An ach-r-ab test strip, method of use and method of manufacture and use

Abstract

An AChR-Ab detection test strip, a use method and a preparation method and purposes; the preparation method comprises the following steps: a detection antibody coupled fluorescent microsphere preparation step, a quality control antibody coupled fluorescent microsphere preparation step, an antibody coupled microsphere combined pad preparation step, a sample pad pretreatment step, a nitrocellulose membrane pretreatment step and a test strip assembly step; the use method is a sample application step: tear sample is added to the sample application hole of the AChR-Ab detection test strip for detection, after the test paper reacts, an immunofluorescence analyzer is used to read the fluorescence signal intensity of the detection line and the quality control line. The scheme can detect the tear specific AChR-Ab, improve the detection sensitivity from 50% of the serum standard to >=80%, and greatly shorten the detection time, and simplify the detection process.

Description

Technical Field

[0001] This invention relates to the field of AChR-Ab test strips, and more particularly to an AChR-Ab test strip, its usage, preparation method, and applications. Background Technology

[0002] Myasthenia gravis (MG) is an acquired autoimmune disease characterized by postsynaptic membrane structural and functional damage at the neuromuscular junction (NMJ) mediated by specific autoantibodies. This ultimately manifests as fluctuating muscle weakness, typically characterized by morning weakness and evening severity, with exacerbations after activity and relief after rest. Unlike other neuromuscular diseases, MG rarely causes irreversible muscle damage, and patients often maintain relatively normal maximum muscle strength during symptom remission. Ocular myasthenia gravis (OMG) is a major clinical subtype of MG, accounting for 51% of MG patients. Its neuromuscular junction lesions affect only the levator palpebrae superioris, extraocular muscles, and orbicularis oculi muscle, with characteristic clinical manifestations including ptosis, diplopia / strabismus, and incomplete eyelid closure. Furthermore, approximately 90% of MG patients will experience symptoms such as ptosis, diplopia / strabismus, or incomplete eyelid closure during the course of the disease.

[0003] Although OMG lesions are limited to the ocular muscles, typical OMG symptoms (such as ptosis, diplopia, and strabismus) can still severely impact a patient's visual function, quality of life, and even life-threatening conditions. Long-term uncontrolled OMG can lead to amblyopia and even permanent visual impairment due to extraocular muscle weakness and atrophy. Persistent eyelid insufficiency can lead to complications such as dry eye and corneal ulcers, potentially causing blindness. Furthermore, ptosis and diplopia can significantly affect a patient's basic daily life, including driving, reading, and walking. More alarmingly, once OMG transforms into generalized myasthenia gravis (GMG), the risk of serious complications such as dysphagia, dysarthria, and respiratory failure increases significantly. Although respiratory muscle involvement is relatively rare in OMG patients, if it occurs, it can rapidly progress to myasthenic crisis, requiring emergency intensive care and mechanical ventilation, greatly increasing the difficulty of treatment and the risk of death. Current evidence-based medicine suggests that early intervention can effectively block the transformation of OMG into GMG. A clinical prediction model developed by Bi et al. showed that early application of immunosuppressive therapy significantly reduced the risk of conversion from 22.6% at baseline to 6.8%. A retrospective study by Teppei Komatsu's team in Japan further confirmed that initiating immunotherapy within the 255-day "golden intervention window" after symptom onset resulted in a sensitivity of 92.9% and a specificity of 91.2% in achieving the "minimal manifestation status" (MMS). This early intervention strategy not only improves disease prognosis but, more importantly, prevents secondary damage caused by persistent ocular myasthenia gravis, including potentially blinding complications such as amblyopia and corneal ulcers. Therefore, the clinical management of OMG should receive the same level of attention as that of GMG.

[0004] Currently, the diagnosis of OMG mainly relies on a comprehensive assessment combining clinical manifestations with serum antibody testing, electrophysiological examination, and pharmacological tests. Acetylcholine receptor antibody (AChR-Ab), as the most valuable specific autoantibody for diagnosing OMG, has been listed as the preferred auxiliary diagnostic test in domestic and international guidelines. However, while the positive rate of serum AChR-Ab can reach 85%-90% in GMG patients, the positive rate in OMG patients is only about 50% due to the limited location of ocular lesions and milder symptoms. Studies show that the misdiagnosis rate of OMG is as high as 46%, highlighting the urgent need to develop novel diagnostic methods that are highly sensitive, non-invasive, and clinically practical.

[0005] The three most commonly used clinical methods for serum AChR-Ab detection are: (1) Radio-immunoprecipitation assay (RIA): Quantification is achieved by combining radiolabeled antigens with serum antibodies to form a complex. It carries the risk of radioactive contamination (using 125I isotope), has a short reagent shelf life (≤1 month), and exhibits low sensitivity in OMG (approximately 50%). (2) Enzyme-linked immunosorbent assay (ELISA): Microplates are coated with linear polypeptide antigens, and antibodies are detected through a colorimetric reaction. Because linear antigens cannot mimic the native conformation of AChR, the sensitivity is lower than RIA, and the strong acid / base chromogenic solutions cause environmental pollution. Furthermore, imported reagent kits are expensive, resulting in high costs per test, which limits clinical application. (3) Cell immunofluorescence assay (CBA): HEK293 cells transfected with AChR subunits are used to capture antibodies. Although it can detect low-affinity antibodies (OMG positive rate increased to 50%), it requires cell culture equipment (detection cycle > 24 hours), is complicated to operate, lacks standardized reagent kits, and is expensive, making it difficult to promote at the grassroots level. The above methods all have the following common shortcomings: (1) Invasive sampling limitations: They rely on venous blood collection, and pediatric patients have poor compliance; (2) Insufficient sensitivity in diagnosing OMG: Serum AChR-Ab cannot reflect the local antibody level in the eye, resulting in an OMG sensitivity of only about 50%, far lower than GMG (85%-90%), leading to a high rate of missed and misdiagnosed cases; (3) High technical thresholds: RIA requires radiation protection qualifications, ELISA has a high false positive rate, and CBA relies on professional cell platforms, resulting in low adoption rates in grassroots medical institutions. In summary, the existing methods are not effective in detecting OMG, and there is an urgent need to develop new non-invasive, highly sensitive, and convenient detection technologies. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing an AChR-Ab test strip, which can produce an AChR-Ab test strip that addresses the shortcomings of existing AChR-Ab tests, such as poor patient compliance in children due to reliance on venous blood collection and inability to reflect local ocular antibody expression. This method can detect tear-specific AChR-Ab.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] A method for preparing an AChR-Ab test strip includes the following steps:

