficiency. Attached Figure Description

[0025] Figure 1 The slides are covered with AChR cell crawlers and pcDNA3.1 cell crawlers.

[0026] Figure 2 The blocking results of the blocking reagents in Table 2 on serum No. 5 are shown.

[0027] Figure 3 The blocking results of the blocking reagents in Table 2 on serum No. 8;

[0028] Figure 4 The blocking results of the blocking reagents in Table 2 on serum No. 19 are shown.

[0029] Figure 5 The blocking results of the blocking reagents in Table 2 on serum No. 22 are shown.

[0030] Figure 6 The results of blocking acetylcholine chloride, acetylcholine bromide, carbacholine, and atracurium besylate blocking reagents on serum No. 5 after high-temperature treatment. Detailed Implementation

[0031] This invention provides the application of a compound in the preparation of a reagent for detecting AChR blocking antibodies, the compound comprising acetylcholine chloride, acetylcholine bromide, carbacholine, or cis-sultracurium besylate.

[0032] In this invention, the compound can bind to the acetylcholine binding site on the acetylcholine receptor, competitively binding to AChR blocking antibodies, and can be used to detect AChR blocking antibodies. The compound of this invention can replace α-BGT, and is non-toxic, inexpensive, and readily available; it can still detect AChR blocking antibodies after high-temperature treatment.

[0033] In a preferred embodiment, the concentration of the acetylcholine chloride solution is 0.2 mg / mL to 100 mg / mL; the concentration of the acetylcholine bromide solution is 0.2 mg / mL to 125 mg / mL; the concentration of the carbacholine solution is 0.01 mg / mL to 100 mg / mL; and the concentration of the atracurium besylate solution is 0.05 mg / mL to 50 mg / mL. The compound solutions of the present invention (such as acetylcholine chloride solution, acetylcholine bromide solution, carbacholine solution, or atracurium besylate solution, etc.) are prepared using working solutions.

[0034] The present invention also provides a kit for detecting AChR blocking antibodies, the kit comprising a working solution, a blocking reagent, an AChR cell smear and a pcDNA3.1 cell smear, wherein the blocking reagent is a solution of the aforementioned compound.

[0035] In this invention, the above-mentioned compounds (such as acetylcholine chloride solution, acetylcholine bromide solution, carbacholine solution, or cissulfonamide solution, etc.) can be used in all experimental methods for detecting AChR blocking antibodies, including but not limited to ELISA, WB, immunofluorescence, flow cytometry, etc., and the compounds can be applied to the selected experimental method based on the blocking principle.

[0036] In this invention, the working solution is preferably PBS or PBST, more preferably PBST. This invention does not have a specific limitation on the dilution ratio, as long as it ensures that when the blocking reagent detects AChR blocking antibodies, the specific fluorescent staining signal of the unblocked group is significantly different from that of the blocking reagent. The preparation methods of the AChR cell crawlers and pcDNA3.1 cell crawlers in this invention are not specifically limited; any preparation method known in the art can be used, such as the preparation method described in patent application number 202111021985.2, entitled "A Cell Crawler for Detecting Acetylcholine Receptor Autoantibodies in Human Fluids and its Preparation Method and Application". In this invention, the AChR cell crawlers and pcDNA3.1 cell crawlers are mounted on a glass slide as follows: Figure 1 As shown, the slide has two sample application areas, sample application area 1 and sample application area 2; sample application area 1 is the sample application area for the non-blocked group, and sample application area 2 is the sample application area for the blocked group; each of sample application area 1 and sample application area 2 has two cell crawling slides attached, the left one is an AChR cell crawling slide and the right one is a pcDNA3.1 cell crawling slide;

[0037] In this invention, the kit preferably includes a washing solution, which is preferably a phosphate buffer, and more preferably PBST or PBS. This invention does not specifically limit the source of PBST or PBS, which can be prepared using commercially available products or preparation methods known in the art.

[0038] This invention provides a method for detecting AChR blocking antibodies based on the CBA method, comprising the following steps:

[0039] Serum diluted with the above working solution was used as the primary antibody for the non-blocking group, and serum diluted with the above blocking reagent was used as the primary antibody for the blocking group. Both were reacted with cell smears in the sample application area. After washing, the fluorescently labeled secondary antibody was incubated with the treated cell smears. The changes in fluorescence signals between the blocking group and the non-blocking group were compared to determine whether the AChR antibody in the serum was a blocking type.

