AChR-Ab detection test strip as well as use method, preparation method and application thereof

Abstract

The invention discloses an AChR-Ab detection test strip as well as a use method, a preparation method and application thereof. The preparation method comprises the following steps: preparing the detection antibody coupling fluorescent microspheres, preparing the quality control antibody coupling fluorescent microspheres, preparing the antibody coupling microsphere combination pad, pretreating the sample pad, pretreating the nitrocellulose membrane and assembling the detection test strip. The use method comprises the sample application steps: adding a tear sample into the sample application hole of the AChR-Ab detection test strip for detection, and after the test strip reacts, reading the fluorescence signal intensity of the detection line and the quality control line by using an immunofluorescence analyzer. According to the scheme, by detecting the tear specificity AChR-Ab, the detection sensitivity is improved from 50% of a serum standard to more than or equal to 80%, the detection time can be greatly shortened, and the detection process is simplified.

Description

Technical Field

[0001] The present invention relates to the field of AChR-Ab detection test strips, and in particular to an AChR-Ab detection test strip, a use method, a preparation method and application thereof. Background Art

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

[0003] Although OMG lesions are confined to the ocular muscles, its typical symptoms (such as ptosis, diplopia, and strabismus) can still severely impact patients' visual function, quality of life, and overall safety. Long-term, uncontrolled OMG can lead to amblyopia and even permanent visual impairment due to weakness and atrophy of the extraocular muscles. Persistent incomplete eyelid closure can lead to complications such as dry eye and corneal ulcers, and in severe cases, blindness. Furthermore, ptosis and diplopia can severely impact patients' basic daily activities, such as driving, reading, and walking. Of particular concern is that once OMG transforms into generalized myasthenia gravis (GMG), patients are significantly more likely to develop serious complications such as dysphagia, dysarthria, and respiratory failure. Although respiratory muscle involvement is rare in OMG, when it occurs, it can rapidly progress to a myasthenic crisis, requiring urgent intensive care and mechanical ventilation, significantly increasing the difficulty of treatment and the risk of death. Existing evidence-based medicine suggests that early intervention can effectively interrupt the progression of OMG to GMG. A clinical prediction model developed by researchers such as Bi et al. showed that early application of immunosuppressive therapy can significantly reduce the risk of transformation from a baseline level of 22.6% to 6.8%. A retrospective study by the team of Japanese scholar Teppei Komatsu further confirmed that initiating immunotherapy within the "golden intervention window" of 255 days after symptom onset has a sensitivity of 92.9% and a specificity of 91.2% for patients reaching "minimal manifestation status" (MMS). This early intervention strategy not only improves disease prognosis but, more importantly, prevents secondary damage caused by persistent ocular muscle weakness, including potentially blinding complications such as amblyopia and corneal ulcers. Therefore, the clinical management of OMG should receive the same level of attention as GMG.

[0004] Currently, the diagnosis of OMG is mainly based on a comprehensive evaluation of clinical manifestations combined with serum antibody testing, electrophysiological examinations, and pharmacological tests. Acetylcholine receptor antibodies (AChR-Ab), as the most diagnostically valuable specific autoantibody for OMG, have been listed as the preferred auxiliary examination for diagnosis in domestic and international guidelines. However, the positive rate of serum AChR-Ab in GMG patients can reach 85%-90%, while in OMG patients, the positive rate is only about 50%, affected by the localization of ocular lesions and mild symptoms. Studies have shown that the misdiagnosis rate of OMG is as high as 46%. This situation urgently requires the development of new diagnostic methods that are highly sensitive, non-invasive, and clinically practical.

[0005] The commonly used clinical methods for serum AChR-Ab detection are mainly the following three: (1) Radioimmunoprecipitation Assay (RIA): quantification is performed by combining radiolabeled antigens with serum antibodies to form a complex. It has the risk of radioactive contamination (using 125I isotope), the reagent has a short shelf life (≤1 month), and the sensitivity is low in OMG (about 50%). (2) Enzyme-linked immunosorbent assay (ELISA): linear peptide antigens are used to coat microplates and antibodies are detected by colorimetric reaction. Since linear antigens cannot simulate the natural conformation of AChR, the sensitivity is not as good as RIA, and the strong acid / strong base colorimetric solution causes environmental pollution. In addition, imported test kits are expensive and the cost of a single test is high, which limits clinical application. (3) Cellular immunofluorescence assay (CBA): HEK293 cells transfected with AChR subunits are used to capture antibodies. Although low-affinity antibodies can be detected (OMG positivity rate increased to 50%), they require cell culture equipment (detection cycle > 24 hours), are complex to operate, and lack standardized reagent kits. They are expensive and 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 for diagnosing OMG: serum AChR-Ab cannot reflect the level of local antibodies in the eye, resulting in an OMG sensitivity of only about 50%, which is much lower than GMG (85%-90%), resulting in a high rate of missed diagnosis and misdiagnosis; (3) High technical barriers: RIA requires radiation protection qualifications, ELISA has a high false positive rate, and CBA relies on a professional cell platform, resulting in a low penetration rate in grassroots medical institutions. In summary, the existing methods are insufficient for the detection of OMG, and there is an urgent need to develop new, non-invasive, highly sensitive and convenient detection technologies. Summary of the Invention

[0006] The present invention aims to provide a method for preparing an AChR-Ab detection test strip, which can produce an AChR-Ab detection test strip. The present invention can address the defects of existing AChR-Ab detection, which relies on venous blood sampling, resulting in poor compliance among pediatric patients and inability to reflect local antibody expression in the eye, by detecting tear-specific AChR-Ab.

