A method for efficiently detecting anti-retinal antibodies in serum and its application

Abstract

The present invention relates to a method for efficiently detecting anti-retinal antibodies (ARAs) in serum, comprising the following steps: (1) extracting venous blood from a clinically suspected AIR patient, centrifuging, and obtaining patient serum; (2) using retinal protein extracted from normal human retinal tissue homogenate as a reaction substrate, using Western blotting to detect the number of antibody-positive bands in the patient serum, that is, the number of ARAs; (3) using a known human recombinant protein antigen as a reaction substrate, using Western blotting to detect and confirm the types of ARAs present in the patient serum; (4) using immunofluorescence to detect ARAs in the patient serum, locating them in normal human retinal tissue sections, and obtaining the location of ARAs in the patient serum in the retinal tissue; (5) according to the patient serum ARAs detection results obtained in steps (2), (3), and (4), determining the number of serum ARAs, the types of serum ARAs, and the location of ARAs in the retinal tissue, and establishing serological indicators for clinically suspected AIR patients. The detection method of the present invention has high sensitivity and specificity, and is suitable for early clinical detection.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a method for efficiently detecting anti-retinal antibodies in serum and an application thereof. Background Art

[0002] Autoimmune retinopathy (AIR) is a type of blinding eye disease mediated by immune inflammation, including paraneoplastic (p-AIR) and non-paraneoplastic (np-AIR). Clinical manifestations of AIR include painless subacute visual loss in both eyes, abnormal ERG, and circulating anti-retinal antibodies (ARAs) in the serum. p-AIR includes cancer-associated retinopathy (CAR) and melanoma-associated retinopathy (MAR). Some benign tumors can also cause CAR-like changes. Systematic examinations of np-AIR patients did not reveal the presence of tumors, but they are often accompanied by autoimmune diseases. The current view is that the pathogenesis of AIR is closely related to the presence of ARAs in the serum. Due to the invasion of external factors such as tumors, viruses, and bacteria, the body produces antibodies similar to the cell structure of a certain retinal tissue and releases them into the serum, destroying the blood-retinal barrier and binding to antigens of specific retinal cells (such as photoreceptor cells) to cause neuronal apoptosis and the disease.

[0003] At present, the international community has not yet established a unified serological detection standard for AIR. There are a large number of potential patients, and delayed treatment or blind treatment due to missed diagnosis or misdiagnosis is common. Based on the limitations of previous studies such as small sample content, small number of ARAs detected, and single detection method, it is necessary to propose a new detection method for ARAs in the serum of patients with clinical suspected AIR. Summary of the invention

[0004] The object of the present invention is to provide a method for efficiently detecting anti-retinal antibodies (ARAs) in serum, comprising the following steps:

[0005] (1) Draw venous blood from patients with clinical suspected AIR and centrifuge it to obtain patient serum;

[0006] (2) using retinal proteins extracted from normal human retinal tissue homogenate as reaction substrates, and detecting the number of antibody-positive bands, i.e., the number of ARAs, in the patient serum extracted in step (1) by Western blotting;

[0007] (3) using known human recombinant protein antigen as a reaction substrate, using Western blotting to detect and confirm the types of ARAs present in the patient serum extracted in step (1);

[0008] (4) using immunofluorescence to detect the ARAs in the patient serum extracted in step (1), and localizing them in normal human retinal tissue sections to obtain the localization of the ARAs in the patient serum in the retinal tissue;

[0009] (5) Based on the patient serum ARAs test results obtained in steps (2), (3), and (4), determine the number of serum ARAs, the types of serum ARAs, and the localization of ARAs in retinal tissue, and establish serological indicators for clinically suspected AIR patients.

[0010] In a preferred technical solution of the present invention, the types of ARAs include any one of recoverin, α-enolase, CA-II, TULP-1, tranducinɑ / β, TRMP-1, CRMP-5, arrestin, HSP-70 or a combination thereof.

[0011] In the preferred technical solution of the present invention, the centrifugation condition in step (1) is 1500-2000 rpm / min and centrifugation for 5-10 min.

