CN118006756A

CN118006756A - Product for evaluating severity of glaucoma and application thereof

Info

Publication number: CN118006756A
Application number: CN202410150237.1A
Authority: CN
Inventors: 鲁芳; 何冲; 袁航; 王金霞; 王左; 修文博; 张高; 陈洋
Original assignee: University of Electronic Science and Technology of China
Current assignee: University of Electronic Science and Technology of China
Priority date: 2024-02-01
Filing date: 2024-02-01
Publication date: 2024-05-10

Abstract

The invention discloses a product for evaluating the severity of glaucoma and application thereof, and relates to the technical field of glaucoma diagnosis. By detecting the AAT content in the sample, the diagnosis of glaucoma can be aided and the severity of glaucoma can be assessed more accurately, with patients with severe glaucoma or end-stage glaucoma having significantly higher AAT levels in the plasma than those with mild glaucoma. Therefore, the detection of AAT levels in the sample is helpful for noninvasive, convenient and rapid monitoring of glaucoma progression or prognosis of glaucoma, so that the intervention treatment of glaucoma is advanced and the life quality of patients is improved.

Description

Product for evaluating severity of glaucoma and application thereof

Technical Field

The invention relates to the technical field of glaucoma diagnosis, in particular to a product for evaluating the severity of glaucoma and application thereof.

Background

Glaucoma is a neurodegenerative disease characterized by optic neuropathy and visual field loss caused by degeneration of Retinal Ganglion Cells (RGCs). Progressive glaucoma is an important risk factor for blindness, reduces the quality of life of patients, and places a burden on society. Current glaucoma diagnosis mainly uses structural and functional combination assessment methods such as Optical Coherence Tomography (OCT) and standard automatic line-of-sight examination, but is sometimes unsuitable for patients due to its time consuming, expensive nature and the inability of patients to fit effectively. These difficulties require clinicians and researchers to explore an easier and more convenient method to identify the severity of glaucoma and monitor the progression of glaucoma. Although elevated ocular pressure is considered a major risk factor for glaucoma, the exact pathogenesis of glaucoma is not completely understood.

Inflammation is one etiological factor in the onset of glaucoma. Inflammatory markers such as neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR) and lymphocyte to monocyte ratio (LMR) levels in glaucoma patients have been reported to be elevated with good accuracy in identifying the severity of glaucoma. Notably, high PLR was found to be associated with the risk of Vision (VF) loss progression in glaucoma patients. These inflammation indices at high levels in glaucoma patients indicate the importance of inflammation in the development of glaucoma and suggest a potential role for anti-inflammatory substances in the disease.

Alpha 1-antitrypsin, also known as AAT, is a glycoprotein synthesized by hepatocytes and secreted into the blood. Notably, AAT exhibits powerful anti-inflammatory properties, affects a range of inflammatory cells, and modulates inflammation caused by host and microbial factors. Furthermore, the anti-inflammatory effects of AAT have progressed from initial pulmonary disease to neurodegenerative diseases such as Alzheimer's Disease (AD). However, the relationship between AAT and glaucoma has not been reported.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a product for evaluating the severity of glaucoma and application thereof to solve the technical problems.

The invention is realized in the following way:

In a first aspect, the invention provides the use of a reagent for detecting the level of a biomarker, AAT, in a sample for the manufacture of a product for assessing the severity of glaucoma.

The inventor researches find that the concentration of AAT in a sample such as blood plasma can be used for assisting in diagnosing glaucoma, and the severity of glaucoma can be accurately estimated, so that patients with serious glaucoma or end-stage glaucoma can have the AAT level in the blood plasma remarkably higher than that of patients with mild glaucoma. Glaucoma patients are prone to severe nerve damage after treatment with unchanged or elevated AAT levels, suggesting that well controlled AAT levels may have a protective effect on nerve function. Thus, detection of AAT levels in a sample can facilitate noninvasive, convenient, and rapid monitoring of glaucoma progression or prognosis. Thereby the glaucoma is intervened and treated in advance, and the life quality of patients is improved.

In a preferred embodiment of the invention, the product is a kit, a chip, a sequencing library, a computer system with a glaucoma severity assessment module, or a device with a glaucoma severity assessment module.

In a preferred embodiment of the application of the present invention, the sample is at least one selected from urine, blood, plasma and liver tissue.