[0007] To achieve this object, the present invention adopts the following technical solutions:

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

[0009] Preparation of detection antibody-coupled fluorescent microspheres: diluting europium fluorescent microspheres with MSE buffer, adding a microsphere labeling activator and a microsphere labeling stabilizer in sequence; centrifuging the formed microsphere suspension, discarding the supernatant, and resuspending the microspheres with MSE buffer; adding AChRα1 recombinant protein to the resuspended microsphere suspension; adding microsphere labeling blocking solution; centrifuging the microsphere suspension, and discarding the supernatant;

[0010] Preparation of quality control antibody-coupled fluorescent microspheres: dilute europium fluorescent microspheres with MSE buffer, add microsphere labeling activator and microsphere labeling stabilizer in sequence; centrifuge the resulting 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 of antibody-coupled microsphere conjugate pad: Mix detection antibody-coupled fluorescent microspheres and quality control antibody-coupled fluorescent microspheres at a volume ratio of (10-1):1 to form a fluorescent microsphere solution, apply the fluorescent microsphere solution on a glass fiber membrane, and dry;

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

[0013] Pretreatment steps of nitrocellulose membrane: Mouse anti-human IgG antibody and DNP-Ag are coated on the surface of nitrocellulose membrane using a gold spray film streak; the mouse anti-human IgG antibody forms the detection line and the DNP-Ag forms the quality control line; after coating, the nitrocellulose membrane is dried;

[0014] Test strip assembly steps: Paste the sample pad, antibody-coupled microsphere conjugate pad, nitrocellulose membrane and absorbent paper to the bottom plate in horizontal order, with adjacent ones overlapping.

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

[0016] Optimally, in the test strip assembly step, the sample pad, antibody-coupled microsphere conjugate pad, nitrocellulose membrane and absorbent paper are sequentially pasted onto the bottom plate in a horizontal order, with the horizontal overlap distance between adjacent ones being 0.5-3 mm.

[0017] Optionally, after the step of preparing the detection antibody-coupled fluorescent microspheres, the detection antibody-coupled fluorescent microspheres are ultrasonically treated using an ultrasonic disruptor;

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

[0019] Optimally, 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 detection test strip is prepared by the above-mentioned preparation method of an AChR-Ab detection test strip.

[0021] An OMG diagnostic kit for detecting tear AChR-Ab comprises the above-mentioned AChR-Ab detection test strip.

[0022] A method for using an AChR-Ab detection test strip, using the above-mentioned AChR-Ab detection test strip, comprising: a sample spotting step;

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

[0024] Optionally, the method may further include: a tear collection step and a tear sample processing step;

[0025] The tear collection steps include gently pulling the patient's lower eyelid conjunctiva and applying moderate pressure backward, and non-invasively placing one end of the Schirmer tear test strip with a yellow indicator line into the conjunctival sac on the outside of the patient's lower eyelid, with the other end of the Schirmer tear test strip naturally hanging outside the lower eyelid; instructing the patient to lightly close their eyes and look slightly upward, maintaining this state to allow the Schirmer tear test strip to fully absorb basal tears;

[0026] The tear sample processing step comprises: mixing the Schirmer tear test paper with the PBS diluent, placing the mixture in an ultrasonic processor, removing solid residues by centrifugation, and taking the supernatant as the tear sample for the spotting step.

[0027] A test strip is used in preparing an OMG diagnostic kit for detecting tear AChR-Ab. The test strip is the above-mentioned AChR-Ab detection test strip.

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

[0029] This proposal provides a method for preparing an AChR-Ab test strip, which can produce an AChR-Ab test strip. This method addresses the shortcomings of existing AChR-Ab testing, which relies on venous blood sampling, resulting in poor compliance among pediatric patients and an inability to reflect local antibody expression in the eye. By detecting tear-specific AChR-Ab, the diagnostic sensitivity of OMG can be increased from 50% of the serum baseline to ≥80%, and the test time can be significantly shortened, simplifying the test process. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a flow chart of tear collection and processing;

[0031] Figure 2 It is CBA to verify the expression of human AChRα1 subunit in HEK 293T cells;