[0009] Preparation steps for antibody-conjugated fluorescent microspheres: Dilute europium fluorescent microspheres with MSE buffer, add microsphere labeling activator and microsphere labeling stabilizer sequentially; centrifuge the formed microsphere suspension, discard the supernatant, and resuspend the microspheres with MSE buffer; add AChRα1 recombinant protein to the resuspended microsphere suspension; add microsphere labeling blocking solution; centrifuge the microsphere suspension and discard the supernatant;

[0010] Steps for preparing quality control antibody-conjugated fluorescent microspheres: Dilute europium fluorescent microspheres with MSE buffer, and add microsphere labeling activator and microsphere labeling stabilizer sequentially; centrifuge the formed microsphere suspension, discard the supernatant, and resuspend the microspheres with MSE buffer; add DNP antibody to the resuspended microsphere suspension; add microsphere labeling blocking solution; centrifuge the microsphere suspension and discard the supernatant;

[0011] Preparation steps of antibody-conjugated microsphere binding pad: Mix the detection antibody-conjugated fluorescent microspheres and the quality control antibody-conjugated fluorescent microspheres at a volume ratio of (10-1):1 to form a fluorescent microsphere solution, apply the fluorescent microsphere solution onto a glass fiber membrane, and dry it;

[0012] Sample pad pretreatment steps: Coat the sample pad buffer evenly with a glass fiber membrane, let the coated sample pad stand at room temperature; dry the sample pad;

[0013] Pretreatment steps for nitrocellulose membranes: Mouse anti-human IgG antibody and DNP-Ag are coated onto the surface of the nitrocellulose membrane using a gold-spraying coating instrument; mouse anti-human IgG antibody forms the detection line, and DNP-Ag forms the control line; after coating, the nitrocellulose membrane is dried.

[0014] Test strip assembly steps: Paste the sample pad, antibody-conjugated microsphere binding pad, nitrocellulose membrane and absorbent paper onto the base plate in a horizontal order, with adjacent pads overlapping.

[0015] Alternatively, in the step of preparing the antibody-conjugated microsphere binding pad, the detection antibody-conjugated fluorescent microspheres and the quality control antibody-conjugated fluorescent microspheres are added to a three-dimensional spraying platform, and the mixed microspheres are sprayed onto the glass fiber membrane at a rate of 1-6 μL / cm using the three-dimensional spraying platform.

[0016] In an optimized manner, during the test strip assembly step, the sample pad, antibody-conjugated microsphere conjugate pad, nitrocellulose membrane, and absorbent paper are sequentially pasted onto the base plate in a horizontal order, with the horizontal overlap between adjacent pads being 0.5-3 mm.

[0017] Alternatively, after performing the step of preparing the detection antibody-conjugated fluorescent microspheres, the detection antibody-conjugated fluorescent microspheres can be ultrasonically treated using an ultrasonic disruptor.

[0018] After performing the step of preparing quality control antibody-conjugated fluorescent microspheres, the quality control antibody-conjugated fluorescent microspheres are ultrasonically treated using an ultrasonic disruptor.

[0019] Alternatively, in the pretreatment step of the nitrocellulose membrane, after coating, the nitrocellulose membrane is dried at 36-38°C for 8-16 hours.

[0020] An AChR-Ab test strip is prepared by the above-described method for preparing an AChR-Ab test strip.

[0021] An OMG diagnostic kit based on the detection of AChR-Ab in tears, comprising: the aforementioned AChR-Ab test strip.

[0022] A method for using an AChR-Ab test strip, comprising: a sample application step;

[0023] The spotting step involves adding the tear sample to the spotting well of the AChR-Ab test strip for detection. After the test strip reacts, the fluorescence signal intensity of the test line and control line is read using an immunofluorescence analyzer.

[0024] Optimally, it may also include: tear collection steps and tear sample processing steps;

[0025] The tear collection procedure is as follows: Gently pull the patient's lower eyelid conjunctiva and apply moderate pressure backward. In a non-invasive manner, gently place one end of the Schirmer tear test strip with the yellow indicator line into the conjunctival sac on the outer side of the patient's lower eyelid, and let the other end of the Schirmer tear test strip hang naturally on the outer side of the lower eyelid. Instruct the patient to gently close their eyes and look slightly upward, maintaining this position to allow the Schirmer tear test strip to fully absorb the basal tears.

[0026] The tear sample processing steps are as follows: Schirmer tear test strips are mixed with PBS diluent, placed in an ultrasonic processor, and then centrifuged to remove solid residues. The supernatant is then used as the tear sample for the spotting step.

[0027] The use of a test strip in the preparation of an OMG diagnostic kit for detecting AChR-Ab in tears, wherein the test strip is the aforementioned AChR-Ab test strip.

[0028] Compared with the prior art, one of the above technical solutions has the following beneficial effects:

[0029] This solution provides a method for preparing AChR-Ab test strips, which can produce AChR-Ab test strips. It addresses the shortcomings of existing AChR-Ab tests, such as poor patient compliance in children due to reliance on venous blood collection and inability to reflect local antibody expression in the eye. By detecting tear-specific AChR-Abs, it can increase the diagnostic sensitivity of OMG from 50% to ≥80% based on serum standards, and significantly shorten the detection time, simplifying the detection process. Attached Figure Description

[0030] Figure 1 This is a flowchart of tear collection and processing;

[0031] Figure 2 The CBA study validated the expression of the human AChRα1 subunit in HEK 293T cells.