[0012] In the preferred technical scheme of the present invention, the method for extracting retinal protein from the normal human retinal tissue homogenate in step (2) is as follows: taking the retina under a dissecting microscope, placing it in a tissue homogenizer, grinding it with 0.5 ml of tissue lysis solution for 5-10 minutes, centrifuging it at 12000 rpm / min at 4°C for 20 minutes, and taking the supernatant to obtain retinal protein; the tissue lysis solution is: 50 mmol / L Tris·Cl with a pH of 8.0, 150 mmol / L NaCl, 0.02% sodium azide, 100 μg / ml PMSF, 1 μg / ml Aprotitin, 1% Triton X-100 or NP-40.

[0013] In the preferred technical solution of the present invention, the method of using Western blotting to detect the number of antibody-positive bands in the patient's serum in step (2) comprises the following steps:

[0014] ① Load normal human retinal protein at a concentration of 6.25 μg / μl into three lanes, 8 μl per well, electrophoresis at 100V*100min, transfer at 100V*60min, and block the transferred PVDF membrane in 5% skim milk powder for 60min;

[0015] ② The blocked PVDF membrane was divided into two pieces according to the lanes, and used as the experimental group and the negative control group, respectively. The experimental group was added with clinically suspected AIR patient serum diluted 200 times with PBS, and the negative control was not added with clinically suspected AIR patient serum but directly added with PBS, and both were submerged in the PVDF membrane and incubated at 4°C overnight. The PBS was a phosphate buffered saline solution;

[0016] ③ Take out each PVDF membrane, rinse it with PBS, then add the secondary antibody (fluorescent anti-human IgG antibody) diluted 2000 times with PBS and submerge the PVDF membrane, incubate at room temperature for 60 minutes, add ECL fluorescent agent, develop by luminescence, and observe the number of positive antibody bands. The total number of positive antibody bands is the number of ARAs in the patient's serum.

[0017] In a preferred technical solution of the present invention, the method of detecting and confirming the target ARAs species present in the patient's serum by Western blotting in step (3) comprises the following steps:

[0018] ① Load recoverin, α-enolase, CA-II, TULP-1, tranducinɑ, TRMP-1, CRMP-5, arrestin, and HSP-70 human recombinant antigens at a concentration of 6.25 μg / μl into different lanes, 8 μl per well, perform electrophoresis at 100V*100min, transfer to the membrane at 100V*60min, and block the transferred PVDF membrane in 5% skim milk powder for 60min;

[0019] ② The blocked PVDF membrane was divided into lanes and loaded with samples as the experimental group, negative control group, and positive control group, respectively, wherein the experimental group was added with clinically suspected AIR patient serum diluted 200 times with PBS; the negative control was added with PBS directly without clinically suspected AIR patient serum; the positive control was added with the corresponding human recombinant antibody (anti-human recombinant antigen) diluted 1000 times with PBS, all of which submerged the PVDF membrane and incubated overnight at 4°C; the human recombinant antibody was a human recombinant antigen, including any one or a combination of the corresponding antibodies of recoverin, α-enolase, carbonic anhydrase II (CAII), TULP-1, TRPM-1, transducin-ɑ / β, arrestin, HSP-70, IRBP, and CRMP-5;

[0020] ③ Take out each PVDF membrane, rinse it with PBS, then add the secondary antibody (fluorescent anti-human IgG antibody) diluted 2000 times with PBS and submerge the PVDF membrane, incubate at room temperature for 60 minutes, add ECL fluorescent agent, and perform luminescent development to determine the types of target ARAs in the patient's serum.

[0021] In the preferred technical scheme of the present invention, the method of detecting ARAs in patient serum by immunofluorescence method described in step (4) comprises the following steps: dropping the patient serum onto a slice of a normal human retina, adding anti-human IgG fluorescent antibody, observing the fluorescent positive staining under a microscope, observing the position of the positive staining in the retinal tissue, and confirming the position of ARAs in the retinal tissue.

[0022] Another object of the present invention is to provide a method for efficiently detecting ARAs in the serum of patients with autoimmune retinopathy for use in preparing a kit for detecting ARAs in the serum of patients with autoimmune retinopathy.

[0023] In the preferred technical solution of the present invention, the kit uses the number and type of positive bands in the patient's serum ARAs and their positions in the human retina to achieve detection.

[0024] In the preferred technical solution of the present invention, when the number of positive bands of the detection antibody is 4, the ability to distinguish AIR from other diseases is the greatest.

[0025] Another object of the present invention is to provide a method for efficiently detecting ARAs in the serum of patients with autoimmune retinopathy for use in screening drugs for preventing or treating autoimmune retinopathy.