[0032] Figure 3 Results of AChR-Ab detection strips (EuNPs-LFIA) for tear fluid 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 tears from OMG patients and HC groups; (B) ROC curve analysis of the sensitivity and specificity of tear EuNPs-LFIA for AChR-Ab detection; (C) Histogram of T-line fluorescence values ​​of AChR-Ab in tears from OMG patients and HC groups, with the dotted line indicating the detection cutoff value (cut-off=1682); (D) Image recording of AChR-Ab detection in tears from OMG patients and healthy controls using EuNPs-LFIA;

[0033] Figure 4 Figure 3. EuNPs-LFIA detection results 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 from OMG patients and HC groups; (B) Receiver operating characteristic (ROC) curve analysis of the sensitivity and specificity of EuNPs-LFIA for detection of serum AChR-Ab; (C) Histogram of T-line fluorescence values ​​of AChR-Ab in serum from OMG patients and HC groups; the dotted line indicates the detection cutoff (cut-off = 1335); (D) Image recording of EuNPs-LFIA detection of serum AChR-Ab in serum from OMG patients and HC groups.

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

[0035] in:

[0036] Sample pad 1, antibody-coupled microsphere binding pad 2, nitrocellulose membrane 3, absorbent paper 4, bottom plate 5. DETAILED DESCRIPTION

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

[0038] For ease of understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. Where specific techniques or conditions are not indicated in the embodiments, they are carried out according to the techniques or conditions described in the literature in this area or according to the product specifications. Where the manufacturer of the reagents or instruments is not indicated, they are all conventional products that can be obtained commercially.

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

[0040] Preparation of detection antibody-coupled fluorescent microspheres: diluting europium fluorescent microspheres with MSE buffer, adding a microsphere labeling activator and a microsphere labeling stabilizer in sequence; centrifuging the formed microsphere suspension, discarding the supernatant, and resuspending the microspheres with MSE buffer; adding AChRα1 recombinant protein to the resuspended microsphere suspension; adding microsphere labeling blocking solution; centrifuging the microsphere suspension, and discarding the supernatant;

[0041] The europium fluorescent microspheres are diluted with MSE buffer. The amount of MSE buffer used can be determined as needed. In general embodiments, the volume ratio of europium fluorescent microspheres to MSE buffer is 1:(30-100), preferably 1:(30-70), and optimally 1:(40-50). After adding the microsphere labeling activator and microsphere labeling stabilizer, after a certain reaction time (e.g., 10-60 minutes), after adding the microsphere labeling activator, the activator will promote the exposure and conversion of the carboxyl groups on the microsphere surface, thereby fully activating the microsphere surface, increasing its reactivity, and providing more binding sites to facilitate the subsequent coupling reaction with the AChRα1 recombinant protein. The microsphere labeling stabilizer helps maintain the stability of the reaction system and prevents the microspheres from aggregating or inactivating during the processing process. In this way, this step can fully activate the microsphere surface and prepare it for coupling with the recombinant protein. AChRα1 recombinant protein is added and incubated for a period of time (e.g., 1-3 hours) to allow the AChRα1 recombinant protein to fully bind to the activated carboxyl groups on the microsphere surface. The amount of AChRα1 recombinant protein added can be determined as needed, preferably in excess relative to the europium fluorescent microspheres. As a specific target, the AChRα1 recombinant protein binds to the activated carboxyl groups on the microsphere surface, completing the antibody coupling, thereby ensuring accurate detection of AChR antibodies in tears during the experiment. The addition of microsphere labeling blocking solution blocks unbound sites on the microsphere surface, reducing nonspecific interactions between other molecules or antibodies and the microspheres, thereby ensuring the accuracy and reliability of the test results.

[0042] Preparation of quality control antibody-coupled fluorescent microspheres: dilute europium fluorescent microspheres with MSE buffer, add microsphere labeling activator and microsphere labeling stabilizer in sequence; centrifuge the resulting 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 fluorescent microspheres coupled to detection antibodies, when preparing fluorescent microspheres coupled to quality control antibodies, europium fluorescent microspheres are diluted with MSE buffer (the amount of MSE buffer can be adjusted as needed). Microsphere labeling activator and microsphere labeling stabilizer fully activate the microsphere surface and prepare it for coupling with recombinant protein. DNP antibody is used to label specific binding sites on the microsphere surface and serves as a standardization control for subsequent quality control testing. The amount of DNP antibody added can be adjusted as needed, generally at a lower level than that of europium fluorescent microspheres, with a preferred volume ratio of europium fluorescent microspheres to DNP antibody of (1-10):1. DNP antibody ensures the effectiveness of the coupling reaction and verifies the functionality of the microspheres, thereby optimizing the coupling efficiency in the experiment. Microsphere labeling blocking buffer reduces nonspecific interactions between other molecules or antibodies and the microspheres.

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

[0045] Sample pad pretreatment steps: evenly apply sample pad buffer on the 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 evenly coated on the surface of the glass fiber membrane, and then the coated glass fiber membrane is left to stand at room temperature overnight 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 caseinate, 1.5-3.5% TritonX-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 adjusting the pH and blocking non-specific sites.