[0032] Figure 3 This is the AChR-Ab test strip (EuNPs-LFIA) result for tear AChR-Ab in OMG patients (n=10) and healthy controls (HC) (n=5). (A) Scatter plot of T-line fluorescence values ​​of tear AChR-Ab in OMG patients and HC group; (B) ROC curve analysis of the sensitivity and specificity of tear EuNPs-LFIA for AChR-Ab detection; (C) Bar chart of T-line fluorescence values ​​of tear AChR-Ab in OMG patients and HC group, with the dashed line representing the detection cutoff value (cut-off = 1682); (D) Image recording of tear EuNPs-LFIA AChR-Ab detection in OMG patients and healthy controls;

[0033] Figure 4 This is the EuNPs-LFIA detection result of serum AChR-Ab in OMG patients (n=10) and healthy controls (HC) (n=5). (A) Scatter plot of T-line fluorescence values ​​of AChR-Ab in serum of OMG patients and HC group; (B) ROC curve analysis of the sensitivity and specificity of EuNPs-LFIA detection of serum AChR-Ab; (C) Bar chart of T-line fluorescence values ​​of AChR-Ab in serum of OMG patients and HC group, with the dashed line representing the detection cutoff value (cut-off=1335); (D) Image recording of EuNPs-LFIA detection of serum AChR-Ab in serum of OMG patients and HC group.

[0034] Figure 5 This is a schematic diagram of the structure of the AChR-Ab test strip.

[0035] in:

[0036] Sample pad 1, antibody-conjugated microsphere binding pad 2, nitrocellulose membrane 3, absorbent paper 4, base plate 5. Detailed Implementation

[0037] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0038] To facilitate understanding of the present invention, a more comprehensive description is provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with techniques or conditions described in the literature in the art or according to product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0039] like Figure 1-5 A method for preparing an AChR-Ab test strip includes the following steps:

[0040] Preparation steps for antibody-conjugated fluorescent microspheres: Dilute europium fluorescent microspheres with MSE buffer, add microsphere labeling activator and microsphere labeling stabilizer sequentially; centrifuge the formed microsphere suspension, discard the supernatant, and resuspend the microspheres with MSE buffer; add AChRα1 recombinant protein to the resuspended microsphere suspension; add microsphere labeling blocking solution; centrifuge the microsphere suspension and discard the supernatant;

[0041] Europium fluorescent microspheres are diluted with MSE buffer. The amount of MSE buffer can be determined as needed. In general examples, the volume ratio of europium fluorescent microspheres to MSE buffer is 1:(30-100), preferably 1:(30-70), and most preferably 1:(40-50). After adding the microsphere labeling activator and microsphere labeling stabilizer, and after a certain reaction time (e.g., 10-60 min), the activator promotes the exposure and transformation of carboxyl groups on the microsphere surface, thereby fully activating the microsphere surface, increasing its reactivity, and providing more binding sites to facilitate subsequent coupling with AChRα1 recombinant protein. The microsphere labeling stabilizer helps maintain the stability of the reaction system and prevents the microspheres from aggregating or becoming inactive during processing. Thus, this step fully activates the microsphere surface and prepares it for coupling with the recombinant protein. Add recombinant AChRα1 protein and incubate for a period of time (e.g., 1-3 hours) to allow the AChRα1 protein to fully bind to the activated carboxyl groups on the microsphere surface. The amount of AChRα1 protein added can be determined as needed, preferably in excess of europium fluorescent microspheres. As a specific target, the AChRα1 protein binds to the activated carboxyl groups on the microsphere surface, completing antibody conjugation and ensuring accurate detection of AChR antibodies in tears during the experiment. Adding a microsphere labeling blocking solution blocks unbound sites on the microsphere surface, reducing non-specific interactions between other molecules or antibodies and the microspheres, thereby ensuring the accuracy and reliability of the test results.

[0042] Steps for preparing quality control antibody-conjugated fluorescent microspheres: Dilute europium fluorescent microspheres with MSE buffer, and add microsphere labeling activator and microsphere labeling stabilizer sequentially; centrifuge the formed microsphere suspension, discard the supernatant, and resuspend the microspheres with MSE buffer; add DNP antibody to the resuspended microsphere suspension; add microsphere labeling blocking solution; centrifuge the microsphere suspension and discard the supernatant;

[0043] Similar to the preparation of antibody-conjugated fluorescent microspheres for detection, europium fluorescent microspheres are diluted with MSE buffer during the preparation of quality control antibody-conjugated fluorescent microspheres. The amount of MSE buffer can be determined as needed. Microsphere labeling activators and stabilizers ensure the microsphere surface is fully activated and ready for conjugation with recombinant proteins. DNP antibody is used to label specific binding sites on the microsphere surface as a standardized control for subsequent quality control detection. The amount of DNP antibody added can be determined as needed, generally less than that of europium fluorescent microspheres; the preferred volume ratio of europium fluorescent microspheres to DNP antibody is (1-10):1. DNP antibody ensures the effectiveness of the conjugation reaction and verifies the functionality of the microspheres, thereby optimizing the conjugation efficiency in the experiment. The microsphere labeling blocking solution reduces non-specific interactions between other molecules or antibodies and the microspheres.

[0044] Preparation steps of antibody-conjugated microsphere binding pad: Mix the detection antibody-conjugated fluorescent microspheres and the quality control antibody-conjugated fluorescent microspheres at a volume ratio of (10-1):1 to form a fluorescent microsphere solution, apply the fluorescent microsphere solution onto a glass fiber membrane, and dry it;

[0045] Sample pad pretreatment steps: Coat the sample pad buffer evenly with a glass fiber membrane, let the coated sample pad stand at room temperature; dry the sample pad;

[0046] The main function of the sample pad is to absorb the sample and regulate its flow rate, thereby ensuring the stability of the detection process. The sample pad buffer is uniformly coated onto the surface of the glass fiber membrane, and then the coated glass fiber membrane is left to stand overnight at room temperature to ensure that the treatment solution is fully adsorbed into the fiber membrane. Next, the glass fiber membrane is dried. After drying, the sample pad can be stored in a constant temperature and humidity drying cabinet. The sample pad buffer is prepared according to the required ratio. For example, the sample pad buffer is prepared as follows: 0.1-1% PEG-4000, 0.1-2% PVP-40000, 0.1-2% BSA, 0.1-2% sodium casein, 1.5-3.5% Triton X-100, and 0.1-1% NaCl are dissolved in 0.01-0.05M borate buffer with a pH of 7-8 by mass percentage to obtain the sample pad buffer. The tear sample first comes into contact with the sample pad, which generally performs a rapid pretreatment of the tear sample, including pH adjustment and blocking of non-specific sites.

[0047] Pretreatment steps for nitrocellulose membranes: Mouse anti-human IgG antibody and DNP-Ag are coated onto the surface of the nitrocellulose membrane using a gold-spraying coating instrument; mouse anti-human IgG antibody forms the detection line, and DNP-Ag forms the control line; after coating, the nitrocellulose membrane is dried.