[0026] In the preferred technical solution of the present invention, the screening method is achieved by utilizing the number and type of positive bands in the patient's serum ARAs and their locations in the human retina.

[0027] Unless otherwise specified, the present invention uses receiver operating characteristic curve (ROC) to evaluate the sensitivity, specificity, and ability of the antibody quantity in distinguishing disease from normal and from other diseases.

[0028] The area under the ROC curve (AUC) is a curve drawn based on a series of different binary classification methods (cutoff values ​​or decision thresholds), with the true positive rate (sensitivity) as the ordinate and the false positive rate (specificity) as the abscissa. The range is 0.5-1, and the larger the value, the higher the discrimination ability.

[0029] The point closest to the upper left corner on the ROC curve is the optimal critical point, and the value at the point is the optimal critical value (cut-off). At this point, the sensitivity and specificity are both higher, and the false positives and false negatives are also the least.

[0030] Youden index is the cutoff value for achieving the maximum value on the ROC curve. Youden index = sensitivity + specificity - 1.

[0031] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between liquids and solids, the percentage is volume / weight percentage; when the present invention relates to the percentage between solids and liquids, the percentage is weight / volume percentage; the rest are weight / weight percentages.

[0032] Compared with the prior art, the present invention has the following beneficial technical effects:

[0033] 1. The present invention uses proteins extracted from normal human donor retinal tissue to comprehensively detect the presence of ARAs in the serum of clinically suspected AIR patients by immunoblotting, including the number, type and location in retinal tissue sections, and determines the number of serum ARAs by specific statistical methods and means. It is beneficial for patients to intervene in treatment in a timely manner, significantly reduce the blindness rate of AIR patients, significantly improve the safety and effectiveness of AIR patient treatment, and enable patients to directly benefit from early detection, prognosis assessment, and guidance of clinical medication.

[0034] 2. The method of the present invention has the advantages of high detection sensitivity, high specificity, good accuracy, and simple operation. The prepared kit can be used for early clinical detection and significantly improve the quality of life of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 ROC curve of antibody quantity

[0036] Figure 2 The serum ARAs test results of a 60-year-old female patient with cancer-related retinopathy (CAR) in Example 2. DETAILED DESCRIPTION

[0037] The present invention is described below with reference to Examples, but the present invention is not limited to the Examples.

[0038] Example 1

[0039] The inclusion and exclusion criteria for patients with clinically suspected AIR were based on the consensus on the diagnosis and treatment of non-paraneoplastic AIR (np-AIR) published by the American Uveitis Society in AJO in 2016 (in addition to malignant tumors, other clinical signs of p-AIR patients are similar to those of p-AIR, see Table 1).

[0040] Table 1

[0041]

[0042] Experimental group: 58 patients with clinical suspected AIR, including 15 patients with 30 eyes of p-AIR and 33 patients with 65 eyes of np-AIR.

[0043] Disease control group: 12 patients with typical primary retinitis pigmentosa (RP) and 15 patients with bilateral uveitis were selected according to the disease diagnostic criteria.

[0044] Normal control group: 10 normal subjects with no obvious retinal lesions and visual function abnormalities were selected.

[0045] The method for efficiently detecting ARAs in the serum of clinically suspected AIR patients comprises the following steps:

[0046] (1) Draw 5 ml of venous blood from the patient, centrifuge the blood at 1500 rpm for 5 min, collect serum, and store it at -80°C for later use;

[0047] (2) Using retinal proteins extracted from normal human retinal tissue homogenate as reaction substrates, the number of antibody-positive bands in the prepared serum was detected by Western blotting, and the specific steps were as follows:

[0048] Human retinal protein is extracted from normal human retinal homogenate. The method for extracting retinal protein from human retinal tissue homogenate is as follows: take the retina under a dissecting microscope, place it in a tissue homogenizer, add 0.5 ml of tissue lysis solution to grind it, centrifuge it at 12000 rpm / min at 4°C for 20 minutes, and take the supernatant, which is the retinal protein. The tissue lysis solution is: 50 mmol / L pH 8.0 Tris·Cl, 150 mmol / L NaCl, 0.02% sodium azide, 100 μg / ml PMSF, 1 μg / ml Aprotitin, 1% Triton X-100 or NP-40.