[0047] Pretreatment steps of nitrocellulose membrane: Mouse anti-human IgG antibody and DNP-Ag are coated on the surface of nitrocellulose membrane using a gold spray film streak; the mouse anti-human IgG antibody forms the detection line and the DNP-Ag forms the quality 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 test line and quality control line pipelines of the gold spray film streak instrument, respectively. The antibody is evenly coated on the surface of the nitrocellulose membrane at a certain rate (for example, 0.5-2 μL / cm), where the mouse anti-human IgG antibody forms the test line (T line) and the DNP-Ag forms the quality control line (C line). When the nitrocellulose membrane is dried, it can promote the binding reaction of the antibody or antigen protein to the membrane surface, ensuring that the antibody or antigen protein is firmly fixed to the membrane through physical adsorption or chemical cross-linking.

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

[0050] The base plate is the substrate of the test strip and can be made of a known material, such as a PVC base plate. The main function of the absorbent paper is to provide capillary force for sample chromatography and absorb excess reagents, thereby preventing excessive liquid diffusion from interfering with the test results. In this step, the antibody-coupled microsphere conjugate pad, nitrocellulose membrane, and absorbent paper are sequentially and horizontally attached to the base plate. The transition areas between the sample pad and the antibody-coupled microsphere conjugate pad overlap, the transition areas between the antibody-coupled microsphere conjugate pad and the nitrocellulose membrane overlap, and the transition areas between the nitrocellulose membrane and the absorbent paper overlap. The overlap between the components is to ensure that the liquid flows evenly on the test strip. Uniform liquid flow is a prerequisite for ensuring an effective immune response. If the fluidity is insufficient, the sample liquid may not fully contact the detection area, resulting in an incomplete signal or increased error. 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 test.

[0051] This proposal provides a method for preparing an AChR-Ab test strip, which can produce an AChR-Ab test strip. This method addresses the shortcomings of existing AChR-Ab testing, which relies on venous blood sampling, resulting in poor compliance among pediatric patients and an inability to reflect local antibody expression in the eye. By detecting tear-specific AChR-Ab, the diagnostic sensitivity of OMG can be increased from 50% of the serum baseline to ≥80%, and the test time can be significantly shortened, simplifying the test process.

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

[0053] Glass fiber membrane has high chemical stability and temperature resistance, which makes it show good stability under various experimental conditions. Although other materials such as nylon membrane, polyester membrane, etc. can also be used as alternatives, glass fiber membrane excels in microsphere adsorption, dispersion uniformity and stability, and rarely has precipitation or unevenness problems, so it was selected as the ideal material for this experiment. In particular, this embodiment uses a three-dimensional spray point platform to spray the microspheres mixed with detection antibody-coupled fluorescent microspheres and quality control antibody-coupled fluorescent microspheres on the glass fiber membrane. This is because the glass fiber membrane has a high porosity, a large specific surface area and good mechanical strength, which can effectively support the adsorption and reaction of the microspheres, and also utilizes the good liquid fluidity of the glass fiber membrane.

[0054] Optimally, in the test strip assembly step, the sample pad, antibody-coupled microsphere conjugate pad, nitrocellulose membrane and absorbent paper are sequentially pasted onto the bottom plate in a horizontal order, with the horizontal overlap distance between adjacent ones being 0.5-3 mm.

[0055] The sample pad, nitrocellulose membrane and absorbent paper are sequentially pasted to the base plate, with the overlap between the components being 0.5-32mm. This range ensures that the liquid flows evenly on the test strip, thereby ensuring the sensitivity and accuracy of the test.

[0056] Optionally, after the step of preparing the detection antibody-coupled fluorescent microspheres, the detection antibody-coupled fluorescent microspheres are ultrasonically treated using an ultrasonic disruptor;

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

[0058] Ultrasonic treatment of the microspheres using an ultrasonic disruptor can ensure uniform dispersion of the microspheres and avoid problems with local concentrations that are too high or too low.

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

[0060] The temperature range of 36-38°C not only helps accelerate water evaporation and ensures that antibodies are firmly fixed to the membrane through physical adsorption or chemical cross-linking. The appropriate temperature and drying time can improve the binding efficiency of antibodies and prevent antibody denaturation caused by excessive temperature, thereby maintaining the detection performance and stability of the nitrocellulose membrane.

[0061] An AChR-Ab detection test strip is prepared by the preparation method of an AChR-Ab detection test strip according to any of the above embodiments.

[0062] like Figure 5The structure of the AChR-Ab detection test strip includes a sample pad 1, an antibody-coupled microsphere binding pad 2, a nitrocellulose membrane 3, an absorbent paper 4 and a bottom plate 5.