[0040] In this invention, the volume of the solution (including primary antibody, secondary antibody and washing solution) added to the above-mentioned sample loading area is preferably 100 μL.

[0041] In this invention, the reaction time is preferably 2-3 hours. Serum diluted with PBST is used as the unblocked group, and serum diluted with the above compound solution is used as the blocked group. AChR cell smears corresponding to the sample application area are incubated separately, serving as a parallel control with the unblocked group. The compound in the blocked group can bind to the acetylcholine binding site of AChR, thereby blocking the binding of AChR blocking antibodies to it. Because the compound in the blocked group blocks the binding of AChR blocking antibodies to AChR expressed on the cell smears, while the AChR blocking antibodies in the unblocked group bind normally to the AChR expressed on the cell smears, the specific fluorescent staining of the blocked group is significantly weakened compared to the specific fluorescent staining of the AChR blocking antibodies in the unblocked group. That is, the positive signal of the AChR blocking antibody is blocked, and therefore the AChR antibody in this serum is a blocking antibody. The results of this invention were observed under a fluorescence microscope, and the results are shown in Table 1.

[0042] Table 1 Results Judgment

[0043]

[0044]

[0045] The present invention determines whether the AChR antibody in serum is a blocking type as follows: (1) If the AChR in the non-blocking group is positive, and the specific fluorescent staining of the AChR cell smear in the blocking group is significantly weakened compared with the non-blocking group, or the blocking group is negative, that is, the positive signal of AChR antibody is blocked, then the AChR antibody in the serum is a blocking type; (2) If the AChR in the non-blocking group is positive, and the specific fluorescent staining of the AChR cell smear in the blocking group is basically unchanged compared with the non-blocking group, that is, the positive signal of AChR antibody is not blocked, then the AChR antibody in the serum is not a blocking type, but a binding type or a regulatory type; (3) If both the non-blocking group and the blocking group are negative for AChR, then there is no AChR antibody in the serum; (4) If the specific fluorescent staining of the AChR cell smear in the non-blocking group is very strong, and the blocking group is not significantly weakened compared with it, the serum can be diluted to a suitable ratio by increasing the content of the compound in the primary antibody or decreasing the content of serum antibody in the primary antibody, and then verified by the above experimental steps. If the fluorescence is significantly weakened, then the AChR antibody in the serum is a blocking type.

[0046] In this invention, the method of increasing the compound content in the primary antibody or decreasing the serum antibody content in the primary antibody can be as follows: First, the serum can be diluted to 1:10 using a higher concentration of blocking reagent within the range, achieving a more significant blocking effect simply by increasing the compound content in the primary antibody. Second, the serum can be diluted with PBST to 1:100, 1:1000, or other ratios (this invention does not specifically limit this dilution ratio, as long as it ensures that when the blocking reagent detects AChR blocking antibodies, the specific fluorescent staining signal of the unblocked group is significantly different from that of the blocking reagent), reducing the concentration of the detected antibody in the serum. Then, the serum can be diluted to 1:10 using the original concentration of blocking reagent, achieving a more significant blocking effect simply by reducing the serum antibody content in the primary antibody. Third, the serum can be diluted to a higher concentration of blocking reagent within the range at a higher dilution ratio, such as 1:100, achieving a more significant blocking effect by increasing the compound content in the primary antibody while simultaneously decreasing the serum antibody content in the primary antibody.

[0047] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0048] In the following embodiments, the test samples used in this invention were 26 AChR autoantibody positive sera, which were numbered 1 to 26. The 26 AChR autoantibody positive sera were detected by the existing radioimmunoassay. Seven of them were AChR autoantibody blocking type, i.e., AChR blocking antibody positive, and their serial numbers were 5, 8, 12, 14, 17, 19, and 22, respectively.

[0049] Example 1: Detection of AChR blocking antibodies using acetylcholine chloride based on the CBA method

[0050] 1. Detection of AChR blocking antibodies using acetylcholine chloride

[0051] Two primary antibodies were prepared from each of the 26 AChR autoantibody-positive sera. The serum was diluted 1:10 with PBST (i.e., 90 μL PBST + 10 μL serum) and used as the primary antibody for the non-blocking group, denoted as primary antibody A. The serum was diluted 1:10 with 2 mg / mL acetylcholine chloride solution and used as the primary antibody for the blocking group, denoted as primary antibody B.