In a preferred embodiment of the application of the present invention, the above-mentioned reagents comprise: a primer pair, probe or antisense nucleotide that specifically binds to a gene of a biomarker; or an antibody, interacting protein, ligand, nanoparticle or aptamer that specifically binds to a protein or peptide fragment of a biomarker.

In a preferred embodiment of the application of the present invention, the above-mentioned reagents comprise: reagents for detecting mRNA levels by polymerase chain reaction, nuclease protection assay, in situ hybridization, nucleic acid microarray, northern blotting;

in an alternative embodiment, the polymerase chain reaction comprises a real-time fluorescent quantitative reverse transcription polymerase chain reaction, a competitive polymerase chain reaction.

In a preferred embodiment of the invention, the product comprises a reagent for detecting protein levels by immunoblotting, enzyme-linked immunosorbent assay, radioimmunoassay, immunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence-activated cell sorting, mass analysis or protein microarray.

In a preferred embodiment of the invention, the level of AAT expression is up-regulated in a population suffering from severe or end-stage glaucoma, as compared to a population suffering from mild glaucoma.

In a second aspect, the invention also provides the use of a reagent for detecting the level of a biomarker, AAT, in a sample for the manufacture of a product for monitoring the progression of glaucoma or prognosis of glaucoma.

In a preferred embodiment of the invention, the product is a kit, a chip, a sequencing library, a computer system with a glaucoma severity assessment module, or a device with a glaucoma severity assessment module;

in an alternative embodiment, the reagent comprises: a primer pair, probe or antisense nucleotide that specifically binds to a gene of a biomarker; or an antibody, interacting protein, ligand, nanoparticle, or aptamer that specifically binds to a protein or peptide fragment of a biomarker;

In an alternative embodiment, a subject is predicted to develop a neurological deficit if the level of AAT in the subject after treatment is unchanged or increased;

In an alternative embodiment, the nerve injury is selected from at least one of the following: the retinal nerve fiber layer thickness is reduced and the cup to disc ratio is increased.

In a third aspect, the present invention also provides a device for assessing the severity of glaucoma, monitoring the progression of glaucoma, or prognosis of glaucoma, the device comprising: a processor; an input module, a computer readable medium containing instructions, and an output module;

the input module is used for inputting the level of a biomarker in the biological sample, wherein the biomarker is AAT;

computer readable medium: the instructions, when executed by the processor, perform an algorithm at an input level of the biomarker;

an output module outputting at least one selected from the following information: glaucoma severity information, glaucoma progression information, and glaucoma prognosis information are evaluated.

The invention has the following beneficial effects:

According to the invention, the analysis of the AAT content in samples such as blood plasma and the like can help to diagnose glaucoma, and the severity of glaucoma can be accurately estimated, so that patients with serious glaucoma or end-stage glaucoma can have the AAT level in blood plasma remarkably higher than those of patients with mild glaucoma. Glaucoma patients are prone to severe nerve damage after treatment with unchanged or elevated AAT levels, suggesting that well controlled AAT levels may have a protective effect on nerve function. Thus, detection of AAT levels in a sample can facilitate noninvasive, convenient, and rapid monitoring of glaucoma progression or prognosis. Thereby the glaucoma is intervened and treated in advance, and the life quality of patients is improved.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing statistical results of the relationship between the serum AAT levels of glaucoma patients and the different disease stages and optic nerve damage ((A) elevated serum AAT levels of glaucoma patients, (B-C) delamination of serum AAT levels of glaucoma patients by H-P-A grading system (B) and AGIS grading system (C) by disease severity. (D-E) delamination of serum AAT levels of glaucoma patients by RNFL (D) and CDR (E), the differences of the parameters using unpaired Wilcoxon rank sum test; P < 0.05; P < 0.01; P < 0.001; P <0.0001; HC: healthy control; AAT: alpha-1 antitrypsin);

FIG. 2 is a graph of statistical results of plasma AAT levels before ocular hypotension surgery versus after about 2 years of follow-up; * p <0:05, < p <0.01, < p <0.001, < p <0.0001;

FIG. 3 is a graph of the results of changes in RNFL thickness versus AAT level in patients with reduced and unchanged/elevated AAT levels; * p <0:05, < p <0.01, < p <0.001, < p <0.0001;

FIG. 4 is a graph of a subject's working characteristics (ROC) curve analysis, based on the H-P-A (A) classification system and the AGIS (B) classification system, to determine the ability to distinguish between healthy controls and patients with different disease severity, and to determine the ability to distinguish between early glaucoma, severe glaucoma, and end-stage glaucoma (C).