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

[0064] In addition to AChR-Ab test strips, the OMG diagnostic kit also includes Schirmer tear strips, EP tubes and PBS solution; Schirmer tear strips are used for tear collection; EP tubes are used to place Schirmer tear strips, and can also be used to prepare PBS solution, or to mix PBS solution with Schirmer tear strips; PBS solution can be prepared into a dilution as needed, for example, according to the ratio of adding 5-50 microliters of PBS solution for every 1 mm of the wet length of the Schirmer tear strip, the PBS dilution can be accurately prepared.

[0065] A method for using an AChR-Ab detection test strip, using an AChR-Ab detection test strip according to any of the above embodiments, comprising: a sample spotting step;

[0066] The spotting step includes adding 90-120 μL of tear sample to the spotting hole of the AChR-Ab test strip for detection. After the test strip reacts, the fluorescence signal intensity of the test line and the quality control line is read using an immunofluorescence analyzer.

[0067] A 90-120 μL tear sample was added to the sample wells of an AChR-Ab test strip for testing; each experiment was repeated three times. After the strip reacted for 15 minutes, the fluorescence signal intensity of the test line (T line) and the control line (C line) was immediately read using a dry-type immunofluorescence analyzer and recorded for subsequent analysis. This project developed a portable lateral flow immunochromatography-based test device, which reduces the test time from 2-48 hours with existing technology to 15 minutes.

[0068] Optionally, the method may further include: a tear collection step and a tear sample processing step;

[0069] The tear collection steps include gently pulling the patient's lower eyelid conjunctiva and applying moderate pressure backward, and non-invasively placing one end of the Schirmer tear test strip with a yellow indicator line into the conjunctival sac on the outside of the patient's lower eyelid, with the other end of the Schirmer tear test strip naturally hanging outside the lower eyelid; instructing the patient to lightly close their eyes and look slightly upward, maintaining this state to allow the Schirmer tear test strip to fully absorb basal tears;

[0070] The tear sample processing step comprises: mixing the Schirmer tear test paper with the PBS diluent, placing the mixture in an ultrasonic processor, removing solid residues by centrifugation, and taking the supernatant as the tear sample for the spotting step.

[0071] This protocol addresses the shortcomings of existing AChR-Ab testing, which relies on venous blood sampling, resulting in poor compliance among pediatric patients and an inability to reflect local antibody expression in the eye. The AChR-Ab test strips used in this protocol establish a non-invasive detection system based on tear samples, overcoming the bottleneck of insufficient sensitivity of OMG serum detection. By detecting tear-specific AChR-Ab in seronegative OMG patients (accounting for 50% of the diagnosed population), the diagnostic sensitivity of OMG is increased from 50% of the serum baseline to ≥80%.

[0072] A test strip is used in preparing an OMG diagnostic kit for detecting tear AChR-Ab, wherein the test strip is an AChR-Ab detection test strip according to any of the above embodiments.

[0073] Example:

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

[0075] (1) Tear sample collection:

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

[0077] 2) Test strip preparation: Use a Schirmer tear test strip and fold it into 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 backward pressure. Non-invasively 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 upward slightly, maintaining this state for 5-10 minutes to ensure that the test strip fully absorbs the basal tear fluid.

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

[0081] (2) Tear sample processing steps:

[0082] 1) Test strip processing: Take out the tear test strip from the -80°C refrigerator and cut it into small pieces using sterile scissors.

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

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

[0085] 4) Centrifugation and Storage: After ultrasonic treatment, remove solid residues by centrifugation. Carefully pipette to mix the supernatant and transfer it to a new sterile EP tube. Store it in a -80°C freezer again until subsequent testing.

[0086] (2) Preparation method of AChR-Ab test strips

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

[0088] 15 μL of europium fluorescent microspheres were diluted with 495 μL of MSE buffer. Subsequently, 5 μL of microsphere labeling activator and 5 μL of microsphere labeling stabilizer were added, followed by reaction at room temperature in the dark for 30 minutes. After activation, the microsphere suspension was centrifuged at 15,000 rpm at 4°C for 30 minutes. The supernatant was discarded, and the microspheres were resuspended in 495 μL of MSE buffer. Subsequently, 12.5 μg of AChRα1 recombinant protein was added to the resuspended microsphere suspension, and the suspension was incubated at room temperature in the dark for 3 hours. After the incubation period, 5 μL of microsphere labeling blocking solution was added, and the suspension was blocked for another 30 minutes at room temperature in the dark. Finally, the microsphere suspension was centrifuged at 15,000 rpm at 4°C for 30 minutes. The supernatant was discarded, and the microspheres were resuspended in 250 μL of microsphere labeling storage solution. The suspension was sonicated to obtain the detection antibody-conjugated fluorescent microspheres.

