CN114689865A - Optic neuromyelitis antibody detection reagent - Google Patents
Optic neuromyelitis antibody detection reagent Download PDFInfo
- Publication number
- CN114689865A CN114689865A CN202011598447.5A CN202011598447A CN114689865A CN 114689865 A CN114689865 A CN 114689865A CN 202011598447 A CN202011598447 A CN 202011598447A CN 114689865 A CN114689865 A CN 114689865A
- Authority
- CN
- China
- Prior art keywords
- antibody
- fluorescent protein
- fluorescence
- reagent
- slide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 37
- 238000001514 detection method Methods 0.000 title claims abstract description 30
- 208000008795 neuromyelitis optica Diseases 0.000 claims abstract description 29
- 108091006047 fluorescent proteins Proteins 0.000 claims abstract description 26
- 102000034287 fluorescent proteins Human genes 0.000 claims abstract description 26
- 102000012002 Aquaporin 4 Human genes 0.000 claims abstract description 12
- 108010036280 Aquaporin 4 Proteins 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 10
- 239000008055 phosphate buffer solution Substances 0.000 claims abstract description 4
- 210000004027 cell Anatomy 0.000 claims description 34
- 239000000523 sample Substances 0.000 claims description 23
- 239000000243 solution Substances 0.000 claims description 10
- 108010043121 Green Fluorescent Proteins Proteins 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 239000012470 diluted sample Substances 0.000 claims description 5
- 239000000499 gel Substances 0.000 claims description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 102100039289 Glial fibrillary acidic protein Human genes 0.000 claims description 4
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 claims description 4
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 claims description 4
- 238000007789 sealing Methods 0.000 claims description 4
- 210000002966 serum Anatomy 0.000 claims description 4
- 241000283707 Capra Species 0.000 claims description 3
- 210000001175 cerebrospinal fluid Anatomy 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000011159 matrix material Substances 0.000 claims description 3
- 210000002381 plasma Anatomy 0.000 claims description 3
- 108010054624 red fluorescent protein Proteins 0.000 claims description 3
- 239000000758 substrate Substances 0.000 claims description 3
- OBYNJKLOYWCXEP-UHFFFAOYSA-N 2-[3-(dimethylamino)-6-dimethylazaniumylidenexanthen-9-yl]-4-isothiocyanatobenzoate Chemical compound C=12C=CC(=[N+](C)C)C=C2OC2=CC(N(C)C)=CC=C2C=1C1=CC(N=C=S)=CC=C1C([O-])=O OBYNJKLOYWCXEP-UHFFFAOYSA-N 0.000 claims description 2
- 239000012099 Alexa Fluor family Substances 0.000 claims description 2
- 241001116389 Aloe Species 0.000 claims description 2
- 241000283074 Equus asinus Species 0.000 claims description 2
- 241000287828 Gallus gallus Species 0.000 claims description 2
- 108010010803 Gelatin Proteins 0.000 claims description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 claims description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 2
- 108010004729 Phycoerythrin Proteins 0.000 claims description 2
- 239000004698 Polyethylene Substances 0.000 claims description 2
- 239000002202 Polyethylene glycol Substances 0.000 claims description 2
- 150000001299 aldehydes Chemical class 0.000 claims description 2
- 235000011399 aloe vera Nutrition 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 claims description 2
- 108091005948 blue fluorescent proteins Proteins 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- KRVSOGSZCMJSLX-UHFFFAOYSA-L chromic acid Substances O[Cr](O)(=O)=O KRVSOGSZCMJSLX-UHFFFAOYSA-L 0.000 claims description 2
- 210000004748 cultured cell Anatomy 0.000 claims description 2
- 108010082025 cyan fluorescent protein Proteins 0.000 claims description 2
- 229910003460 diamond Inorganic materials 0.000 claims description 2
- 239000010432 diamond Substances 0.000 claims description 2
- CMMUKUYEPRGBFB-UHFFFAOYSA-L dichromic acid Chemical compound O[Cr](=O)(=O)O[Cr](O)(=O)=O CMMUKUYEPRGBFB-UHFFFAOYSA-L 0.000 claims description 2
- 238000010790 dilution Methods 0.000 claims description 2
- 239000012895 dilution Substances 0.000 claims description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims description 2
- AWJWCTOOIBYHON-UHFFFAOYSA-N furo[3,4-b]pyrazine-5,7-dione Chemical compound C1=CN=C2C(=O)OC(=O)C2=N1 AWJWCTOOIBYHON-UHFFFAOYSA-N 0.000 claims description 2
- 239000008273 gelatin Substances 0.000 claims description 2
- 229920000159 gelatin Polymers 0.000 claims description 2
- 235000019322 gelatine Nutrition 0.000 claims description 2
- 235000011852 gelatine desserts Nutrition 0.000 claims description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 2