Detailed Description

Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait, eds., 1984); animal cell Culture (ANIMAL CELL Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (academic Press Co., ltd. (ACADEMIC PRESS, inc.)), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C. Blackwell, inc.), gene transfer Vectors for mammalian cells (GENE TRANSFER vector for MAMMALIAN CELLS) (J.M.Miller and M.P.Calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, 1987), polymerase chain reaction (PCR: the Polymerase Chain Reaction) (Mullis et al, 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which are expressly incorporated herein by reference.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The term "sample" or "test sample" refers to a biological specimen obtained or derived from an individual of interest, the source of which may be a fresh, frozen and/or preserved organ or tissue sample or solid tissue resulting from a biopsy or primer; blood or any blood component. The term "sample" or "test sample" includes biological samples that have been manipulated in any manner after they have been obtained, such as by reagent treatment, stabilization, or enrichment for certain components (e.g., proteins or polynucleotides), or embedding in a semi-solid or solid matrix for sectioning purposes. In one embodiment of the present invention, plasma is used as the sample.

The term "biomarker" broadly refers to any detectable compound present in or derived from a sample, such as a protein, peptide, proteoglycan, glycoprotein, lipoprotein, carbohydrate, lipid, nucleic acid (e.g., DNA, such as cDNA or amplified DNA, or RNA, such as mRNA), organic or inorganic chemical, natural or synthetic polymers, small molecules (e.g., metabolites), or distinguishing molecules or distinguishing fragments of any of the foregoing. For example, detection of a particular cDNA may indicate the presence of a particular RNA transcript in a sample. As another example, detection of a particular antibody or binding to a particular antibody may indicate the presence of a particular antigen (e.g., protein) in a sample. Herein, a distinguishing molecule or fragment is a molecule or fragment that, when detected, indicates the presence or abundance of a compound identified above. The biomarker may be isolated from the sample, measured directly in the sample, or detected or determined in the sample, for example. Biomarkers may, for example, be functional, partially functional or nonfunctional.

Alpha-antitrypsin (alpha 1-antitrypsin, alpha 1AT or AAT), also known as alpha-1-protease inhibitor (alpha 1-protease inhibit or, alpha 1-PI), is an acute phase response protein with protease inhibition. Alpha antitrypsin is a single chain glycoprotein, the peptide chain consists of 394 amino acid residues.

Biomarkers include genes and their encoded proteins and their homologs, mutations, and isoforms. The term encompasses full length, unprocessed biomarkers, as well as any form of biomarker derived from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of the biomarker. The gene ID is available at https:// www.ncbi.nlm.nih.gov/gene.

The inventors found that: by detecting the AAT content in a sample such as plasma, the diagnosis of glaucoma can be assisted, and the severity of glaucoma can be evaluated more accurately, with patients with severe glaucoma or end-stage glaucoma having significantly higher AAT levels in plasma than those with mild glaucoma. Glaucoma patients are prone to severe nerve damage after treatment with unchanged or elevated AAT levels, suggesting that well controlled AAT levels may have a protective effect on nerve function. Thus, detection of AAT levels in a sample can facilitate noninvasive, convenient, and rapid monitoring of glaucoma progression or prognosis. Thereby the glaucoma is intervened and treated in advance, and the life quality of patients is improved.

In alternative embodiments, binding agents for proteins include, but are not limited to, peptides, peptidomimetics, aptamer, spiegelmer, darpin, ankyrin repeat proteins, kunitz-type domains, antibodies, single domain antibodies, and monovalent antibody fragments.

Such agents include, but are not limited to, anti-AAT antibodies or functional fragments thereof, interacting proteins, and the like.

The term "primer" refers to a short nucleic acid sequence having a short free 3 hydroxyl group capable of forming base pairs with a complementary template that serves as an origin for replication of the template strand. The primers may induce DNA synthesis in the presence of reagents for polymerization (i.e., DNA polymerase or reverse transcriptase) and the different 4 nucleoside triphosphates in appropriate buffers and temperatures.