[0052] Experimental steps:

[0053] (1) Take the detection material for detecting AChR blocking antibodies, wherein the detection material is a glass slide with AChR cell crawlers and pcDNA3.1 cell crawlers attached, such as... Figure 1 As shown;

[0054] (2) Primary antibody incubation: Add primary antibody A to sample loading area 1 and primary antibody B to sample loading area 2, and incubate for 1 hour;

[0055] (3) Primary antibody elution: Wash three times with PBST for 5 min each time to obtain the treated cell smears;

[0056] (4) Secondary antibody incubation: The cells were incubated with FITC-labeled secondary antibody for 30 min.

[0057] (5) Secondary antibody elution: Wash three times with PBST for 5 min each time;

[0058] (6) Mounting: Mount the slide with glycerin;

[0059] (7) Observation results: Under the fluorescence microscope, compared with the specific fluorescence staining of AChR antibody in the unblocked group, the specific fluorescence staining of the blocked group was significantly weakened, that is, the positive signal of AChR antibody was blocked, and the AChR antibody in the serum was the blocking type.

[0060] Experimental results: Among the 26 AChR autoantibody positive sera, compared with the non-blocking group, the fluorescence intensity of the specific staining on AChR cell smears was significantly reduced in 7 sera (serum numbers 5, 8, 12, 14, 17, 19, and 22), while the remaining sera showed little change.

[0061] 2. Detection of AChR blocking antibodies using acetylcholine chloride treated at high temperature.

[0062] Two primary antibodies were prepared from each of the 26 AChR autoantibody-positive sera detected by radioimmunoassay. The serum was diluted 1:10 with PBST and designated as primary antibody A for the non-blocking group. The serum was diluted 1:10 with acetylcholine chloride solution (2 mg / mL, treated at 100℃ for 30 min) and designated as primary antibody B for the blocking group.

[0063] Experimental steps: Same as the above experimental steps.

[0064] Experimental results: Among the 26 AChR autoantibody positive sera, compared with the non-blocking group, the fluorescence intensity of the specific staining on AChR cell smears was significantly reduced in 7 sera (serum numbers 5, 8, 12, 14, 17, 19, and 22), while the remaining sera showed little change.

[0065] Experimental conclusion: In the CBA method, acetylcholine chloride solution has a blocking effect on AChR blocking antibodies, and it can be detected in all 7 AChR blocking antibody positive sera. After high-temperature treatment, it still has a blocking effect on these 7 sera and can be detected in all of them.

[0066] Example 2: Detection of AChR blocking antibodies using acetylcholine bromide based on the CBA method

[0067] 1. Detection of AChR blocking antibodies using acetylcholine bromide

[0068] Two primary antibodies were prepared from each of the 26 existing AChR autoantibody-positive sera. The serum was diluted 1:10 with PBST and designated as primary antibody A for the non-blocking group. The serum was diluted 1:10 with 2 mg / mL acetylcholine bromide solution and designated as primary antibody B for the blocking group.

[0069] Experimental steps: Same as in Example 1.

[0070] Experimental results: Among the 26 AChR autoantibody positive sera, compared with the non-blocking group, the fluorescence intensity of the specific staining on AChR cell smears was significantly reduced in 7 sera (serum numbers 5, 8, 12, 14, 17, 19, and 22), while the remaining sera showed little change.

[0071] 2. Detection of AChR blocking antibodies using acetylcholine bromide treated at high temperature.

[0072] Two primary antibodies were prepared from each of the 26 existing AChR autoantibody-positive sera. The serum was diluted 1:10 with PBST and used as the primary antibody for the non-blocking group, designated as primary antibody A. The serum was diluted 1:10 with acetylcholine bromide (2 mg / mL) after being treated at 100℃ for 30 min and used as the primary antibody for the blocking group, designated as primary antibody B.

[0073] Experimental steps: Same as in Example 1.

[0074] Experimental results: Among the 26 AChR autoantibody positive sera, compared with the non-blocking group, the fluorescence intensity of the specific staining on AChR cell smears was significantly reduced in 7 sera (serum numbers 5, 8, 12, 14, 17, 19, and 22), while the remaining sera showed little change.