- 239000010931 gold Substances 0.000 claims description 2
- 229910052737 gold Inorganic materials 0.000 claims description 2
- 239000005090 green fluorescent protein Substances 0.000 claims description 2
- 229960002523 mercuric chloride Drugs 0.000 claims description 2
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 2
- 239000002480 mineral oil Substances 0.000 claims description 2
- 235000010446 mineral oil Nutrition 0.000 claims description 2
- 239000012285 osmium tetroxide Substances 0.000 claims description 2
- 229910000489 osmium tetroxide Inorganic materials 0.000 claims description 2
- 239000012188 paraffin wax Substances 0.000 claims description 2
- OXNIZHLAWKMVMX-UHFFFAOYSA-N picric acid Chemical compound OC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-N 0.000 claims description 2
- 229920001223 polyethylene glycol Polymers 0.000 claims description 2
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 claims description 2
- 238000010791 quenching Methods 0.000 claims description 2
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 238000012360 testing method Methods 0.000 claims description 2
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 claims description 2
- 108091005957 yellow fluorescent proteins Proteins 0.000 claims description 2
- 239000000834 fixative Substances 0.000 claims 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 9
- 239000002953 phosphate buffered saline Substances 0.000 description 9
- 239000011521 glass Substances 0.000 description 4
- 230000000903 blocking effect Effects 0.000 description 3
- 230000002757 inflammatory effect Effects 0.000 description 3
- 208000016192 Demyelinating disease Diseases 0.000 description 2
- 210000001130 astrocyte Anatomy 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- 238000002073 fluorescence micrograph Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000004451 qualitative analysis Methods 0.000 description 2
- 230000004393 visual impairment Effects 0.000 description 2
- 201000004569 Blindness Diseases 0.000 description 1
- 101100298998 Caenorhabditis elegans pbs-3 gene Proteins 0.000 description 1
- 206010012305 Demyelination Diseases 0.000 description 1
- 206010015958 Eye pain Diseases 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 206010033799 Paralysis Diseases 0.000 description 1
- 206010033892 Paraplegia Diseases 0.000 description 1
- 206010046996 Varicose vein Diseases 0.000 description 1
- 206010047571 Visual impairment Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000024203 complement activation Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000010339 dilation Effects 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000004438 eyesight Effects 0.000 description 1
- 108010021843 fluorescent protein 583 Proteins 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000007837 multiplex assay Methods 0.000 description 1
- 210000003733 optic disk Anatomy 0.000 description 1
- 210000001328 optic nerve Anatomy 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 210000003019 respiratory muscle Anatomy 0.000 description 1
- 210000001525 retina Anatomy 0.000 description 1
- 238000012764 semi-quantitative analysis Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 210000005070 sphincter Anatomy 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 208000020431 spinal cord injury Diseases 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 208000027185 varicose disease Diseases 0.000 description 1
- 208000029257 vision disease Diseases 0.000 description 1
- 230000009978 visual deterioration Effects 0.000 description 1
- 230000004382 visual function Effects 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/285—Demyelinating diseases; Multipel sclerosis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- General Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Rheumatology (AREA)
- Rehabilitation Therapy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides an antibody detection reagent for neuromyelitis optica, which comprises a slide reagent, a fluorescence-labeled secondary antibody and a phosphate buffer solution, and is characterized in that the slide reagent comprises AQP4 transfected cells for expressing labeled fluorescent protein and blank vector transfected cells for expressing the labeled fluorescent protein; the fluorescence of the fluorescently labeled secondary antibody and the fluorescence of the transfected cells expressing the fluorescent protein are fluorescence of different color systems. The method is applied to judging whether the sample contains the target antibody or not through fluorescence of different color systems and fluorescence combination.