The term "probe" refers to a nucleic acid fragment, such as RNA or DNA, corresponding to several bases to hundreds of bases capable of specifically binding mRNA. Being labeled, it is possible to confirm whether or not a specific mRNA is present. The probe can be produced in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, or the like. In the present invention, hybridization is performed using a probe complementary to the AAT gene, and the expression level of the above gene can be diagnosed by whether hybridization is performed. The selection and hybridization conditions for the appropriate probes may be modified based on techniques well known in the art, and are not particularly limited in the present invention.

The primers or probes of the invention may be chemically synthesized using a phosphoramidite solid support method or other well known methods. Such nucleic acid sequences may be deformed by a variety of means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution of one or more homologs of the natural nucleotide, and modifications between nucleotides, for example, modifications to uncharged linkers (e.g., methyl phosphonate, phosphotriester, phosphoramidate, carbamate, etc.) or charged linkers (e.g., phosphorothioate, phosphorodithioate, etc.).

Antisense nucleotides include, but are not limited to, antisense DNA or antisense RNA, where antisense RNA refers to an RNA molecule having a sequence complementary to a target RNA.

The aptamer is an oligonucleotide sequence which is screened from a random oligonucleotide library and has high specificity and affinity for a target substance, and can be combined with the corresponding ligand with high affinity and strong specificity.

In an alternative embodiment, the nanoparticles are selected from the group consisting of organic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

In a preferred embodiment of the application of the present invention, the above-mentioned reagents comprise: reagents for detecting mRNA levels by polymerase chain reaction, nuclease protection assay, in situ hybridization, nucleic acid microarray, northern blotting.

In one embodiment, the reagent is a reagent for detecting the amount of RNA, in particular mRNA, transcribed by the biomarker AAT. In yet another embodiment, the reagent is a reagent that detects the amount of cDNA complementary to the mRNA.

In one embodiment, the above-described diagnostic aid or diagnostic product further comprises total RNA extraction reagents, reverse transcription reagents, and/or second generation sequencing reagents.

The above product for evaluating the severity of glaucoma is an in vitro evaluation product.

The above "kit" is an article of manufacture (e.g., a package or container) containing probes for specifically detecting the biomarker genes or proteins of the present invention. In certain embodiments, the article of manufacture is promoted, distributed, or sold as a unit for carrying out the methods of the present invention.

Such kits may comprise carrier means compartmentalized to closely hold in confinement one or more container means (e.g., vials, tubes, etc.), each of the container means comprising one of the separate components to be used in the method. For example, one of the container means may comprise a probe that is or may be detectably labeled. Such probes may be polynucleotides specific for polynucleotides comprising one or more genes characteristic of gene expression. Where the kit utilizes nucleic acid hybridization to detect a target nucleic acid, the kit may also have a container containing one or more nucleic acids for amplifying the target nucleic acid sequence and/or a container containing a reporter means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzyme, fluorescent or radioisotope label.

The kit will typically comprise the above-described container and one or more other containers containing materials that are commercially and consumer-desirable, including buffers, diluents, filters, needles, syringes and package insert containing instructions for use. A label may be present on the container to indicate that the composition is for a particular therapeutic or non-therapeutic application, and may also indicate the direction of in vivo or in vitro use, such as those described above. Other optional components of the kit include one or more buffers (e.g., blocking buffer, wash buffer, substrate buffer, etc.), other reagents (e.g., substrates chemically altered by enzymatic labeling), epitope retrieval solutions, control samples (positive and/or negative controls), control sections, and the like.

In an alternative embodiment, the reagent comprises: a primer pair, probe or antisense nucleotide that specifically binds to a gene of a biomarker; or an antibody, interacting protein, ligand, nanoparticle or aptamer that specifically binds to a protein or peptide fragment of a biomarker.

In an alternative embodiment, the reagent comprises: reagents for detecting mRNA levels by polymerase chain reaction, nuclease protection assay, in situ hybridization, nucleic acid microarray, northern blotting.