[0049] Human retinal protein with a concentration of 6.25 μg / μl was loaded into three lanes, 8 μl was loaded into each well, electrophoresed at 100 V*100 min, and transferred to the membrane at 100 V*60 min;

[0050] The transferred PVDF membrane was placed in 5% skimmed milk powder for blocking for 60 minutes; the blocked PVDF membrane was cut into two pieces according to the lanes, and the samples were loaded as the experimental group and the negative control group respectively.

[0051] The experimental group was added with serum from a clinically suspected AIR patient diluted 200 times with PBS and submerged in the PVDF membrane.

[0052] The negative control was not added with serum from clinically suspected AIR patients, but directly added with PBS to submerge the PVDF membrane.

[0053] Incubate overnight at 4°C; take out each PVDF membrane, rinse it with PBS, then add the secondary antibody (fluorescent anti-human IgG antibody) diluted 2000 times with PBS to submerge the PVDF membrane, incubate at room temperature for 60 minutes, add ECL fluorescent agent, develop by luminescence, and observe the number of positive antibody bands, which is the number of ARAs in the patient's serum.

[0054] (3) Using known human recombinant protein antigens as reaction substrates, Western blotting was used to detect and confirm the target antibodies present in the prepared serum. The specific steps were as follows: recoverin, α-enolase, CA-II, TULP-1, tranducinɑ, TRMP-1, CRMP-5, arrestin, and HSP-70 human recombinant antigens at a concentration of 6.25 μg / μl were loaded into different lanes, 8 μl was loaded into each well, electrophoresis was performed at 100 V*100 min, and transfer was performed at 100 V*60 min. The PVDF membrane after transfer was placed in 5% skimmed milk powder for blocking for 60 min;

[0055] The blocked PVDF membrane was divided into lanes and used as the experimental group, negative control group, and positive control group.

[0056] In the experimental group, serum from clinically suspected AIR patients diluted 200 times with PBS was added to submerge the PVDF membrane;

[0057] The negative control group did not add serum from clinically suspected AIR patients but directly added PBS to submerge the PVDF membrane;

[0058] The positive control group was a human recombinant protein antibody (corresponding to the human recombinant protein) diluted 1000 times with PBS and flooded with the PVDF membrane.

[0059] All were incubated at 4°C overnight; the human recombinant protein antibody is a human recombinant protein antigen, including any one or a combination of the corresponding antibodies of recoverin, α-enolase, carbonic anhydrase II (CAII), TULP-1, TRPM-1, transducin-ɑ / β, arrestin, HSP-70, IRBP, and CRMP-5;

[0060] Each PVDF membrane was taken out, rinsed with PBS, and then the secondary antibody (fluorescent anti-human IgG antibody) diluted 2000 times with PBS was added to submerge the PVDF membrane. The membrane was incubated at room temperature for 60 minutes, and ECL fluorescent agent was added for luminescent development to determine the type of positive antibody, which was the type of ARAs in the serum.

[0061] (4) The patient's serum is dripped onto a slice of a normal human retina, and then anti-human IgG fluorescent antibody is added. The fluorescent positive staining is observed under a microscope to observe the location of the positive staining in the retinal tissue and confirm the location of ARAs in the retinal tissue.

[0062] (5) Count the ARA test results of the clinically suspected patients, disease control patients and normal subjects in the above steps, determine the number of serum ARAs in clinically suspected AIR patients, the types of target antibodies, and the localization of ARAs in retinal tissue, and compare and analyze them with the disease control group and the normal control group, and test the ability to distinguish, and establish a serological detection system and serological detection indicators for AIR patients.

[0063] The statistical analysis software used was SAS9.4, and the research data were quantitative or counting data.

[0064] (6) Experimental results:

[0065] In the serum of patients with p-AIR, anti-recoverin antibodies were 20% (3 / 15). Antibodies to ɑ-enolase were 100%, and antibodies to CAII were 60% (9 / 15). Other ARAs included CRMP-5 (22%), HSP-70 (10%), TRMP-1 (22%), and S-arrestin (10%). Transduction-ɑ and TULP-1 were not detected. In the serum of patients with np-AIR, ɑ-enolase was 60% (20 / 33), CAII was 48% (15 / 33), and others included TRMP-1 (38%), CRMP-5 (6%), S-arrestin (6%), and TULP-1 (6%). Antibodies to recoveirin, HSP70, and Transduction-ɑ were not found.