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

[0090] 15 μL of europium fluorescent microspheres were diluted with 495 μL of MSE buffer. Subsequently, 5 μL of microsphere labeling activator and 5 μL of microsphere labeling stabilizer were added, followed by reaction at room temperature in the dark for 30 minutes. After activation, the microsphere suspension was centrifuged at 15,000 rpm at 4°C for 30 minutes. The supernatant was discarded, and the microspheres were resuspended in 495 μL of MSE buffer. Subsequently, 3 μg of DNP antibody was added to the resuspended microsphere suspension, and the suspension was incubated at room temperature in the dark for 3 hours. After the incubation period, 5 μL of microsphere labeling blocking solution was added, and the blocking reaction was continued at room temperature in the dark for 30 minutes. Finally, the microsphere suspension was centrifuged at 15,000 rpm at 4°C for 30 minutes. The supernatant was discarded, and the microspheres were resuspended in 250 μL of microsphere labeling storage solution. The suspension was sonicated to obtain quality control antibody-conjugated fluorescent microspheres.

[0091] (3) Preparation steps of antibody-coupled microsphere conjugate pad:

[0092] Cut the glass fiber membrane into 1 x 30 cm strips using a wide-type strip cutter. Mix the test antibody-conjugated fluorescent microspheres and the control antibody-conjugated fluorescent microspheres in a 3:1 (v:v) ratio to create a fluorescent microsphere solution. Add the solution to the three-dimensional spraying platform. Use the three-dimensional spraying platform to evenly spray the mixed microspheres onto the glass fiber membrane at a rate of 3 μL / cm. Dry the solution in a 37°C forced air drying oven for 12 hours. Once the antibody-conjugated microsphere conjugate pad is completely dry, store it in a constant temperature and humidity drying cabinet until needed.

[0093] (4) Sample pad pretreatment steps:

[0094] Evenly coat the surface of a 20 x 30 cm glass fiber membrane with 50 mL of sample pad buffer. Allow the coated membrane to stand overnight at room temperature to ensure adequate absorption of the treatment solution. Transfer the membrane to a 37°C forced-air drying oven and dry for 6 hours to achieve uniform drying and stable performance. After drying, use a wide-width strip cutter to cut the membrane into 2 x 30 cm strips.

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

[0096] (5) Pretreatment steps of nitrocellulose membrane:

[0097] 1 mg / mL mouse anti-human IgG antibody and 1 mg / mL DNP-Ag were added to the test line and quality control line of the film gold labeling machine, respectively. The antibodies were evenly coated on the surface of the nitrocellulose membrane using the film gold labeling machine at a rate of 1 μL / cm, with the mouse anti-human IgG antibody forming the test line (T line) and the DNP-Ag forming the quality control line (C line). After coating, the nitrocellulose membrane was transferred to a 37°C forced air drying oven and dried for 12 hours. After the membrane strip was completely dry, it was sealed and stored for later use.

[0098] (6) Preparation of absorbent paper

[0099] Use a wide-type strip cutter to cut H2 absorbent paper into 2.5 × 30 cm strips, ensuring that the cutting process is uniform and the size is accurate to meet the experimental requirements.

[0100] (7) Test strip assembly steps:

[0101] Affix the sample pad, antibody-conjugated microsphere conjugate pad, nitrocellulose membrane, and absorbent paper to a PVC substrate in the order specified, with a 2mm overlap. Use a programmable strip cutter to cut individual strips of 3.9mm width and place them in a plastic card holder.

[0102] (8) Sample spotting steps:

[0103] 90-120 μL of tear fluid sample was added to the sample well of the AChR-Ab test strip for testing. Each experiment was repeated three times. After the test strip reacted for 15 minutes, the fluorescence signal intensity of the test line (T line) and the quality control line (C line) was immediately read using a dry immunofluorescence analyzer, and the data was recorded for subsequent analysis.

[0104] Experiment 1: Tear AChR-Ab validation based on cell-based assay:

[0105] 1. Cell Preparation

[0106] Observe the cell status: Take out 10cm from the 37℃, 5% CO2 incubator 2 Observe the cell growth status under a microscope to ensure that the cells are growing well and are free of contamination.

[0107] Cell digestion: Aspirate 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 to evenly cover the cell layer, and incubate in a 37°C cell culture incubator for 2 minutes. During this period, observe under a microscope that the cells become round and begin to fall off to avoid over-digestion; add 4 mL of culture medium containing FBS to terminate the action of trypsin and mix gently 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 the cell suspension, and count the cell density and number using a hemocytometer.

[0110] Cell plating: take 5×10 5 Cells / well were evenly plated in a six-well cell culture plate. 2 mL of HEK293T cell culture medium preheated to 37°C was added to each well to ensure that the cells were evenly distributed.

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

[0112] 2. Cell transfection

[0113] Based on Lipofectamine TM According to the instructions of the 3000 transfection kit, all reagents should be equilibrated to room temperature before use and operated under sterile conditions in a clean bench to ensure the accuracy and reproducibility of the experiment. The specific steps are as follows (taking each well of a six-well plate as an example):

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

[0115] Preparation of plasmid DNA dilution solution: Take 125 μL Opti-MEM to a sterile centrifuge tube; add 2.5 μg plasmid DNA (pAChRα1-pcDNA3.1(+), purchased from Qingke Biotechnology Co., Ltd.); add 5 μL P3000 TM Mix the reagents by gently pipetting.