In an alternative embodiment, the product for monitoring the progression of glaucoma or prognosis of glaucoma comprises a reagent for detecting protein levels by immunoblotting, enzyme-linked immunosorbent assay, radioimmunoassay, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence activated cell sorting, mass analysis or protein microarray.

in an alternative embodiment, the nerve injury is selected from at least one of the following: retinal Nerve Fiber Layer (RNFL) thickness is reduced and cup to disc ratio is increased. The cup-disc ratio is also called C/D clinically, which means the area of the optic cup to the area of the upper optic disc, the optic disc means the optic nerve disc, which is an important structure of the fundus, and the optic cup is the fovea in the center of the optic disc. Normal individuals typically have a C/D of no more than 0.6, typically at 0.3 or 0.4, and if the C/D exceeds 0.6, this indicates a possibly glaucomatous optic disc change.

The processor may be an integrated circuit chip having signal processing capabilities. The processor may be a general-purpose processor including a central processing unit (Central Processing Unit, CPU), a network processor (Network Processor, NP), etc.; but may also be a digital signal processor (DIGITAL SIGNAL Processing, DSP), application SPECIFIC INTEGRATED Circuit (ASIC), field-Programmable gate array (Field-Programmable GATE ARRAY, FPGA) or other Programmable logic device, discrete gate or transistor logic device, discrete hardware components.

A processor may execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as a memory. Instructions may be directed to a processor, which may then be programmed or otherwise configured to implement the present disclosure. Examples of operations performed by a processor may include reading, decoding, performing, and writing back.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

The present example provides the use of a reagent for detecting AAT levels in a plasma sample in the preparation of a kit for assessing the severity of glaucoma.

1. Sample recruitment:

Glaucoma patients and age, sex matched healthy controls were recruited and blood samples were left, as follows.

1. 163 Glaucoma patients were recruited in this example. All subjects signed informed consent at admission and provided corresponding clinical information. The ophthalmologist performs a comprehensive ophthalmic examination to diagnose glaucoma. The subject is required to be free of inflammatory diseases, autoimmune diseases, neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease, and the like.

2. This example recruits 111 healthy physical examination persons. Healthy controls had neither a trend toward glaucoma nor a family history. The exclusion criteria included: there is clinical evidence of glaucoma or family history of glaucoma, ocular discomfort, elevated IOP (. Gtoreq.21 mmHg), any recent history of surgery, and any other neurodegenerative disease. And ensure that healthy controls are matched to glaucoma patient groups for gender and age without statistical differences.

3. Each subject was bled from the anterior elbow vein after 8 hours of fasting in the early morning. EDTA anticoagulation vacuum container CPT blood collection tube is centrifugated at the speed of 3000 rpm, and the supernatant is immediately frozen in-80 ℃ ultralow temperature refrigerator after being sucked until analysis.

Table 1: clinical information form of case

Table 1, clinical information table of cases. Data are expressed as mean ± standard deviation. The age difference of glaucoma patients and healthy control groups adopts a t test of double-tailed unpaired students, and the sex difference adopts a chi-square test. p <0.05 is statistically significant for differences. Abbreviations HPA: hodapp, parish, and Anderson; AGIS: advanced glaucoma intervention study score.

2. And preparing a corresponding reagent for AAT detection, and carrying out AAT detection.

1. Preparation of washing liquid

30Ml of 25 Xwashing solution was added to 720ml of ddH ₂ O to prepare 750ml of 1 Xwashing solution, which was then left stand after being thoroughly mixed until a large number of bubbles were present as normal.

2. Preparation of standard substance

The source of the standard is provided for ELISA kits (alpha 1-ANTITRYPSIN ELISA kit accession No: E-EL-H0965c, elabscience).

7 New 1.5ml centrifuge tubes, labeled 100,50,25,12.5,6.25,3.125,0, were removed. Mu.l of standard & sample diluent was added to each EP tube. The standard tube (hereinafter referred to as zero tube) containing the powder was placed in a centrifuge pre-cooled in advance, and parameters were adjusted to 10000rpm,4℃for 1min and centrifuged. Taking out after centrifugation, sucking 1ml of standard substance and sample diluent, adding into a zero pipe, standing for 2min, and fully and uniformly mixing. Then, 500. Mu.l of the standard solution in the zero line was added to the EP line of the standard 100, after thoroughly mixing, 500. Mu.l of the standard solution in the EP line of the standard 100 was added to the EP line of the standard 50, after thoroughly mixing, 500. Mu.l of the standard solution in the EP line of the standard 50 was added to the EP line of the standard 25, and so on. Note that there is only standard & sample diluent in the EP tube labeled 0.