[0048] 10-50 μg / mL of mouse anti-human IgG antibody and 10-50 μg / mL of DNP-Ag can be added to the detection line and control line channels of a gold-sprayed membrane scrubbing apparatus, respectively. Using the apparatus, the antibodies are uniformly coated onto the nitrocellulose membrane surface at a specific rate (e.g., 0.5-2 μL / cm), with the mouse anti-human IgG antibody forming the detection line (T line) and the DNP-Ag forming the control line (C line). After the nitrocellulose membrane dries, it promotes the binding reaction between the antibody or antigen protein and the membrane surface, ensuring that the antibody or antigen protein is firmly immobilized on the membrane through physical adsorption or chemical cross-linking.

[0049] Test strip assembly steps: Attach the sample pad, antibody-conjugated microsphere binding pad, nitrocellulose membrane, and absorbent paper horizontally to the base plate, overlapping adjacent pads. After this step, a programmable strip cutter can be used to cut the assembled strips into 3.9mm ± 1mm ​​widths to obtain the AChR-Ab test strips.

[0050] The base plate serves as the substrate for the test strip and can be made of known materials, such as PVC. The main function of the absorbent paper is to provide capillary action for sample chromatography and absorb excess reagent, thus preventing excessive liquid diffusion from interfering with the test results. In this step, the antibody-conjugated microspheres, nitrocellulose membrane, and absorbent paper are sequentially and horizontally attached to the base plate. The transition areas between the sample pad and the antibody-conjugated microspheres overlap, as do the transition areas between the antibody-conjugated microspheres and the nitrocellulose membrane, and the transition areas between the nitrocellulose membrane and the absorbent paper. This overlap between components ensures uniform liquid flow on the test strip. Uniform liquid flow is a prerequisite for an effective immune reaction. If the flowability is insufficient, the sample liquid may not fully contact the detection area, leading to incomplete signals or increased errors. Therefore, by rationally designing the overlapping areas of the test strip components, smooth liquid flow can be ensured, thereby ensuring the sensitivity and accuracy of the detection.

[0051] This solution provides a method for preparing AChR-Ab test strips, which can produce AChR-Ab test strips. It addresses the shortcomings of existing AChR-Ab tests, such as poor patient compliance in children due to reliance on venous blood collection and inability to reflect local antibody expression in the eye. By detecting tear-specific AChR-Abs, it can increase the diagnostic sensitivity of OMG from 50% to ≥80% based on serum standards, and significantly shorten the detection time, simplifying the detection process.

[0052] Alternatively, in the step of preparing the antibody-conjugated microsphere binding pad, the detection antibody-conjugated fluorescent microspheres and the quality control antibody-conjugated fluorescent microspheres are added to a three-dimensional spraying platform, and the mixed microspheres are sprayed onto the glass fiber membrane at a rate of 1-6 μL / cm using the three-dimensional spraying platform.

[0053] Glass fiber membranes possess high chemical stability and temperature resistance, resulting in excellent stability under various experimental conditions. While other materials such as nylon and polyester membranes can be used as alternatives, glass fiber membranes exhibit superior performance in microsphere adsorption, dispersion uniformity, and stability, with fewer issues of precipitation or unevenness, making them the ideal material for this experiment. In particular, this embodiment utilizes a three-dimensional spraying platform to spray a mixture of antibody-coupled fluorescent microspheres and quality control antibody-coupled fluorescent microspheres onto the glass fiber membrane. This is because the glass fiber membrane has high porosity, a large specific surface area, and good mechanical strength, effectively supporting the adsorption and reaction of the microspheres, while also utilizing its good liquid flowability.

[0054] In an optimized manner, during the test strip assembly step, the sample pad, antibody-conjugated microsphere conjugate pad, nitrocellulose membrane, and absorbent paper are sequentially pasted onto the base plate in a horizontal order, with the horizontal overlap between adjacent pads being 0.5-3 mm.

[0055] The sample pad, nitrocellulose membrane, and absorbent paper are attached to the base plate in sequence, with an overlap of 0.5-32 mm between each component. This range ensures that the liquid flows evenly on the test strip, thereby ensuring the sensitivity and accuracy of the detection.

[0056] Alternatively, after performing the step of preparing the detection antibody-conjugated fluorescent microspheres, the detection antibody-conjugated fluorescent microspheres can be ultrasonically treated using an ultrasonic disruptor.

[0057] After performing the step of preparing quality control antibody-conjugated fluorescent microspheres, the quality control antibody-conjugated fluorescent microspheres are ultrasonically treated using an ultrasonic disruptor.

[0058] Using an ultrasonic disruptor to sonicate microspheres ensures uniform dispersion of the microspheres, avoiding problems such as excessively high or low local concentrations.

[0059] Alternatively, in the pretreatment step of the nitrocellulose membrane, after coating, the nitrocellulose membrane is dried at 36-38°C for 8-16 hours.

[0060] A temperature range of 36-38℃ not only helps accelerate moisture evaporation, ensuring that antibodies are firmly fixed on the membrane through physical adsorption or chemical cross-linking, but also, with appropriate temperature and drying time, improves antibody binding efficiency and prevents antibody denaturation due to excessively high temperatures, thereby maintaining the detection performance and stability of the nitrocellulose membrane.

[0061] An AChR-Ab test strip is prepared by any of the above-described methods for preparing an AChR-Ab test strip.

[0062] like Figure 5The AChR-Ab test strip consists of a sample pad 1, an antibody-conjugated microsphere binding pad 2, a nitrocellulose membrane 3, absorbent paper 4, and a base plate 5.

[0063] An OMG diagnostic kit based on the detection of tear AChR-Ab includes: an AChR-Ab test strip according to any of the above embodiments.

[0064] In addition to the AChR-Ab test strip, the OMG diagnostic kit may also include Schirmer tear test strips, EP tubes, and PBS solution. The Schirmer tear test strips are used for tear collection. The EP tubes are used to hold the Schirmer tear test strips and can also be used to prepare the PBS solution or mix the PBS solution with the Schirmer tear test strips. The PBS solution can be prepared as a dilution as needed, for example, by adding 5-50 μL of PBS solution for every 1 mm of wet length of the Schirmer tear test strip.