[0075] Experimental conclusion: In the CBA method, acetylcholine bromide has a blocking effect on AChR blocking antibodies, and it can be detected in all 7 AChR group blocking antibody-positive sera. After high-temperature treatment, it still has a blocking effect on these 7 sera and can be detected in all of them.

[0076] Example 3: Detection of AChR blocking antibodies using carbacholine based on the CBA method

[0077] 1. Detection of AChR blocking antibodies using carbachol

[0078] Two primary antibodies were prepared from each of the 26 existing AChR autoantibody-positive sera. The serum was diluted 1:10 with PBST and designated as primary antibody A for the non-blocking group. The serum was diluted 1:10 with 2 mg / mL carbachol and designated as primary antibody B for the blocking group.

[0079] Experimental steps: Same as in Example 1.

[0080] Experimental results: Among the 26 AChR autoantibody positive sera, compared with the non-blocking group, the fluorescence intensity of the specific staining on AChR cell smears was significantly reduced in 6 sera (serum numbers 5, 12, 14, 17, 19, and 22), while the remaining sera showed little change.

[0081] 2. Detection of AChR blocking antibodies using carbachol that has undergone high-temperature treatment.

[0082] Two primary antibodies were prepared from each of the 26 existing AChR autoantibody-positive sera. The serum was diluted 1:10 with PBST and designated as primary antibody A. The serum was diluted 1:10 with carbacholine solution (2 mg / mL, treated at 100℃ for 30 min) and designated as primary antibody B.

[0083] Experimental steps: Same as in Example 1.

[0084] Experimental results: Among the 26 AChR autoantibody positive sera, compared with the non-blocking group, the fluorescence intensity of the specific staining on AChR cell smears was significantly reduced in 6 sera (serum numbers 5, 12, 14, 17, 19, and 22), while the remaining sera showed little change.

[0085] Experimental conclusion: In the CBA method, carbachol has a blocking effect on AChR blocking antibodies, and 6 cases of AChR blocking antibody positive serum can be detected; after high temperature treatment, the blocking effect is still present on these 6 cases of serum, and they can all be detected.

[0086] Example 4: Detection of AChR blocking antibodies using cis-atracurium besylate based on the CBA method

[0087] 1. Detection of AChR blocking antibodies using atracurium besylate

[0088] Two primary antibodies were prepared from each of the 26 existing AChR autoantibody-positive sera. The serum was diluted 1:10 with PBST and used as the primary antibody for the non-blocking group, denoted as primary antibody A. The serum was diluted 1:10 with 2 mg / mL atracurium besylate and used as the primary antibody for the blocking group, denoted as primary antibody B.

[0089] Experimental steps: Same as in Example 1.

[0090] Experimental results: Among the 26 AChR autoantibody positive sera, compared with the non-blocking group, the fluorescence intensity of specific staining on AChR cell smears was significantly reduced in 5 sera (serum numbers 5, 12, 14, 17, and 22), while the remaining sera showed little change.

[0091] 2. Detection of AChR blocking antibodies using cis-atracurium besylate treated at high temperature.

[0092] Two primary antibodies were prepared from each of the 26 existing AChR autoantibody-positive sera. The serum was diluted 1:10 with PBST and used as the primary antibody for the non-blocking group, designated as primary antibody A. The serum was diluted 1:10 with a 2 mg / mL atracurium besylate solution that had been treated at 100℃ for 30 min and used as the primary antibody for the blocking group, designated as primary antibody B.

[0093] Experimental steps: Same as in Example 1.

[0094] Experimental results: Among the 26 AChR autoantibody positive sera, compared with the non-blocking group, the fluorescence intensity of specific staining on AChR cell smears was significantly reduced in 5 sera (serum numbers 5, 12, 14, 17, and 22), while the remaining sera showed little change.

[0095] Experimental conclusion: In the CBA method, atracurium besylate has a blocking effect on AChR blocking antibodies, and 5 cases of AChR blocking antibody positive serum can be detected; after high temperature treatment, the blocking effect is still present on these 5 cases of serum, and they can all be detected.

[0096] Example 5

[0097] The difference between this embodiment and Example 1 is that in this embodiment, the serum was diluted 1:10 with 0.2 mg / mL acetylcholine chloride solution and designated as primary antibody B for the blocking group. The remaining experimental steps are the same as in Example 1.