3. Preparation of biotin antibodies

The total volume of the sample size calculation working solution is first determined. The calculation method is (number of samples +2) x 100. Mu.l to obtain the total volume of the required working solution. Dilution with biotin antibody dilutions were performed according to 1:100 dilution of 100X biotin antibody and thorough mixing (e.g., 100. Mu.l biotin antibody was added to 9900. Mu.l biotin antibody dilution).

Preparation of HRP-streptavidin conjugate (SABC)

The preparation method refers to the preparation of biotin antibody working solution.

5. Specific procedure for determining AAT levels in standards and samples

5.1 The kit (E-EL-H0965 c, elabscience) was removed in advance about 20min before the experiment and equilibrated to room temperature. And taking out the ELISA plates required by the experiment, and putting the rest ELISA plates into a sealing bag to be frozen in a refrigerator at the temperature of minus 20 ℃ for preservation.

5.2 Adding 100. Mu.l of sample and standard substance into the enzyme-labeled well, and incubating the enzyme-labeled plate in a 37℃incubator for 90min after coating. The gun head does not touch the bottom of the hole or the wall of the hole to destroy the surface antibody and avoid bubbling out during sample application.

5.3 Preparing biotin antibody working solution 10min in advance. After the incubation, the coating is removed, the liquid in the wells is thrown out, 100 mu l of biotin antibody working solution is added into each enzyme-labeled well, the coating is placed in a 37 ℃ incubator for incubation for 60min.

5.4 Preparation of HRP-streptavidin conjugate (SABC) working solution 10min in advance. And after the incubation is finished, removing the coating, throwing away the liquid in the hole, sucking the washing liquid by using a 300-mu l row gun, adding the washing liquid into the enzyme-labeled hole, standing for 1min, throwing away the washing liquid in the hole, and repeating the steps for 3 times. And after the last washing is finished, the ELISA plate is reversely buckled on the absorbent paper to be beaten with moderate force, and all washing liquid is removed. Then, 100. Mu.l of enzyme conjugate working solution was added to each of the wells, covered, and incubated in a 37℃incubator for 30 minutes.

And 5.5, after the incubation is finished, removing the coating film, washing the plate for 5 times according to the method of the step 5.4, and reversely buckling the ELISA plate on the absorbent paper to perform medium-strength beating after the last washing is finished, so as to remove all washing liquid. Then adding 90 mu l of TMB substrate solution into the enzyme-labeled hole, coating a film, and placing the mixture in a 37 ℃ incubator for incubation for about 15 minutes, wherein the incubation time can be shortened or prolonged according to actual conditions, but cannot exceed 30 minutes. When a clear blue gradient appears in the front four wells of the standard, the reaction can be terminated.

5.6 After the incubation, 50. Mu.l of reaction termination solution was added to the wells, and the visible color changed from blue to yellow immediately, noting that the order of addition of the reaction termination solution should be the same as the order of addition of TMB substrate solution.

And 5.7, opening the microplate reader 15min in advance for preheating. Immediately after the addition of the stop solution, the absorbance was measured at the absorbance at 450nm of the microplate reader, and the OD450 value was read. Curve Expert software is used for drawing a Curve according to the concentration of the standard substance and the value of OD ₄₅₀, and the value of the sample OD ₄₅₀ is substituted into the Curve to calculate the concentration.

The AAT concentration levels in the serum samples of the patient and control population can be obtained according to the above procedure.

Example 2

In this example, differential analysis was performed using the sample plasma AAT concentration detected in example 1.

Analysis of the difference in expression levels of aat in healthy controls versus glaucoma patients.

Plasma AAT was determined as in example 1 using a commercially available AAT ELISA kit (cat# E-EL-H0965c, elabscience). Statistical analysis was performed using the R language (version number: 4.1.3), and data normal distribution was checked with Kolmogorov-Smirnov, and after analysis, the data was found to be non-normal distribution. The difference analysis was performed using a Mann-Whitney U nonparametric test, with p <0.05 indicating a difference in the results.

As a result, referring to a in fig. 1, glaucoma patients had significantly higher plasma AAT levels than healthy controls compared to p <0.001 for glaucoma versus control.

Analysis of the differences in the expression levels of aat in glaucoma patients of varying severity.