[0065] A method of using an AChR-Ab test strip, using an AChR-Ab test strip of any of the above embodiments, includes: a sample application step;

[0066] The spotting step is as follows: 90-120 μL of tear fluid sample is added to the spotting well of the AChR-Ab test strip for detection. After the test strip reacts, the fluorescence signal intensity of the test line and control line is read using an immunofluorescence analyzer.

[0067] Add 90-120 μL of tear fluid sample to the well of the AChR-Ab test strip for detection; each experiment is repeated 3 times; after the test strip has reacted for 15 minutes, immediately use a dry immunofluorescence analyzer to read the fluorescence signal intensity of the test line (T line) and control line (C line) and record the data for subsequent analysis. This protocol develops a portable detection device based on lateral flow immunochromatography, reducing the detection time from 2-48 hours in the current technology to 15 minutes.

[0068] Optimally, it may also include: tear collection steps and tear sample processing steps;

[0069] The tear collection procedure is as follows: Gently pull the patient's lower eyelid conjunctiva and apply moderate pressure backward. In a non-invasive manner, gently place one end of the Schirmer tear test strip with the yellow indicator line into the conjunctival sac on the outer side of the patient's lower eyelid, and let the other end of the Schirmer tear test strip hang naturally on the outer side of the lower eyelid. Instruct the patient to gently close their eyes and look slightly upward, maintaining this position to allow the Schirmer tear test strip to fully absorb the basal tears.

[0070] The tear sample processing steps are as follows: Schirmer tear test strips are mixed with PBS diluent, placed in an ultrasonic processor, and then centrifuged to remove solid residues. The supernatant is then used as the tear sample for the spotting step.

[0071] This protocol addresses the shortcomings of existing AChR-Ab tests, which rely on venous blood collection, leading to poor patient compliance in children and failing to reflect local ocular antibody expression. The proposed AChR-Ab test strips establish a non-invasive testing system based on tear samples, overcoming the bottleneck of insufficient sensitivity in OMG serum testing. For serum-negative OMG patients (accounting for 50% of the diagnosed population), by detecting tear-specific AChR-Abs, the diagnostic sensitivity for OMG is increased from 50% of the serum baseline to ≥80%.

[0072] The use of a test strip in the preparation of an OMG diagnostic kit for detecting AChR-Ab in tears, wherein the test strip is an AChR-Ab test strip of any of the above embodiments.

[0073] Example:

[0074] (I) Tear collection and processing, the general process is as follows: Figure 1 :

[0075] (1) Tear sample collection:

[0076] 1) Operation preparation: Operators must wear medical gloves to ensure that the entire operation process meets aseptic requirements.

[0077] 2) Test strip preparation: Use Schirmer tear test strips and fold them at a right angle along the preset yellow indicator line.

[0078] 3) Test strip placement: Gently pull the patient's lower eyelid conjunctiva and apply moderate pressure backward. In a non-invasive manner, gently place the end of the test strip with the yellow indicator line into the conjunctival sac at the outer 1 / 3 of the patient's lower eyelid, allowing the other end to hang naturally on the outside of the lower eyelid.

[0079] 4) Sample collection: Instruct the patient to gently close their eyes and look slightly upward, maintaining this position for 5-10 minutes to ensure that the test strip fully absorbs the basal tear fluid.

[0080] 5) Sample preservation: Quickly and carefully remove the test strip, place it in a sterile EP tube, and immediately freeze it in an ultra-low temperature freezer at -80°C for subsequent analysis.

[0081] (2) Tear sample processing steps:

[0082] 1) Test strip preparation: Remove the tear test strip from the -80℃ freezer and cut it into small pieces using sterile scissors.

[0083] 2) Preparation of diluent: Prepare diluent by adding 10 μL of PBS solution for every 1 mm of wet length of the test strip.

[0084] 3) Ultrasonic treatment: Mix the shredded test paper with an appropriate amount of PBS solution, place it in an ultrasonic processor, set the frequency to 80Hz and the power to 100%, and run for 10 minutes to achieve thorough physical breaking and mixing.

[0085] 4) Centrifugation and storage: After ultrasonic treatment, remove solid residue by centrifugation, carefully mix the supernatant by blowing and transferring it to a new sterile EP tube, and store it again in a -80℃ freezer for subsequent testing.

[0086] (II) Preparation method of AChR-Ab test strips

[0087] (1) Preparation steps of antibody-conjugated fluorescent microspheres:

[0088] 15 μL of europium fluorescent microspheres were diluted with 495 μL of MSE buffer, followed by the addition of 5 μL of microsphere labeling activator and 5 μL of microsphere labeling stabilizer. The mixture was reacted for 30 minutes at room temperature in the dark. After activation, the microsphere suspension was centrifuged at 15,000 rpm for 30 minutes at 4°C, the supernatant was discarded, and the microspheres were resuspended in 495 μL of MSE buffer. Then, 12.5 μg of AChRα1 recombinant protein was added to the resuspended microsphere suspension, and the mixture was incubated for 3 hours at room temperature in the dark. After incubation, 5 μL of microsphere labeling blocking buffer was added, and the mixture was blocked for another 30 minutes at room temperature in the dark. Finally, the microsphere suspension was centrifuged at 15,000 rpm for 30 minutes at 4°C, the supernatant was discarded, and the microspheres were resuspended in 250 μL of microsphere labeling preservation buffer. The suspension was then processed using an ultrasonic homogenizer to obtain the antibody-conjugated fluorescent microspheres.

[0089] (2) Preparation steps of quality control antibody-conjugated fluorescent microspheres:

[0090] 15 μL of europium fluorescent microspheres were diluted with 495 μL of MSE buffer, followed by the addition of 5 μL of microsphere labeling activator and 5 μL of microsphere labeling stabilizer. The mixture was reacted at room temperature in the dark for 30 minutes. After activation, the microsphere suspension was centrifuged at 4°C and 15,000 rpm for 30 minutes, the supernatant was discarded, and the microspheres were resuspended in 495 μL of MSE buffer. Then, 3 μg of DNP antibody was added to the resuspended microsphere suspension, and the mixture was incubated at room temperature in the dark for 3 hours. After incubation, 5 μL of microsphere labeling blocking buffer was added, and the mixture was blocked for another 30 minutes at room temperature in the dark. Finally, the microsphere suspension was centrifuged at 4°C and 15,000 rpm for 30 minutes, the supernatant was discarded, and the microspheres were resuspended in 250 μL of microsphere labeling preservation buffer. The suspension was then processed using an ultrasonic homogenizer to obtain the final quality control antibody-conjugated fluorescent microspheres.