[0098] Example 6

[0099] The difference between this embodiment and Example 1 is that in this embodiment, the serum is diluted 1:10 with 100 mg / mL acetylcholine chloride solution and designated as primary antibody B for the blocking group. The remaining experimental steps are the same as in Example 1.

[0100] Example 7

[0101] The difference between this embodiment and Embodiment 2 is that in this embodiment, the serum was diluted 1:10 with 0.2 mg / mL acetylcholine bromide solution and designated as primary antibody B for the blocking group. The remaining experimental steps are the same as in Embodiment 2.

[0102] Example 8

[0103] The difference between this embodiment and Embodiment 2 is that in this embodiment, the serum was diluted 1:10 with 100 mg / mL acetylcholine bromide solution and designated as primary antibody B for the blocking group. The remaining experimental steps are the same as in Embodiment 2.

[0104] Example 9

[0105] The difference between this embodiment and Example 3 is that in this embodiment, the serum was diluted 1:10 with 0.01 mg / mL carbachol solution and designated as primary antibody B for the blocking group. The remaining experimental steps are the same as in Example 3.

[0106] Example 10

[0107] The difference between this embodiment and embodiment 3 is that in this embodiment, the serum is diluted 1:10 with 100 mg / mL carbachol solution and designated as primary antibody B for the blocking group. The remaining experimental steps are the same as in embodiment 3.

[0108] Example 11

[0109] The difference between this embodiment and embodiment 4 is that in this embodiment, the serum was diluted 1:10 with 0.05 mg / mL atracurium besylate solution and designated as primary antibody B for the blocking group. The remaining experimental steps are the same as in embodiment 4.

[0110] Example 12

[0111] The difference between this embodiment and embodiment 4 is that in this embodiment, serum is diluted 1:10 with 50 mg / mL atracurium besylate solution and designated as primary antibody B for the blocking group. The remaining experimental steps are the same as in embodiment 4.

[0112] Comparative Example 1: Detection of AChR blocking antibodies using α-BGT based on the CBA method

[0113] 1. Detection of AChR blocking antibodies using commercially available α-BGT

[0114] Two primary antibodies were prepared from each of the 26 existing AChR autoantibody-positive sera. The serum was diluted 1:10 with PBST and designated as primary antibody A for the non-blocking group. The serum was diluted 1:10 with 0.1 mg / mL commercial α-BGT and designated as primary antibody B for the blocking group.

[0115] Experimental procedure: Same as in Example 1.

[0116] Experimental results: Among the 26 AChR autoantibody positive sera, compared with the non-blocking group, the fluorescence intensity of specific staining on AChR cell smears was significantly reduced in 5 sera (serum numbers 5, 8, 12, 14, 17, and 19), while the remaining sera showed little change.

[0117] 2. Detection of AChR blocking antibodies using recombinant α-BGT obtained through prokaryotic expression.

[0118] Two primary antibodies were prepared from each of the 26 existing AChR autoantibody-positive sera. The serum was diluted 1:10 with PBST and designated as primary antibody A for the non-blocking group. The serum was diluted 1:10 with 0.1 mg / mL recombinant α-BGT and designated as primary antibody B for the blocking group.

[0119] Experimental steps: Same as the above experimental steps.

[0120] Experimental results: Among the 26 AChR autoantibody positive sera, compared with the non-blocking group, the fluorescence intensity of specific staining on AChR cell smears was significantly reduced in 5 sera (serum numbers 5, 8, 12, 14, 17, and 19), while the remaining sera showed little change.

[0121] 3. Detection of AChR blocking antibodies using α-BGT that has undergone high-temperature treatment.

[0122] Two primary antibodies were prepared from each of the 26 existing AChR autoantibody-positive sera. The serum was diluted 1:10 with PBST and used as the primary antibody for the non-blocking group, denoted as primary antibody A. The serum was diluted 1:10 with α-BGT treated at 100℃ for 30 min with 2 mg / mL and used as the primary antibody for the blocking group, denoted as primary antibody B.

[0123] Experimental steps: Same as the above experimental steps.

[0124] Experimental results: In these 26 cases of AChR autoantibody positive serum, the fluorescence intensity of the positive signal was consistent in the AChR cell smears after specific staining, with the blocking group and the non-blocking group being the same.