[0116] Mix transfection reagent: add plasmid DNA dilution to Lipofectamine TM Gently pipette to mix thoroughly in a 3000 μL solution. Let stand at room temperature for 15 minutes to allow lipid-nucleic acid complexes to form and enhance transfection efficiency.

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

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

[0119] Continue culturing: Place in a 37°C, 5% CO2 cell culture incubator and incubate for 4-6 hours.

[0120] Replace 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, check the cell status: observe the cell growth and ensure good cell viability with no obvious cell death or floating phenomenon.

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

[0123] 1) Cell inoculation and culture: First, transfected HEK293T cells were cultured at 5×10 4 Cells were seeded at a density of 100 cells / well on slides in a 24-well plate 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 remove the cell culture medium and wash the cells with PBS for 3 times, 3 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 for 3 times, 3 minutes each time.

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

[0128] 6) Washing and blocking solution: Wash the cells with PBS for 3 times, 3 minutes each time.

[0129] 7) Primary antibody incubation: 100 μL of the corresponding samples (serum samples and tear samples of OMG and HC) were added to the wells and incubated in a 4°C refrigerator overnight.

[0130] 8) Washing the primary antibody: The next day, remove the primary antibody and wash the cells with PBS three times, each wash lasting 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 the secondary antibody: Wash the cells with PBS for 3 times, each wash lasting 10 minutes.

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

[0134] 12) Final wash: Wash the cells with PBS five times, each wash lasting 10 minutes.

[0135] 13) Sealing and Observation: Finally, the slides were sealed with an anti-fluorescence quencher, and the cells were observed and images were collected 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 from 3 AChR-Ab positive OMG patients and 3 healthy controls were collected and analyzed by CBA method to verify the presence of AChR-Ab in tears. The results showed that the serum of OMG patients ( Figure 2 A) and tears ( Figure 2 B) After incubation, transfected cells exhibited significantly enhanced green fluorescence signals on their cell membranes, indicating the presence of AChR-Ab in both patient serum and tear samples. In contrast, no detectable green fluorescence signals were observed in transfected cells incubated with serum and tears from a healthy control group.

[0138] 5. Conclusion

[0139] (1) This example clearly detected for the first time that AChR-Ab in the tears of OMG patients could specifically bind to the AChRα1 protein on the transfected cell membrane, confirming the presence of AChR-Ab in tears.

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

[0141] Experiment 2: Application of Tear AChR-Ab Lateral Flow Immunochromatographic Test Strips

[0142] 1. Experimental Design

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

[0144] 2. Experimental results

[0145] (1) Tear EuNPs-LFIA results

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

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

[0148] The results showed that the T-line fluorescence values ​​of the OMG group were not significantly different from those of the HC group ( Figure 4 A), indicating that the detection results of this method in serum samples cannot effectively distinguish OMG patients from healthy individuals. To further evaluate the diagnostic efficacy of the serum EuNPs-LFIA method, this experiment drew the ROC curve based on all the samples included ( 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 showed that the optimal cut-off value of 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 according to the optimal critical value determined by the ROC curve. There was no positive sample in the healthy control group, while OMG patient samples 7, 9, 11, and 14 were positive ( Figure 4 C).

[0149] (3) This experiment evaluated the results of the EuNPs-LFIA test strips based on the optimal critical value determined by the above ROC curve, and analyzed the consistency between the EuNPs-LFIA test strip results and the clinical diagnosis. For the EuNPs-LFIA test of tear AChR-Ab, the results showed that the negative consistency rate was 80% (healthy donor group) and the positive consistency rate was also 80% (OMG patient group). This shows that the tear AChR-Ab test has a 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 consistency rate reached 100%, the positive consistency rate was only 40% (see Table 2). This means that although the serum test performed well in excluding healthy controls, it had a lower accuracy in identifying OMG patients. These analysis results highlight the potential advantages of tear AChR-Ab testing in the diagnosis of OMG, especially in improving the positive consistency rate, showing better diagnostic efficacy than serum AChR-Ab testing.

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

[0151]

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

[0153]

[0154] 3. Conclusion

[0155] The results of this experiment suggest that tears may be a more reliable biological sample for the noninvasive and differential diagnosis of OMG compared to serum samples. EuNPs-LFIA detection of tear AChR-Ab showed a high positive coincidence rate, significantly better than the results obtained with serum samples. This finding suggests that tear testing has significant advantages in improving the accuracy and efficiency of OMG diagnosis. As a noninvasive and easily accessible biological sample, tears have the potential to become an important tool for OMG diagnosis, especially in situations where rapid and simple screening and differential diagnosis are required.