[0091] (3) Preparation steps of antibody-conjugated microsphere binding pads:

[0092] The glass fiber membrane was cut into 1×30cm pieces using a wide strip cutter. The detection antibody-conjugated fluorescent microspheres and the quality control antibody-conjugated fluorescent microspheres were mixed at a 3:1 (V:V) ratio to form a fluorescent microsphere solution, which was then added to a three-dimensional spraying platform. The mixed microspheres were uniformly sprayed onto the glass fiber membrane at a rate of 3 μL / cm using the three-dimensional spraying platform, and then dried in a 37℃ forced-air drying oven for 12 hours. After the antibody-conjugated microsphere binding pads were completely dry, they were stored in a constant temperature and humidity drying cabinet for later use.

[0093] (4) Sample pad pretreatment steps:

[0094] A 50 mL sample pad buffer solution was evenly coated onto the surface of a 20 × 30 cm glass fiber membrane. The coated membrane was then left to stand overnight at room temperature to ensure the treatment solution was fully adsorbed into the fiber membrane. The glass fiber membrane was then transferred to a 37°C forced-air drying oven and dried for 6 hours to ensure uniform drying and stable performance. After drying, the glass fiber membrane was cut into 2 × 30 cm strips using a wide strip cutter.

[0095] The sample pad buffer is prepared as follows: 0.5% PEG-4000, 1% PVP-40000, 1% BSA, 1% sodium caseinate, 2.5% Triton X-100 and 0.5% NaCl are dissolved in 0.02M borate buffer with pH 7.4 by mass percentage to obtain the sample pad buffer.

[0096] (5) Pretreatment steps for nitrocellulose membranes:

[0097] Add 1 mg / mL of mouse anti-human IgG antibody and 1 mg / mL of DNP-Ag to the detection line and control line channels of the streak sprayer, respectively. Using the streak sprayer, evenly spread the antibodies onto the nitrocellulose membrane surface at a rate of 1 μL / cm, with the mouse anti-human IgG antibody forming the detection line (T line) and the DNP-Ag forming the control line (C line). After coating, transfer the nitrocellulose membrane to a 37°C forced-air drying oven and dry for 12 hours. Once the membrane strip is completely dry, seal and store for later use.

[0098] (6) Preparation of absorbent paper

[0099] The H2 absorbent paper was cut into strips of 2.5×30cm using a wide strip cutter to ensure that the cutting process was uniform and the dimensions were accurate and met the experimental requirements.

[0100] (7) Test strip assembly steps:

[0101] The sample pad, antibody-conjugated microsphere binding pad, nitrocellulose membrane, and absorbent paper are sequentially attached to the PVC base plate, with each component overlapping by 2 mm. The assembled strips are then cut into 3.9 mm wide individual test strips using a programmable strip cutter and placed into plastic cartridges.

[0102] (8) Spotting steps:

[0103] Add 90-120 μL of tear fluid sample to the spotting well of the AChR-Ab test strip for detection; repeat each experiment 3 times; after the test strip has reacted for 15 minutes, immediately use a dry immunofluorescence analyzer to read the fluorescence signal intensity of the test line (T line) and the control line (C line) and record the data for subsequent analysis.

[0104] Experiment 1: Validation of tear AChR-Ab based on cell detection method:

[0105] 1. Cell preparation

[0106] Observe cell state: Remove 10cm cells from a 37℃, 5% CO2 incubator. 2 Cell culture dishes are used to observe cell growth under a microscope to ensure that cells are growing well and free from contamination.

[0107] Cell digestion: Discard the culture medium and use Ca-free... 2+ / Mg 2+ Gently rinse the cells 1-2 times with PBS to remove residual culture medium; add 1 mL of 0.25% trypsin-EDTA solution preheated to 37°C, evenly cover the cell layer, and incubate in a 37°C cell culture incubator for 2 minutes. During this time, observe the cells under a microscope as they become round and begin to detach to avoid over-digestion; add 4 mL of culture medium containing FBS to stop the trypsin effect and gently mix by pipetting.

[0108] Cell collection: Transfer the cell suspension to a 15 mL sterile centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant.

[0109] Cell counting: Resuspend the cell pellet in 1 mL of fresh culture medium, take 10 μL of cell suspension, and use a hemocytometer to count the cell density and calculate the cell number.

[0110] Cell plating: Take 5 × 10 5 10 cells / well, evenly seeded in a six-well cell culture plate. Add 2 mL of HEK293T cell culture medium preheated to 37°C to each well to ensure uniform cell distribution.

[0111] Waiting for transfection: Place the culture plate in a 37℃, 5% CO2 cell culture incubator for 12-24 hours until the cell confluence reaches 80%-90%, then prepare for transfection.

[0112] 2. Cell transfection

[0113] According to Lipofectamine TM The instructions for the 3000 transfection kit state that all reagents should be equilibrated to room temperature before use and handled under aseptic conditions in a laminar flow hood to ensure the accuracy and reproducibility of the experiment. The specific steps are as follows (using one well of a six-well plate as an example):

[0114] Lipofectamine TM Preparation of 3000 dilution: Transfer 125 μL of Opti-MEM (serum-free medium) to a sterile centrifuge tube. Add 5 μL of Lipofectamine. TM 3000, gently blow and mix well, let stand for 5 minutes for later use.

[0115] Plasmid DNA dilution preparation: Transfer 125 μL of Opti-MEM to a sterile centrifuge tube; add 2.5 μg of plasmid DNA (pAChRα1-pcDNA3.1(+), purchased from Qingke Biotechnology Co., Ltd.). Add 5 μL of LP3000... TM Mix the reagents by gently blowing them into the container.

[0116] Mixed transfection reagent: Add plasmid DNA dilution buffer to Lipofectamine TM In a 3000 mL solution, gently mix by pipetting. Let stand at room temperature for 15 minutes to allow the lipid-nucleic acid complex to form, thus enhancing transfection efficiency.

[0117] Change the culture medium: Remove the culture plate, discard the old culture medium, and replace it with 1.5 mL of fresh Opti-MEM culture medium (to improve transfection efficiency).