The present invention employs two methods, HPA and AGIS, to separately determine the severity of glaucoma. According to the HPA grading system, the severity of glaucoma is primarily judged by mean deviation of field (MD), which is indicated earlier to be greater than-6 dB; medium, -12db to-6 db; seriously, not more than-12 dB.

The AGIS grading system judges the severity of glaucoma according to the VF score, wherein the early stage and the visual field score are between 3 and 5 points; medium, 6 to 12 minutes; severe, 13 to 18 minutes; end stage, 18 to 20 minutes. The score is calculated by adding 1 score if a nasal defect or nasal step occurs; if the sensitivity is <12dB in 4 or more of the 6 nasal sites, then 1 point is added; in each half visual field, 1 cluster or more forms visual field defect by 3 continuous collapse points, if 3-5 defect sites exist, 1 minute is added; if the number of the components is 6-12, adding 2 points; if 13-20 are provided, adding 3 points; if the number of the components is more than 20, adding 4 points; if the sensitivity of one half or more defect sites is reduced by 28dB or more in one half field of view, adding 5 points; the reduction of 24dB-27dB is carried out, and 4 minutes are added; the method has the advantages that the method is reduced by 20dB-23dB, added with 3 points, reduced by 16dB-19dB and added with 2 points; the reduction is 12dB-15dB, and 1 minute is added; at most 5 points can be added to each half field defect; if there are no cluster of 3 adjacent pinch points in the half field of view, but 2 adjacent pinch points are included, and the sensitivity is reduced by more than 12dB, plus 1 minute.

Results referring to fig. 1B and 1C, both classification system results showed that as the severity of glaucoma increased, so did the concentration of plasma AAT. Furthermore, based on the HPA classification system (fig. 1B) and the AGIS system (fig. 1C), it was shown that AAT levels in healthy control groups were significantly different from those in patients with different glaucoma severity. Under the HPA classification system, AAT levels in plasma of patients with mild glaucoma are significantly lower than AAT levels in plasma of patients with severe glaucoma. AAT levels in plasma of patients with mild glaucoma are significantly lower than those of patients with severe glaucoma and patients with end-stage glaucoma under AGIS fractionation.

Relationship of aat content to glaucomatous nerve damage.

This example delaminates patients according to RNFL (retinal nerve fiber layer) thickness and C/D (cup to disc ratio).

The results showed that AAT levels increased as RNFL thinned (fig. 1D). However, although an increase in AAT levels corresponds to an increase in CDRs, this association does not appear to be statistically significant since CDRs may have a relationship with genetic factors (fig. 1E).

To further monitor the dynamic progression of glaucoma, this example recruited 25 subjects receiving pre-ocular hypotensive surgery and evaluated ophthalmic examination data in follow-up examinations.

The results showed that the plasma AAT levels decreased and the differences were statistically significant after the patients had undergone the hypotensive surgery for about 2 years (fig. 2).

Next, this example also investigated the relationship between changes in plasma AAT levels and nerve damage. After their ophthalmic study was examined, 19 subjects were eventually enrolled (6 out of 25 subjects refused to receive the study or performed poorly) and 12 subjects were found to have decreased AAT levels and 7 subjects had unchanged/increased AAT levels (fig. 3).

Among 7 subjects with unchanged/elevated AAT levels, 5 subjects had a reduced RNFL thickness and 3 subjects had an increased CDR during the course of the study. Whereas of the 12 subjects with reduced AAT levels, only 2 subjects had a thinned RNFL thickness and increased CDRs (RNFL: p=0.0342; CDR: p= 0.2111, chi-square test).

The results show that patients with unchanged/elevated AAT levels are prone to severe nerve damage following ocular hypotension surgery, suggesting that well controlled AAT levels may have a protective effect on nerve function.

Example 3

This example uses the sample of example 1 for subject curve analysis (ROC) and is analyzed for diagnostic efficacy of AAT for glaucoma severity. Subject operating characteristic (ROC) curve analysis and area under ROC curve (AUC) were used to determine diagnostic capability. The accuracy of the diagnosis was assessed by calculating the about index (sensitivity + specificity-1).