[0125] Experimental conclusions: The commercially available α-BGT purchased and the recombinant α-BGT expressed and purified in our laboratory showed consistent blocking activity against AChR blocking antibodies in the CBA method, and both were detectable in 5 AChR blocking antibody-positive sera. However, after high-temperature treatment, α-BGT showed no blocking activity in these 5 sera, and therefore was not detected in any of them.

[0126] Comparative Example 2: Detection of AChR blocking antibodies using vecuronium bromide choline based on the CBA method

[0127] 1. Detection of AChR blocking antibodies using vecuronium bromide

[0128] Two primary antibodies were prepared from each of the 26 existing AChR autoantibody-positive sera. The serum was diluted 1:10 with PBST and designated as primary antibody A for the non-blocking group. The serum was diluted 1:10 with 2 mg / mL vecuronium bromide solution and designated as primary antibody B for the blocking group.

[0129] Experimental steps: Same as in Example 1.

[0130] Experimental results: Among the 26 AChR autoantibody positive sera, compared with the non-blocking group, the fluorescence intensity of the specific staining on AChR cell smears was significantly reduced in 3 sera (serum numbers 5, 14, and 17), while the remaining sera showed little change.

[0131] 2. Detection of AChR blocking antibodies using vecuronium bromide that has undergone high-temperature treatment.

[0132] Two primary antibodies were prepared from each of the 26 existing AChR autoantibody-positive sera. The serum was diluted 1:10 with PBST and used as the primary antibody for the non-blocking group, denoted as primary antibody A. The serum was diluted 1:10 with vecuronium bromide solution (2 mg / mL, treated at 100℃ for 30 min) and used as the primary antibody for the blocking group, denoted as primary antibody B.

[0133] Experimental steps: Same as in Example 1.

[0134] Experimental results: Among the 26 AChR autoantibody positive sera, compared with the non-blocking group, the fluorescence intensity of the specific staining on AChR cell smears was significantly reduced in 3 sera (serum numbers 5, 14, and 17), while the remaining sera showed little change.

[0135] Experimental conclusion: In the CBA method, vecuronium bromide has a blocking effect on AChR blocking antibodies, and three cases of AChR blocking antibody-positive serum can be detected; after high-temperature treatment, the blocking effect on these three serums is still present, and they can all be detected.

[0136] Comparative Example 3: Detection of AChR blocking antibodies using acetylcholine iodide based on the CBA method

[0137] 1. Detection of AChR blocking antibodies using acetylcholine iodide

[0138] Two primary antibodies were prepared from each of the 26 existing AChR autoantibody-positive sera. The serum was diluted 1:10 with PBST and designated as primary antibody A for the non-blocking group. The serum was diluted 1:10 with 2 mg / mL acetylcholine iodide solution and designated as primary antibody B for the blocking group.

[0139] Experimental steps: Same as in Example 1.

[0140] Experimental results: Among the 26 AChR autoantibody positive sera, compared with the non-blocking group, the fluorescence intensity of specific staining on AChR cell smears was significantly reduced in 2 sera (serum numbers 17 and 22), while the remaining sera showed little change.

[0141] 2. Detection of AChR blocking antibodies using acetylcholine iodide that has undergone high-temperature treatment.

[0142] Two primary antibodies were prepared from each of the 26 existing AChR autoantibody-positive sera. The serum was diluted 1:10 with PBST and used as the primary antibody for the non-blocking group, denoted as primary antibody A. The serum was diluted 1:10 with iodinated acetylcholine solution that had been treated at 100℃ for 30 min and used as the primary antibody for the blocking group, denoted as primary antibody B.

[0143] Experimental steps: Same as in Example 1.

[0144] Experimental results: Among the 26 AChR autoantibody positive sera, compared with the non-blocking group, the fluorescence intensity of specific staining on AChR cell smears was significantly reduced in 2 sera (serum numbers 17 and 22), while the remaining sera showed little change.

[0145] Experimental conclusion: In the CBA method, acetylcholine iodide has a blocking effect on AChR blocking antibodies, and two cases of AChR blocking antibody positive serum can be detected; after high temperature treatment, the blocking effect is still present on these two serums, and they can be detected.