[0118] Plasmid transfection: Slowly add the transfection mixture dropwise into the cell culture wells and gently shake the culture plate to ensure even distribution.

[0119] Continue culturing: Incubate at 37°C in a 5% CO2 cell culture incubator for 4-6 hours.

[0120] Change the culture medium: After 4-6 hours, discard the Opti-MEM medium, add 2 mL of fresh HEK293T cell culture medium, and continue culturing.

[0121] 24 hours after transfection, the cell status was checked: cell growth was observed to ensure good cell viability and no obvious cell death or floating phenomenon.

[0122] 3. Cell-Based Assay (CBA)

[0123] 1) Cell seeding and culture: First, the transfected HEK293T cells were seeded at a rate of 5 × 10⁶ cells / year. 4 Cells were seeded at a density of 1 cell / well on crawling slides in 24-well plates and initially cultured for 6 hours in DMEM medium containing 10% fetal bovine serum, 1% penicillin-streptomycin solution, and 1% glutamine. After cell attachment, immunofluorescence staining was performed to detect AChRα1 expression.

[0124] 2) Cell washing: Carefully aspirate the cell culture medium and wash the cells with PBS three times for three minutes each time.

[0125] 3) Cell fixation: Add 1 mL of freshly prepared 75% ethanol solution to each well and fix the cells for 20 minutes.

[0126] 4) Wash again: Wash the cells with PBS three times, for three minutes each time.

[0127] 5) Blocking treatment: Add 100 μL of goat serum stock solution to each well and block for 1 hour. Incubate at room temperature to block the cells, thereby blocking non-specific binding and reducing staining background.

[0128] 6) Washing and blocking solution: Wash cells with PBS, three times, for three minutes each time.

[0129] 7) Primary antibody incubation: Add 100 μL of the corresponding sample (serum and tear samples of OMG and HC) to the well and incubate overnight at 4°C.

[0130] 8) Washing primary antibody: The next day, remove the primary antibody and wash the cells with PBS three times, each time for 10 minutes.

[0131] 9) Secondary antibody incubation: Add goat anti-human FITC solution (1:200 dilution) to each well and incubate at room temperature in the dark for 2 hours.

[0132] 10) Washing secondary antibody: Wash cells with PBS, three times, each time for 10 minutes.

[0133] 11) Nuclear staining: Add 50 μL of Hoechst nuclear staining solution (1:500 dilution) and incubate at room temperature in the dark for 10 minutes.

[0134] 12) Final washing: Wash the cells with PBS 5 times, each time for 10 minutes.

[0135] 13) Mounting and observation: Finally, mount the slides with an anti-fluorescence quencher and observe the cells and acquire images using a laser confocal microscope.

[0136] 4. Experimental Results

[0137] (1) Verification of the presence of AChR-Ab in tears: Mixed serum samples and paired mixed tear samples were collected from 3 AChR-Ab positive OMG patients and 3 healthy controls. The presence of AChR-Ab in tears was verified by CBA analysis. Results showed that serum from OMG patients ( Figure 2 A) and tears ( Figure 2 B) The transfected cells after incubation all exhibited significantly enhanced green fluorescence on their cell membranes, indicating the presence of AChR-Ab in both patient serum and tear samples. In contrast, no detectable green fluorescence was observed in transfected cells incubated with serum and tears from healthy controls.

[0138] 5. Conclusion

[0139] (1) This embodiment is the first to clearly detect that AChR-Ab in the tears of OMG patients can specifically bind to AChRα1 protein on the transfected cell membrane, confirming the presence of AChR-Ab in tears.

[0140] (2) The AChR-Ab level in the tears of patients with OMG was significantly higher than that in the healthy control group, and the immunofluorescence colocalization was clear, indicating that the AChR-Ab detection in tears has good specificity for the diagnosis of OMG.

[0141] Experiment 2: Application of tear AChR-Ab lateral flow immunochromatographic test strip

[0142] 1. Experimental Design

[0143] In this embodiment, tear samples and matched serum samples from 10 clinically diagnosed OMG patients were collected as the positive group, and tear samples and matched serum samples from 5 healthy controls (HC) were collected as the negative control group.

[0144] 2. Experimental Results

[0145] (1) Results of EuNPs-LFIA in tear fluid

[0146] Data shows that the T-line fluorescence value of the OMG group was generally higher than that of the HC group. Figure 3 A) indicates elevated AChR-Ab levels in the tear fluid of patients with OMG. To further evaluate the diagnostic efficacy of the EuNPs-LFIA method, ROC curves were plotted based on all enrolled samples in this study. Figure 3 B). The analysis results showed that the area under the ROC curve (AUC) was 0.80 (95% confidence interval: 0.65-0.95, p = 0.066), indicating that it has certain clinical application value in diagnostic efficacy, especially in differentiating OMG patients from healthy controls. However, since the p-value was slightly higher than 0.05 and the confidence interval was relatively wide, further verification of the stability and reliability of this method is needed in future practical applications. Based on ROC curve analysis, the optimal cut-off value of T-line fluorescence value was determined to be 1682, with a sensitivity of 80% (95% confidence interval: 49.02%-96.45%) and a specificity of 80% (95% confidence interval: 37.55%-98.97%). Figure 3 C). Based on the optimal cutoff value determined by the ROC curve, EuNPs-LFIA images of tear fluids from OMG patients and healthy controls were analyzed. Sample 1 from the healthy control group and samples 6, 8, 10, 12, 13, 14, and 15 from OMG patients were positive. Figure 3 D).

[0147] (2) Serum EuNPs-LFIA test results

[0148] The results showed that the T-line fluorescence value of the OMG group was not significantly different from that of the HC group. Figure 4 A) indicates that this method cannot effectively distinguish between OMG patients and healthy individuals in serum samples. To further evaluate the diagnostic efficacy of the serum EuNPs-LFIA method, ROC curves were plotted based on all enrolled samples. Figure 4 B) The results showed that the area under the ROC curve (AUC) was 0.51 (95% confidence interval: 0.2–0.82, p = 0.951). ROC curve analysis indicated that the optimal cut-off value for the T-line fluorescence value was 1335, with a sensitivity of 40% (95% confidence interval: 16.82%–68.73%) and a specificity of 100% (95% confidence interval: 56.55%–100.0%). Figure 4C). Serum EuNPs-LFIA images of OMG patients and healthy controls were analyzed based on the optimal cutoff value determined by the ROC curve. No positive samples were found in the healthy control group, while samples 7, 9, 11, and 14 from OMG patients were positive. Figure 4 C).