Description
Technical Field
The invention belongs to the field of immunodetection, and particularly relates to an antibody detection reagent for neuromyelitis optica.
Background
Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system that involves the optic nerve and spinal cord primarily selectively. The primary manifestations are rapid and severe visual deterioration of both eyes (eyes develop several hours, days or even weeks apart), and relatively rare eye pain; some patients develop edema of the optic papilla, varicose veins of the retina, dilation and perioptic papillary exudation; visual function recovery is poor and most patients leave behind severe visual impairment of both eyes or at least one eye (final vision below 0.1). The acute spinal cord injury of NMO can occur before, after, or even simultaneously with vision loss, which can be separated by days, weeks, months, or years, manifested as paraplegia, sensory and sphincter dysfunction, and can lead to respiratory muscle paralysis in severe cases.
The pathogenesis of NMO has not been fully elucidated, and the autoantibody aquaporin 4 (AQP 4) -IgG is now thought to play a dominant role. The combination of AQP4-IgG and AQP4 on the foot processes of astrocytes starts the classical complement activation pathway, produces direct toxic effect on astrocytes, releases a series of inflammatory factors to recruit granulocytes, macrophages and the like, and aggravates inflammatory injury and demyelination of the central nervous system, and NMO other related antibodies also comprise MOG, MBP, GFAP and the like.
Monochromatic fluorescence is adopted during the detection and judgment of the neuromyelitis antibody at present, and the monochromatic fluorescence is easy to cause false positive during detection and has low accuracy.
Disclosure of Invention
In order to solve the problems in the detection and improve the accuracy of the detection of the neuromyelitis optica antibody, the invention provides a reagent for detecting the neuromyelitis optica antibody. Whether the fluorescence spots of different color systems are superposed or not and whether the fluorescence characteristics are consistent or not are judged through fluorescence and fluorescence combination of different color systems, and the method is applied to judging whether a sample contains a target antibody or not, so that the accuracy of antibody detection is improved, and qualitative or semi-quantitative analysis can be carried out on the neuromyelitis optica antibody.
The neuromyelitis optica antibody detection reagent comprises a slide glass reagent, a fluorescence-labeled secondary antibody and Phosphate Buffered Saline (PBS), and is characterized in that the slide glass reagent comprises AQP4 transfected cells for expressing labeled fluorescent protein and blank carrier transfected cells for expressing the labeled fluorescent protein; the fluorescence of the fluorescently labeled secondary antibody and the fluorescence of the transfected cells expressing the fluorescent protein are fluorescence of different color systems.
Furthermore, AQP4 transfected cells expressing labeled fluorescent protein and blank vector transfected cells expressing labeled fluorescent protein are fixed on different areas of the same surface of the slide respectively.
Further, the slide reagent also comprises one or more of MOG, MBP and GFAP transfected cells expressing a labeled fluorescent protein.
Further, the fluorescence-labeled secondary antibody is selected from goat anti-human IgG, mouse anti-human IgG, rabbit anti-human IgG, donkey anti-human IgG, horse anti-human IgG or chicken anti-human IgG.
Further, the fluorescent substance used in the fluorescently labeled secondary antibody is selected from fluorescein isothiocyanate, rhodamine, tetramethylrhodamine isothiocyanate, Texas Red, phycoerythrin, propidium iodide, Alexa Fluor series, Dylight series, iFluor series, PE or Cy series.