[0066] The number of ARAs in p-AIR patients was 5.077±2.05 (2-9), and that in np-AIR was 4.63±2.21 (1-8); that in RP patients was 2±0.91, that in uveitis patients was 2.9±1.63, and that in normal subjects was 1.2±0.91. Statistical analysis (combined with age and gender factors) showed that the number of bands was significantly different among the groups (P<0.05). The number of bands in p-AIR and np-AIR patients was significantly greater than that in the disease control group and the normal control group (P<0.01). On this basis, the sensitivity and specificity of the antibody positive bands were analyzed.

[0067] Figure 1The ROC curve of the number of antibodies has an AUC value of 0.835 and a cut-off value of 3.5. The detection method has high sensitivity and specificity. According to the DeLong test analysis, when the number of antibody positive bands is 4, the Youden index is 0.459, indicating that when the number of antibody positive bands is 4, the ability to distinguish AIR from other diseases is the greatest (Table 2).

[0068] Table 2

[0069]

[0070] Example 2

[0071] The treatment plan for the 58 patients tested in Example 1 is as follows:

[0072] Of the 15 p-AIR patients, two patients with newly diagnosed lung cancer received chemotherapy, and 13 p-AIR patients had already received tumor-directed treatment (including tumor resection, chemotherapy, or radiotherapy) at the time of presentation. After testing, 12 patients received local or / and systemic glucocorticoids, and 3 patients chose observation.

[0073] Among the 33 np-AIR patients, 8 had systemic autoimmune diseases. After testing, 26 patients started or continued to receive local and / or systemic glucocorticoids or cyclosporine treatment, and 7 np-AIR patients chose observation.

[0074] Among the treated patients, visual acuity improved in 30% (26 / 85) of eyes, decreased in 23% (16 / 85) of eyes, and remained stable in 53% (46 / 85) of eyes during the observation period.

[0075] Two types of retinopathy responded well to intravitreal injection of triamcinolone acetonide (TA) or dexamethasone injectable implant (Ozurdex): one with macular edema and the other showing a prominent inflammatory infiltrate. After treatment, macular edema was reduced in all 12 eyes and retinal inflammation subsided. Visual acuity improved significantly in the short term after TA injection, but half of the eyes became blind due to optic atrophy after repeated injections.

[0076] Regardless of the disease stage, nearly 90% of patients received more than one treatment, and only 5 patients with np-AIR chose to be observed. Visual function was maintained in more than 50% of patients, and improved in 1 / 3 of patients. Intravitreal injection of TA or OZURDEX alone can still improve visual function in patients with macular edema or retinal inflammatory infiltrates. During the 5-year observation period, visual function was stable in 2 of the 5 np-AIR patients who did not receive specific treatment. The visual prognosis of patients with acute-onset CAR or np-AIR is usually poor after one year of follow-up, and timely treatment has significant improvement.

[0077] Figure 2 The results of serum ARAs test were obtained using the method of Example 1 for a 60-year-old female patient with cancer-associated retinopathy (CAR) suffering from lung cancer. A total of 11 antibody-positive bands (ARAs) were detected, among which recoverin, ɑ-enolase and CAII were confirmed to be positive. The patient was found to have undiagnosed small cell lung cancer due to this test. After the diagnosis was confirmed by this test, the patient received treatment for the tumor and 0.7 mg of dexamethasone sustained-release agent (OZURDEX) was injected into the vitreous cavity of both eyes. After three months of follow-up, the patient's binocular vision improved from manual / anterior to 0.1 before treatment.

[0078] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art may make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of protection of the claims of the present invention.