[0156] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for preparing an AChR-Ab detection test strip, characterized in that: The following steps are involved: Preparation of detection antibody-coupled fluorescent microspheres: diluting europium fluorescent microspheres with MSE buffer, adding a microsphere labeling activator and a microsphere labeling stabilizer in sequence; centrifuging the formed microsphere suspension, discarding the supernatant, and resuspending the microspheres with MSE buffer; adding AChRα1 recombinant protein to the resuspended microsphere suspension; adding microsphere labeling blocking solution; centrifuging the microsphere suspension, and discarding the supernatant; Preparation of quality control antibody-coupled fluorescent microspheres: dilute europium fluorescent microspheres with MSE buffer, add microsphere labeling activator and microsphere labeling stabilizer in sequence; centrifuge the resulting 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 of antibody-coupled microsphere conjugate pad: Mix detection antibody-coupled fluorescent microspheres and quality control antibody-coupled fluorescent microspheres at a volume ratio of (10-1):1 to form a fluorescent microsphere solution, apply the fluorescent microsphere solution on a glass fiber membrane, and dry; Sample pad pretreatment steps: evenly apply sample pad buffer on the glass fiber membrane, let the coated sample pad stand at room temperature; dry the sample pad; Pretreatment steps for nitrocellulose membrane: Mouse anti-human IgG antibody and DNP-Ag are added to the test line and quality control line pipelines of the film gold labeling machine, respectively, and the antibodies are coated on the surface of the nitrocellulose membrane using the film gold labeling machine; the mouse anti-human IgG antibody forms the test line, and the DNP-Ag forms the quality control line; After coating, the nitrocellulose membrane was dried; Pretreatment steps for nitrocellulose membrane: Mouse anti-human IgG antibody and DNP-Ag are coated on the surface of nitrocellulose membrane using a gold spray film streak; the mouse anti-human IgG antibody forms the detection line, and the DNP-Ag forms the quality control line; After coating, the nitrocellulose membrane was dried; Test strip assembly steps: Paste the sample pad, antibody-coupled microsphere conjugate pad, nitrocellulose membrane and absorbent paper to the bottom plate in horizontal order, with adjacent ones overlapping.

2. The method for preparing an AChR-Ab detection test strip according to claim 1, wherein In the step of preparing the antibody-coupled microsphere conjugate pad, the detection antibody-coupled fluorescent microspheres and the quality control antibody-coupled fluorescent microspheres are added to a three-dimensional spraying platform, and the mixed microspheres are sprayed on the glass fiber membrane at 1-6 μL / cm using the three-dimensional spraying platform.

3. The method for preparing an AChR-Ab detection test strip according to claim 1, wherein In the test strip assembly step, the sample pad, the antibody-coupled microsphere binding pad, the nitrocellulose membrane and the absorbent paper are sequentially pasted onto the bottom plate in a horizontal order, with the horizontal overlap distance between adjacent ones being 0.5-3 mm.

4. The method for preparing an AChR-Ab detection test strip according to claim 1, wherein After performing the step of preparing the detection antibody-coupled fluorescent microspheres, ultrasonically treating the detection antibody-coupled fluorescent microspheres using an ultrasonic disruptor; After performing the step of preparing the quality control antibody-coupled fluorescent microspheres, the quality control antibody-coupled fluorescent microspheres are ultrasonically treated using an ultrasonic disruptor.

5. The method for preparing an AChR-Ab detection test strip according to claim 1, wherein In the pretreatment step of the nitrocellulose membrane, after coating, the nitrocellulose membrane is dried at 36-38° C. for 8-16 hours.

6. An AChR-Ab test strip, characterized in that: The test strip is prepared by the preparation method of the AChR-Ab detection test strip according to claims 1-5.

7. An OMG diagnostic kit based on the detection of tear AChR-Ab, characterized in that: include: An AChR-Ab detection test strip according to claim 6.

8. A method for using an AChR-Ab test strip, characterized in that: The AChR-Ab detection test strip according to claim 6 comprises: a sample spotting step; The spotting step includes adding the tear sample to the spotting hole of the AChR-Ab test strip for detection, and after the test strip reacts, using an immunofluorescence analyzer to read the fluorescence signal intensity of the test line and the quality control line.

9. The method for using the AChR-Ab detection test strip according to claim 8, wherein: Also includes: Tear collection steps and tear sample processing steps; The tear collection steps include gently pulling the patient's lower eyelid conjunctiva and applying moderate pressure backward, and non-invasively placing one end of the Schirmer tear test strip with a yellow indicator line into the conjunctival sac on the outside of the patient's lower eyelid, with the other end of the Schirmer tear test strip naturally hanging outside the lower eyelid; instructing the patient to lightly close their eyes and look slightly upward, maintaining this state to allow the Schirmer tear test strip to fully absorb basal tears; The tear sample processing step comprises: mixing the Schirmer tear test paper with the PBS diluent, placing the mixture in an ultrasonic processor, removing solid residues by centrifugation, and taking the supernatant as the tear sample for the spotting step.

10. Use of a test strip in preparing an OMG diagnostic kit for detecting tear AChR-Ab, characterized in that: The test strip is the AChR-Ab detection test strip according to claim 6.

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