Further, the fluorescent protein is selected from red fluorescent protein, orange fluorescent protein, yellow fluorescent protein, green fluorescent protein, cyan fluorescent protein or blue fluorescent protein.
Further, the preparation method of the neuromyelitis optica antibody detection reagent comprises the following steps:
1) respectively culturing AQP4 transfected cells expressing a labeled fluorescent protein and blank vector transfected cells expressing the labeled fluorescent protein on a flat substrate;
2) removing the culture medium from the cultured cells, and washing with a phosphate buffer solution;
3) adding a fixing solution to fix the cells;
4) sealing the fixed cells with a sealing liquid, and drying;
5) the dried cell-loaded plate matrix was transferred to a slide and fixed with a shadowless gel.
Further, the fixing solution is a solution prepared from one or more of aldehydes, methanol, ethanol, acetone, osmium tetroxide, picric acid, chloroform, chromic acid, mercuric chloride and dichromic acid.
Further, the mounting solution is selected from one or more of polyethylene glycol, BSA, SlowFade Diamond, SlowFade Gold, glycerol, paraffin, gelatin, aloe gel, anti-quenching agent and mineral oil.
The detection method of the neuromyelitis optica antibody detection reagent comprises the following steps:
1) preparing a serum, plasma or cerebrospinal fluid sample, taking the diluted sample and adding the diluted sample into a slide reaction zone;
2) incubation at 37 ℃ or overnight at 4 ℃;
3) washing with PBS;
4) fully draining PBS, adding a fluorescence-labeled secondary antibody into a slide reaction zone, and incubating at 37 ℃;
5) washing with PBS;
6) adding PBS into the reaction zone;
7) the slide was placed under a fluorescent microscope for microscopic examination.
Further, a fluorescence microscope is used for observing the fluorescence spots and the combination of the fluorescence spots, whether the positions of the fluorescence spots are overlapped and whether the fluorescence characteristics are consistent or not is judged, whether the sample contains the anti-AQP 4 antibody or not is judged, and qualitative or semi-quantitative detection and analysis are carried out on the anti-AQP 4 antibody in the sample through the dilution multiple of serum, plasma or cerebrospinal fluid of a subject and the intensity of the spots, so that a basis is provided for clinically judging the severity of the neuromyelitis optica patients.
Furthermore, the detection reagent is in a slide glass form, and the sample amount and the PBS amount can be adjusted according to the structure of the slide glass.
For example, the red fluorescent protein is selected from mCherry, mBanana, mOrange, dTomato, mTangerine, or mStrawberry.
The invention provides a diagnosis method for neuromyelitis optica by using a double-fluorescence positioning interpretation method as a quantitative or qualitative analysis method for neuromyelitis optica.
The dual fluorescence used in the invention is different color systems, so that the color systems of protein fluorescence and labeled fluorescence can be accurately distinguished when a fluorescence interpretation device or naked eyes are used for identification.
The detection reagent of the invention can adopt fluorescence interpretation equipment or naked eye identification when judging the position and the intensity of fluorescence, and preferably adopts fluorescence interpretation equipment for interpretation when carrying out quantitative detection.
The cells used in the invention are selected from HEK293T cells, and can be preserved.
The invention has at least the following beneficial effects:
1) the antibody detection reagent prepared by the invention can detect the AQP4 antibody, has strong specificity, high sensitivity and good repeatability of experimental results, can be popularized and applied, and has extremely important significance clinically. 2) In addition, aiming at the problem that misjudgment is easily caused under the condition of low fluorescence signals in the prior art, the invention reduces the problem that misjudgment is easily caused under the condition of low fluorescence signals by expressing fluorescent protein by cells, adding a fluorescence-labeled secondary antibody and observing whether the positions of fluorescent cells emitted by different color systems can be overlapped and the characteristics of fluorescence.