Claims

1. A method for efficiently detecting anti-retinal antibodies in serum, comprising the following steps: (1) Draw venous blood from patients with clinical suspected AIR, centrifuge it, and obtain patient serum; (2) Using retinal proteins extracted from normal human retinal tissue homogenate as the reaction substrate, the number of antibody-positive bands in the patient serum extracted in step (1), i.e., the number of ARAs, was detected by Western blotting; The method for extracting retinal protein from normal human retinal tissue homogenate is as follows: placing the retina in a tissue homogenizer, grinding with 0.5 mL of tissue lysis solution for 5-10 minutes, centrifuging at 12000 rpm / min at 4°C for 20 minutes, and taking the supernatant to obtain retinal protein; the tissue lysis solution is: 50 mmol / L Tris·Cl with pH 8.0, 150 mmol / L NaCl, 0.02% sodium azide, 100 µg / mL PMSF, 1 µg / mL Aprotitin, 1% Triton X-100 or NP-40; the concentration of normal human retinal protein used in Western blotting is 6.25 μg / μL; (3) Using known human recombinant protein antigen as the reaction substrate, the Western blotting method is used to detect and confirm the types of ARAs present in the patient serum extracted in step (1); (4) using immunofluorescence to detect the ARAs in the patient's serum obtained in step (1), localizing them in normal human retinal tissue sections, and obtaining the localization of the ARAs in the patient's serum in the retinal tissue; (5) Based on the patient serum ARAs test results obtained in steps (2), (3), and (4), determine the number of serum ARAs, the types of serum ARAs, and the location of ARAs in retinal tissue, and establish serological indicators for clinically suspected AIR patients; The method of using Western blotting to detect the number of antibody positive bands in the patient's serum in step (2) comprises the following steps: ① Load normal human retinal protein at a concentration of 6.25 μg / μL into three lanes, 8 μL per well, electrophoresis at 100V*100min, transfer at 100V*60min, and block the transferred PVDF membrane in 5% skim milk powder for 60min; ② The blocked PVDF membrane was divided into two pieces according to the lanes, which were used as the experimental group and the negative control group respectively. The experimental group was added with serum of clinically suspected AIR patients diluted 200 times with PBS, and the negative control was directly added with PBS without serum of clinically suspected AIR patients, and both were submerged in the PVDF membrane and incubated at 4°C overnight. The PBS was a phosphate buffered saline solution. ③ Take out each PVDF membrane, rinse it with PBS, then add the secondary antibody diluted 2000 times with PBS to submerge the PVDF membrane, incubate at room temperature for 60 minutes, add ECL fluorescent agent, develop by luminescence, and observe the number of positive antibody bands. The total number of positive antibody bands is the number of ARAs in the patient's serum; The method for detecting ARAs in patient serum by immunofluorescence comprises the following steps: dropping the patient serum onto a slice of a normal human retina, adding anti-human IgG fluorescent antibody, observing the fluorescent positive staining under a microscope, observing the location of the positive staining in the retinal tissue, and confirming the location of ARAs in the retinal tissue.

2. The method according to claim 1, wherein the ARAs are selected from any one of recoverin, α-enolase, CA-II, TULP-1, tranducin ɑ / β, TRMP-1, CRMP-5, arrestin, HSP-70 or a combination thereof.

3. The method according to claim 1, wherein the centrifugation condition in step (1) is 1500-2000 rpm / min for 5-10 min.

4. The method according to claim 1, wherein the method for detecting and confirming the target ARAs species present in the patient's serum by Western blotting comprises the following steps: ① Load recoverin, α-enolase, CA-II, TULP-1, tranducin ɑ, TRMP-1, CRMP-5, arrestin, and HSP-70 human recombinant antigens at a concentration of 6.25 μg / μL into different lanes, 8 μL per well, electrophoresis at 100 V*100 min, transfer to the membrane at 100 V*60 min, and block the transferred PVDF membrane in 5% skim milk powder for 60 min; ② The blocked PVDF membrane was divided into lanes and loaded with samples as the experimental group, negative control group, and positive control group, respectively. The experimental group was added with clinically suspected AIR patient serum diluted 200 times with PBS; the negative control was added with PBS directly without clinically suspected AIR patient serum; the positive control was added with corresponding human recombinant antibodies diluted 1000 times with PBS, all of which submerged the PVDF membrane and incubated overnight at 4°C; the human recombinant antibodies were human recombinant antigens including recoverin, α-enolase, carbonicanhydrase II, TULP-1, TRPM-1, transducin-ɑ / β, arrestin, HSP-70, IRBP, CRMP-5, or any one or a combination thereof; ③ Take out each PVDF membrane, rinse it with PBS, then add the secondary antibody diluted 2000 times with PBS to submerge the PVDF membrane, incubate at room temperature for 60 minutes, add ECL fluorescent agent, and perform luminescent development to determine the types of target ARAs in the patient's serum.

5. Use of the method for efficiently detecting anti-retinal antibodies in serum as claimed in any one of claims 1 to 4 for screening drugs for preventing or treating autoimmune retinopathy.

6. The use as claimed in claim 5, wherein the use is achieved based on the number and type of positive bands in the patient's serum ARAs and their positions in the human retina.

Images