[0149] (3) This experiment evaluated the EuNPs-LFIA test strip results based on the optimal cutoff value determined by the aforementioned ROC curve, and analyzed the concordance rate between the EuNPs-LFIA test strip results and clinical diagnosis. For the EuNPs-LFIA test of tear AChR-Ab, the results showed a negative concordance rate of 80% (healthy donor group) and a positive concordance rate of 80% (OMG patient group). This indicates that the tear AChR-Ab test has high accuracy in distinguishing OMG patients from healthy controls (see Table 1). In contrast, the EuNPs-LFIA test results of serum AChR-Ab showed that although its negative concordance rate reached 100%, its positive concordance rate was only 40% (see Table 2). This means that although the serum test performed well in excluding healthy controls, its accuracy in identifying OMG patients was low. These analytical results highlight the potential advantages of the tear AChR-Ab test in the diagnosis of OMG, especially in improving the positive concordance rate, showing better diagnostic efficacy compared to the serum AChR-Ab test.

[0150] Table 1 - Concordance rate between EuNPs-LFIA results of tear AChR-Ab and clinical diagnosis

[0151]

[0152] Table 2 - Concordance rate between EuNPs-LFIA results of serum AChR-Ab and clinical diagnosis

[0153]

[0154] 3. Conclusion

[0155] The results of this experiment indicate that tears may be a more reliable biological sample for the non-invasive diagnosis and differential diagnosis of OMG compared to serum samples. EuNPs-LFIA detection of tear AChR-Ab showed a high positive concordance rate, significantly superior to the results obtained from serum samples. This finding demonstrates that tear testing has significant advantages in improving the accuracy and efficiency of OMG diagnosis. As a non-invasive and readily available biological sample, tears hold promise as an important tool for OMG diagnosis, especially in cases requiring rapid and convenient screening and differential diagnosis.

[0156] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The use of a test strip in the preparation of an OMG diagnostic kit for detecting tear AChR-Ab, characterized in that, The test strip is an AChR-Ab test strip; The preparation method of the AChR-Ab test strip includes the following steps: Preparation steps for antibody-conjugated fluorescent microspheres: Dilute europium fluorescent microspheres with MSE buffer, add microsphere labeling activator and microsphere labeling stabilizer sequentially; centrifuge the formed microsphere suspension, discard the supernatant, and resuspend the microspheres with MSE buffer; add AChRα1 recombinant protein to the resuspended microsphere suspension; add microsphere labeling blocking solution; centrifuge the microsphere suspension and discard the supernatant; Steps for preparing quality control antibody-conjugated fluorescent microspheres: Dilute europium fluorescent microspheres with MSE buffer, and add microsphere labeling activator and microsphere labeling stabilizer sequentially; centrifuge the formed microsphere suspension, discard the supernatant, and resuspend the microspheres with MSE buffer; add DNP antibody to the resuspended microsphere suspension; add microsphere labeling blocking solution; centrifuge the microsphere suspension and discard the supernatant; Preparation steps of antibody-conjugated microsphere binding pad: Mix the detection antibody-conjugated fluorescent microspheres and the quality control antibody-conjugated fluorescent microspheres at a volume ratio of (10-1):1 to form a fluorescent microsphere solution, apply the fluorescent microsphere solution onto a glass fiber membrane, and dry it; Sample pad pretreatment steps: Coat the sample pad buffer evenly with a glass fiber membrane, let the coated sample pad stand at room temperature; dry the sample pad; Pretreatment steps for nitrocellulose membranes: Mouse anti-human IgG antibody and DNP-Ag are added to the detection line and control line channels of the gold-spraying membrane coating machine, respectively. The antibody is then coated onto the surface of the nitrocellulose membrane using the gold-spraying membrane coating machine. Mouse anti-human IgG antibody forms the detection line, and DNP-Ag forms the control line. After coating, the nitrocellulose membrane is dried. Test strip assembly steps: Paste the sample pad, antibody-conjugated microsphere binding pad, nitrocellulose membrane and absorbent paper onto the base plate in a horizontal order, with adjacent ones overlapping; The OMG diagnostic kit includes: AChR-Ab test strips, Schirmer tear test strips, EP tubes, and PBS solution; The Schirmer tear test strip is used for tear collection; The EP tube is used to hold Schirmer tear test strips, to prepare PBS solutions, and to mix PBS solutions with Schirmer tear test strips. The PBS solution was prepared into a diluent by adding 5-50 microliters of PBS solution for every 1 mm of wet length of the Schirmer tear test strip.

2. The use of the test strip according to claim 1 in the preparation of an OMG diagnostic kit for detecting tear AChR-Ab, characterized in that, In the step of preparing the antibody-conjugated microsphere binding pad, the detection antibody-conjugated fluorescent microspheres and the quality control antibody-conjugated fluorescent microspheres are added to a three-dimensional spraying platform, and the mixed microspheres are sprayed onto the glass fiber membrane at a rate of 1-6 μL / cm using the three-dimensional spraying platform.

3. The use of the test strip according to claim 1 in the preparation of an OMG diagnostic kit for detecting tear AChR-Ab, characterized in that, In the test strip assembly step, the sample pad, antibody-conjugated microsphere conjugate pad, nitrocellulose membrane and absorbent paper are pasted onto the base plate in a horizontal order, with the horizontal overlap between adjacent pads being 0.5-3 mm.

4. The use of the test strip according to claim 1 in the preparation of an OMG diagnostic kit for detecting tear AChR-Ab, characterized in that, After the step of preparing the detection antibody-conjugated fluorescent microspheres, the detection antibody-conjugated fluorescent microspheres were subjected to ultrasonic treatment using an ultrasonic disruptor. After performing the step of preparing quality control antibody-conjugated fluorescent microspheres, the quality control antibody-conjugated fluorescent microspheres are ultrasonically treated using an ultrasonic disruptor.

5. The use of the test strip according to claim 2 in the preparation of an OMG diagnostic kit for detecting tear AChR-Ab, characterized in that, In the pretreatment step of the nitrocellulose membrane, after coating, the nitrocellulose membrane is dried at 36-38℃ for 8-16 hours.

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