Drawings
Fig. 1-2: fluorescence image of sample under microscope detected by neuromyelitis optica antibody detection reagent (sample containing anti-AQP 4 antibody)
Fig. 3 to 4: fluorescence image of sample detected by neuromyelitis optica antibody detection reagent under microscope (sample does not contain anti-AQP 4 antibody)
Detailed Description
Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples are given for purposes of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
According to the embodiment of the invention, the anti-AQP 4 antibody detection reagent is prepared, and the anti-AQP 4 antibody in the sample is detected by using the detection reagent, so that whether the sample contains the anti-AQP 4 antibody or not is judged, and a basis is provided for a clinician to diagnose the severity of the neuromyelitis optica.
In the examples, red fluorescence and green fluorescence are used, where mCherry emits red fluorescence and Dylight 488 emits green fluorescence. The fluorescence of different color systems is selected to ensure that the fluorescence color difference of the two can be easily distinguished by naked eyes.
Example 1: preparation of neuromyelitis optica antibody detection reagent
1) Culturing AQP4 transfected cells expressing a marker mCherry and blank vector transfected cells expressing the marker mCherry on a plate substrate, observing the growth condition of the cells under a microscope, and waiting until the cells expand to nearly 100%;
2) removing the culture medium, and washing with 1 × PBS;
3) adding 50 mul of 4% paraformaldehyde fixing solution to fix the cells for 60min, discarding the fixing solution, and adding 50 mul of 1 XPBS to wash for 2 times;
4) adding 50 mul of 5% BSA blocking solution for blocking for 20min, discarding the blocking solution, adding 50 mul of 1 XPBS for washing for 2 times, and drying;
5) the dried cell-loaded plate matrix was transferred to a slide and fixed with a shadowless gel.
Example 2: neuromyelitis optica antibody detection reagent for sample detection
The reagent prepared in example 1 was used for the detection of the anti-AQP 4 antibody as follows:
1) preparing a serum sample, adding 30-50 mul of the diluted sample into a slide reaction area, and keeping the sample moist in a dark place;
2) incubation at 37 ℃ for 1 hour or overnight at 4 ℃;
3) wash 3 times with 1 × PBS for 3 minutes each;
4) fully draining 1 XPBS, adding 30-50 mul of goat anti-human secondary antibody marked by fluorescent Dylight 488 into a slide reaction area, incubating for 30 minutes at 37 ℃, and keeping moisture in a dark place;
5) 1 × PBS 3 washing, each time 3 minutes;
6) adding 30-50 mu l of 1 XPBS into the reaction area, and keeping the reaction area in a dark place for moisture;
7) the slide was placed under a fluorescent microscope for microscopic examination.
Example 3: use of microscopy for interpretation of anti-AQP 4 antibodies in samples
Example 2 interpretation of the treated slides was as follows:
1) observing the emitted fluorescent spots by using a fluorescence microscope at the wavelength of 518nm and the wavelength of 610nm respectively, and observing whether the green fluorescent spots are emitted or not;
2) comparing the position and the characteristics of the green fluorescent spot with the position of the red fluorescent spot to determine whether the positions and the characteristics of the green fluorescent spot are overlapped;
3) if the positions emitting green fluorescent spots and the positions emitting red fluorescent spots can be overlapped and have basically the same characteristics, the sample can be judged to contain the anti-AQP 4 antibody;
4) if the green fluorescent spot can not be emitted; or the position emitting the green fluorescent spot and the position emitting the red fluorescent spot cannot be overlapped; or the position emitting green fluorescent spots and the position emitting red fluorescent spots can overlap, but the features are greatly different; the sample can be judged to be free of anti-AQP 4 antibody.
FIG. 1 shows the red spot overlaps and has substantially the same characteristics as the green spot of FIG. 2, indicating that the sample contains anti-AQP 4 antibody; FIG. 3 position of red fluorescent spots in FIG. 4 there are no fluorescent spots, indicating that the sample does not contain anti-AQP 4 antibody.
Similarly, a multiplex assay test reagent may be prepared as described in example 1, and the reagent may include one or more of cells transfected with MOG, MBP, GFAP and labeled with fluorescent protein in addition to cells transfected with AQP4 labeled with fluorescent protein, so that a plurality of antibodies in a sample can be simultaneously detected and interpreted.