[0146] Comparative Example 4: Detection of AChR blocking antibodies using other irrelevant proteins based on the CBA method

[0147] We collected some AChR-independent proteins from our laboratory, including CV2, Ma2, Titin, PKC, Recoveryin, GAD65, Zic4, SOX1, Tr, and S100B. For each of the 26 AChR autoantibody-positive serum samples, we prepared two primary antibodies. The serum was diluted 1:10 with PBST and designated as primary antibody A for the non-blocking group. The serum was diluted 1:10 with 2 mg / mL of CV2, Ma2, Titin, PKC, Recoveryin, GAD65, Zic4, SOX1, Tr, and S100B and designated as primary antibody B for the blocking group.

[0148] Experimental steps: Same as in Example 1.

[0149] Experimental results: In 26 AChR autoantibody-positive sera, the fluorescence intensity of all proteins in the AChR cell smears showed essentially no change compared to the non-blocked group.

[0150] Experimental conclusion: Compared with the example, other irrelevant proteins do not compete with AChR blocking antibodies for binding sites and have no blocking effect on AChR blocking antibodies. They were not detected in any of the 7 AChR blocking antibody positive sera. Therefore, other irrelevant proteins cannot be used to detect AChR blocking antibodies.

[0151] Comparative Example 5: Detection of AChR blocking antibodies using salt solutions of the same molar concentration based on the CBA method.

[0152] Two primary antibodies were prepared from each of the 26 existing AChR autoantibody-positive sera. The serum was diluted 1:10 with PBST and designated as primary antibody A for the non-blocking group. The serum was diluted 1:10 with 0.64 mg / mL NaCl and designated as primary antibody B for the blocking group.

[0153] Experimental steps: Same as in Example 1.

[0154] Experimental results: In 26 AChR autoantibody-positive sera, the fluorescence intensity of specific staining on AChR cell smears in the blocking group and the non-blocking group showed almost no change.

[0155] Experimental conclusion: Compared with Example 1, NaCl of the same molar concentration does not compete with AChR blocking antibodies for binding sites and has no blocking effect on AChR blocking antibodies. All 7 AChR blocking antibody positive sera were undetectable. Therefore, other irrelevant proteins cannot be used to detect AChR blocking antibodies.

[0156] Summary of experimental conclusions:

[0157] All substances in the examples and comparative examples were detected using the CBA method in 26 existing AChR autoantibody-positive sera. The detection results of AChR blocking antibodies are shown in Table 2.

[0158] Table 2. Detection of AChR blocking antibodies by different compounds

[0159]

[0160]

[0161] Table 2 shows the blocking results of the blocking agents on some serum samples. Figure 2 , 3 As shown in Figures 4 and 5, among which Figure 2 This is serum number 5. Figure 3This is serum number 8. Figure 4 This is serum number 19. Figure 5 This refers to serum sample 22. The blocking results of some of the blocking reagents listed in Table 2 (such as acetylcholine chloride, acetylcholine bromide, carbachol, and atracurium besylate) on serum sample 5 after high-temperature treatment are shown below. Figure 6 As shown.

[0162] From Table 2 and Figures 2-5 It is known that different choline derivatives can compete with AChR blocking antibodies for AChR binding sites, but their competitive abilities differ. Therefore, they also have different blocking effects on sera containing AChR blocking antibodies. Some of these compounds can detect more sera than α-BGT and can replace α-BGT in detecting AChR blocking antibodies, resulting in more accurate detection results. Examples include acetylcholine chloride, acetylcholine bromide, and carbachol. However, some of these compounds do not have a significant blocking effect on sera that can detect some α-BGT and cannot completely replace α-BGT. Examples include vecuronium bromide and acetylcholine iodide.

[0163] like Figure 6 As shown, acetylcholine chloride, acetylcholine bromide, carbacholine, and atracurium besylate still have blocking effects after high-temperature treatment.

[0164] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a compound in the preparation of a reagent for detecting AChR blocking antibodies, characterized in that, The compound is acetylcholine bromide.

2. The application according to claim 1, characterized in that, The concentration of the acetylcholine bromide solution is 0.2 mg / mL to 125 mg / mL.

3. The use of a compound in the preparation of a kit for detecting AChR blocking antibodies, characterized in that, The kit includes a working solution, a blocking reagent, AChR cell smears, and pcDNA3.1 cell smears, wherein the blocking reagent is a compound as described in claim 1 or 2.

4. The application according to claim 3, characterized in that, The working solution is PBST or PBS.

5. The application according to claim 3, characterized in that, The kit also includes a washing solution, which is a phosphate buffer.

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