AAT was evaluated for its ability to healthy controls and glaucoma of varying severity and to differentiate the severity of glaucoma. In the HPA classification system, AAT distinguished healthy controls from patients with early, moderate, and severe glaucoma with AUROC values of 0.654, 0.673, and 0.713, respectively (shown as a in fig. 4). Whereas in the AGIS system, AAT differentiated healthy controls from early, moderate, severe and end-stage glaucoma patients with AUROC values of 0.628, 0.679, 0.760 and 0.739, respectively (fig. 4B). The results indicate that the discrimination of AAT increases with the severity of glaucoma.

Table 2 and fig. 4B show that AAT (early vs. severity: auc=0.616, about log index=0.237; according to the HPA classification system; early vs. severity: auc=0.763, about log index=0.567; early vs end-stage: auc=0.660, about log index=0.244) was found to exhibit sufficient accuracy in distinguishing early glaucoma patients from late glaucoma patients, indicating that the index can help distinguish (or evaluate) the severity of the disease.

The above results demonstrate that AAT has better distinguishing efficacy against glaucoma of varying severity.

Table 2 plasma AAT identifies healthy controls and ability to stratify glaucoma patients by severity of the condition and identify patients with different severity of glaucoma.

In summary, the examples of the present invention demonstrate for the first time the differences in expression of AAT in healthy controls and glaucoma of varying severity, AAT having a better ability to differentiate between glaucoma of varying severity. AAT has been suggested to be useful in the auxiliary diagnosis and assessment of glaucoma and its severity, providing a novel non-invasive detection method for the assessment of glaucoma severity, the detection of glaucoma progression or the prognosis of glaucoma.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of an agent for detecting the level of a biomarker in a sample, wherein the biomarker is AAT, in the manufacture of a product for assessing the severity of glaucoma.

2. The use according to claim 1, wherein the product is a kit, a chip, a sequencing library, a computer system with a glaucoma severity assessment module or a device with a glaucoma severity assessment module.

3. The use according to claim 1, wherein the sample is selected from at least one of urine, blood, plasma and liver tissue.

4. The use according to claim 1, wherein the agent comprises: a primer pair, probe or antisense nucleotide that specifically binds to a gene of the biomarker; or an antibody, an interacting protein, a ligand, a nanoparticle or an aptamer that specifically binds to a protein or peptide fragment of the biomarker.

5. The use according to claim 4, wherein the agent comprises: reagents for detecting mRNA levels by polymerase chain reaction, nuclease protection assay, in situ hybridization, nucleic acid microarray, northern blotting;

Preferably, the polymerase chain reaction comprises a real-time fluorescent quantitative reverse transcription polymerase chain reaction, a competitive polymerase chain reaction.

6. The use according to claim 4, wherein the product comprises a reagent for detecting protein levels by immunoblotting, enzyme-linked immunosorbent assay, radioimmunoassay, immunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence activated cell sorting, mass analysis or protein microarray.

7. The use according to claim 1, wherein the expression level of AAT is up-regulated in a population suffering from severe or end-stage glaucoma compared to a population suffering from mild glaucoma.

8. Use of an agent for detecting the level of a biomarker in a sample for the manufacture of a product for monitoring the progression of glaucoma or prognosis of glaucoma, wherein the biomarker is AAT.

9. The use of claim 8, wherein the product is a kit, a chip, a sequencing library, a computer system with a glaucoma severity assessment module, or a device with a glaucoma severity assessment module;

Preferably, the reagent comprises: a primer pair, probe or antisense nucleotide that specifically binds to a gene of the biomarker; or an antibody, an interacting protein, a ligand, a nanoparticle or an aptamer that specifically binds to a protein or peptide fragment of the biomarker;

Preferably, if the level of AAT in the subject after treatment is unchanged or increased, then a neurological impairment of the subject is predicted;

Preferably, the nerve injury is selected from at least one of: the retinal nerve fiber layer thickness is reduced and the cup to disc ratio is increased.

10. A device for assessing the severity of glaucoma, monitoring the progression of glaucoma, or prognosis of glaucoma, the device comprising: a processor; an input module, a computer readable medium containing instructions, and an output module;

The input module is used for inputting the level of a biomarker in a biological sample, wherein the biomarker is AAT;

The computer readable medium: the instructions, when executed by the processor, perform an algorithm at an input level of a biomarker;

The output module outputs at least one selected from the following information: glaucoma severity information, glaucoma progression information, and glaucoma prognosis information are evaluated.