All technical features disclosed in the present specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.
Furthermore, from the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes to the invention to adapt it to various usages and conditions, and therefore such changes are intended to fall within the scope of the appended claims.
Claims (10)
1. The reagent for detecting the neuromyelitis optica antibody comprises a slide reagent, a fluorescence-labeled secondary antibody and a phosphate buffer solution, and is characterized in that the slide reagent comprises AQP4 transfected cells for expressing labeled fluorescent protein and blank vector transfected cells for expressing the labeled fluorescent protein; the fluorescence of the fluorescently labeled secondary antibody and the fluorescence of the transfected cells expressing the fluorescent protein are fluorescence of different color systems.
2. The reagent for detecting neuromyelitis optica antibody of claim 1, wherein AQP4 transfected cells expressing a labeled fluorescent protein and blank vector transfected cells expressing a labeled fluorescent protein are immobilized on the same surface of a slide at different regions, respectively.
3. The reagent for detecting neuromyelitis optica antibody of claim 1, wherein said slide reagent further comprises one or more of cells transfected with MOG, MBP, GFAP expressing labeled fluorescent protein.
4. The reagent of claim 1, wherein the fluorescently labeled secondary antibody is selected from goat anti-human IgG, mouse anti-human IgG, rabbit anti-human IgG, donkey anti-human IgG, horse anti-human IgG, and chicken anti-human IgG.
5. The reagent for the detection of neuromyelitis optica antibody as claimed in claim 1, wherein said fluorescently labeled secondary antibody uses a fluorescent substance selected from the group consisting of fluorescein isothiocyanate, rhodamine, tetramethylrhodamine isothiocyanate, Texas Red, phycoerythrin, propidium iodide, Alexa Fluor series, Dylight series, iFluor series, PE and Cy series.
6. The reagent for detecting neuromyelitis optica antibody of claim 1, wherein said fluorescent protein is selected from the group consisting of red fluorescent protein, orange fluorescent protein, yellow fluorescent protein, green fluorescent protein, cyan fluorescent protein and blue fluorescent protein.
7. The method for preparing an neuromyelitis optica antibody detection reagent of any one of claims 1 to 6, comprising:
1) respectively culturing AQP4 transfected cells expressing a labeled fluorescent protein and blank vector transfected cells expressing the labeled fluorescent protein on a flat substrate;
2) removing the culture medium from the cultured cells, and washing with a phosphate buffer solution;
3) adding a fixing solution to fix the cells;
4) sealing the fixed cells with a sealing liquid, and drying;
5) the dried cell-loaded plate matrix was transferred to a slide and fixed with a shadowless gel.
8. The method according to claim 7, wherein the fixative is selected from the group consisting of solutions prepared from one or more of aldehydes, methanol, ethanol, acetone, osmium tetroxide, picric acid, chloroform, chromic acid, mercuric chloride, dichromic acid; the confining liquid is one or more selected from polyethylene glycol, BSA, SlowFade Diamond, SlowFade Gold, glycerol, paraffin, gelatin, aloe gel, anti-quenching agent and mineral oil.
9. The method for detecting an neuromyelitis optica antibody detection reagent of any one of claims 1 to 6, comprising the steps of:
1) preparing a serum, plasma or cerebrospinal fluid sample, taking the diluted sample and adding the diluted sample into a slide reaction zone;
2) incubation at 37 ℃ or overnight at 4 ℃;
3) washing with PBS;
4) fully draining PBS, adding a fluorescence-labeled secondary antibody into a slide reaction zone, and incubating at 37 ℃;
5) washing with PBS;
6) adding PBS into the reaction zone;
7) the slide was placed under a fluorescent microscope for microscopic examination.
10. The method for detecting an neuromyelitis optica antibody test reagent of claim 9, wherein a fluorescent spot and a combination of fluorescent spots are observed with a fluorescence microscope, and whether or not the anti-AQP 4 antibody is contained in the sample is determined by determining whether or not the positions of the fluorescent spots overlap and whether or not the fluorescent characteristics are identical; and carrying out qualitative or semi-quantitative detection analysis on the anti-AQP 4 antibody in the sample through the dilution multiple of the sample and the strength of the spots.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011598447.5A CN114689865A (en) | 2020-12-30 | 2020-12-30 | Optic neuromyelitis antibody detection reagent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011598447.5A CN114689865A (en) | 2020-12-30 | 2020-12-30 | Optic neuromyelitis antibody detection reagent |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114689865A true CN114689865A (en) | 2022-07-01 |
Family
ID=82132305
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011598447.5A Pending CN114689865A (en) | 2020-12-30 | 2020-12-30 | Optic neuromyelitis antibody detection reagent |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114689865A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102388307A (en) * | 2008-11-12 | 2012-03-21 | 耶达研究与发展有限公司 | Diagnosis of multiple sclerosis |
| CN106318974A (en) * | 2016-08-29 | 2017-01-11 | 南方医科大学南方医院 | Cell immobilization technology and AQP4 antibody detection kit prepared through same |
-
2020
- 2020-12-30 CN CN202011598447.5A patent/CN114689865A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102388307A (en) * | 2008-11-12 | 2012-03-21 | 耶达研究与发展有限公司 | Diagnosis of multiple sclerosis |
| CN106318974A (en) * | 2016-08-29 | 2017-01-11 | 南方医科大学南方医院 | Cell immobilization technology and AQP4 antibody detection kit prepared through same |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20170253918A1 (en) | Combining protein barcoding with expansion microscopy for in-situ, spatially-resolved proteomics | |
| JP5555846B2 (en) | Prognosis determination method for acute central nervous system disorder | |
| EP0565541B1 (en) | Diagnostic method for detecting the rupture of fetal membranes and test kit employing the method | |
| US8476026B2 (en) | Biomarkers of ovarian cancer | |
| Waters et al. | Detection methods for neural autoantibodies | |
| CN110361547B (en) | Reagent for chemiluminescence quantitative detection of fecal occult blood, detection method thereof and application of reagent in detection of lower digestive tract health | |
| CA1340973C (en) | Immunometric assay kit and method applicable to whole cells | |
| KR100689995B1 (en) | Composition comprising aldolase and methof for diagnosing retinal vascular disease | |
| US20220137047A1 (en) | Borrelia immunoassays and materials therefor | |
| CN117405897B (en) | Immunochromatography detection card for detecting acute myocardial infarction and preparation method and application thereof | |
| WO2016175406A1 (en) | Method for diagnosing sjogren's syndrome by using antibody reaction test specific to sjogren's syndrome | |
| CN114689865A (en) | Optic neuromyelitis antibody detection reagent | |
| WO2014157926A1 (en) | Marker for diagnosing age-related macular degeneration, and method for diagnosing age-related macular degeneration by using same | |
| CN107449914A (en) | The application of UCHL1 autoantibody | |
| CN114689866A (en) | Optic neuromyelitis antibody detection reagent | |
| US11579146B2 (en) | Detection of haptoglobin for gastrointestinal cancer determination | |
| CN114689863A (en) | Reagent for combined detection of encephalitis | |
| CN108414478A (en) | A kind of detection method of biological sample | |
| CN114689861A (en) | Encephalitis joint inspection reagent | |
| JP6488236B2 (en) | A sensitive multiplex immunoassay for soluble fibroblast growth factor receptor. | |
| FR3062919A1 (en) | IMMUNOLOGICAL ASSAY METHOD FOR VIRUS ANTIGEN-SPECIFIC ANTIBODIES OF THE POXVIRIDAE FAMILY | |
| CN114689862A (en) | anti-NMDAR antibody detection reagent | |
| CN114689860A (en) | anti-NMDAR antibody detection reagent | |
| CN114689864A (en) | Antibody detection reagent for membranous nephropathy | |
| US6379906B1 (en) | Compositions and methods for detecting adult Taenia solium |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |