CN107022548B - Anti- AQP4 autoantibody detection material of a kind of human body and preparation method thereof - Google Patents

Anti- AQP4 autoantibody detection material of a kind of human body and preparation method thereof Download PDF

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CN107022548B
CN107022548B CN201710175642.9A CN201710175642A CN107022548B CN 107022548 B CN107022548 B CN 107022548B CN 201710175642 A CN201710175642 A CN 201710175642A CN 107022548 B CN107022548 B CN 107022548B
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闫亚平
柴单单
李科
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Shaanxi Mai Yuan Biotechnology Co Ltd
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Abstract

The present invention provides anti-AQP4 autoantibody detection materials of a kind of human body and preparation method thereof, first obtain the AQP4 overall length target gene of M1, M23 hypotype, it is contaminated in HEK293 cell, constructs the HEK293/pCI-neo-M1-AQP4 cell and HEK293/pCI-neo-M23-AQP4 cell of expression M1, M23 hypotype AQP4；The AQP4- cell membrane complexes in above-mentioned cell are extracted again；Finally AQP4- cell membrane complexes are solidificated on carrier and detect material to get to the anti-AQP4 autoantibody of human body.The present invention is using the AQP4- cell membrane complexes extracted as detection material, the intrinsic protein amount for causing background coloration can be lowered, relatively detection sensitivity and specificity are further increased using the method that cell is the Immunofluorescence test that detection material carries out at present, it is more easier testing result interpretation clear, simply and rapidly can be used to detect patient's AQP4 autoantibody.

Description

Anti- AQP4 autoantibody detection material of a kind of human body and preparation method thereof
Technical field
The invention belongs to biomedicine field, it is related to a kind of anti-AQP4 autoantibody detection material of human body and its preparation side Method, detection material obtained can be used in detecting the anti-AQP4 autoantibody of human body.
Background technique
Important blood serum designated object of the human body autoantibody as autoimmune disease, quickly detection is for autoimmunity Diagnosis, remedy measures and the prognosis of property disease have important application value.The sensitivity and specificity detected with autoantibody It improves, the diagnostic criteria of associated autoimmune disease also updates adjustment corresponding.
Neuromyelitis optica (neuromyelitis, NMO) is a kind of serious central nervous system demyelinating disease, about Patient's aquaporin 4 (aquaporin, AQP4) antibody positive of 90% NMO and more than half.Aquaporin in 2004 The discovery of 4 antibody becomes independent disease for NMO and provides objective evidence, and the NMO diagnosis mark of correcting was included into 2015 It is quasi-.
Currently, the AQP4 autoantibody detection method in patients serum or cerebrospinal fluid includes the following categories: 1, using high table Neural tissue slice up to the antigen carries out immunological experiment (indirect immunofluorescence, Indirect Immunofluorescence Assay, IIF), to detect the autoantibody for AQP4.The method is due to neural tissue slice Prepare more complex, differing greatly between batch, it is difficult to Quality Control；And to the more demanding of reviewer, need reviewer There is nervous physiology abundant to gain knowledge, can just accurately identify the position in slice where positive signal and meaning, therefore realize quotient The difficulty of product is larger.2, Immunofluorescence test or flow cytometry (cell-based are carried out using the cell of overexpression protection original assay,CBA).But the major defect of such detection method is: (1) intracellular multiple protein may cause background coloration and ask Topic.It in specific detection process, is compared in spite of idle running control cell, when external source antigen presentation amount is lower, part is detected As a result still it is not easy clear interpretation.(2) operating process is complicated, and whether exact operations directly affect testing result to each step, this is very The experience for relying on monitoring personnel, when being additionally carried out immunofluorescence or flow cytometry tests, needs specific apparatus, such as fluorescence microscopy Mirror or flow cytometer.3, enzyme linked immunosorbent assay (ELISA) is carried out using recombinant antigen protein.AQP4 is as memebrane protein, just The space conformation structure that true cross-film is formed is most important to the identification of autoantibody, during the expression of recombinant protein often because It is incorrect to fold translation, the conception of mistake is formed, the specificity of detection is influenced.In addition, recombinant protein especially memebrane protein Isolate and purify that work is relatively time consuming, effort.In addition there are the methods of several detection AQP4 autoantibodies, no longer enumerate herein.
Currently, IIF and CBA method is the common detection methods of anti-AQP4 autoantibody.IIF method is compared with CBA method sensibility and spy It is anisotropic relatively low, thus it is speculated that may be because there are the differences between kind to cause for its reaction substrate.And CBA method stablizes height by building The cell strain of people AQP4 is expressed as substrate, the difference between kind is on the one hand avoided, improves sensibility and the spy of the method The opposite sex, the acquisition on the other hand detecting material are also easy relatively.
Summary of the invention
The purpose of the present invention is to provide anti-AQP4 autoantibody detection material of a kind of human body and preparation method thereof, the detections Material can further decrease nonspecific signals, make to examine under the premise of based on cell detection method high sensitivity with specificity It surveys result interpretation to be more easier to define, simply and rapidly can be used to detect patient's AQP4 autoantibody.
In order to achieve the above objectives, the technical solution adopted by the present invention are as follows:
A kind of preparation method of the anti-AQP4 autoantibody detection material of human body, comprising the following steps:
1) the AQP4 overall length target gene for obtaining M1, M23 hypotype, is distinguished target gene by pCI-neo plasmid vector It is transfected into HEK293 cell, constructs HEK293/pCI-neo-M1-AQP4 cell and the expression of expression M1 hypotype AQP4 gene The HEK293/pCI-neo-M23-AQP4 cell of M23 hypotype AQP4 gene；
2) AQP4- of HEK293/pCI-neo-M1-AQP4 cell and HEK293/pCI-neo-M23-AQP4 cell is extracted Cell membrane complexes；
3) the AQP4- cell membrane complexes of extraction are solidificated on carrier, obtain the anti-AQP4 autoantibody detection material of human body Material.
The step 1) specifically includes the following steps:
1.1) the AQP4 overall length target gene of M1, M23 hypotype is obtained by artificial synthesized or PCR method, and in purpose base Because both ends add NotI/NheI restriction enzyme site；
1.2) two kinds of target gene with restriction enzyme site are inserted into respectively in pCI-neo plasmid vector, insertion point is NotI/NheI obtains two kinds of plasmids for having target gene, is respectively designated as pCI-neo-M1-AQP4 and pCI-neo-M23- AQP4；
1.3) pCI-neo-M1-AQP4 plasmid and pCI-neo-M23-AQP4 plasmid are taken respectively, are transfected by PEI transfection reagent Method transfects cultured HEK293 cell, obtains the HEK293/pCI-neo-M1-AQP4 cell of expression M1 hypotype AQP4 gene With the HEK293/pCI-neo-M23-AQP4 cell of expression M23 hypotype AQP4 gene.
The specific steps of the step 2) are as follows: HEK293/pCI-neo-M1-AQP4 cell is removed by centrifugation respectively first With the nucleus of HEK293/pCI-neo-M23-AQP4 cell, detergent is then added, 4 DEG C act on 30~60 minutes, then are centrifuged Undissolved part is removed, supernatant is soluble AQP4- cell membrane complexes；Wherein detergent is 5~20mmol/L's The Triton X-100 that CHAPS or volume fraction are 0.5~1%.
The specific steps of the step 2) are as follows: first pass through physical grinding, ultrasound or low temperature freeze thawing makes HEK293/pCI- respectively The cellular membrane disruption of neo-M1-AQP4 cell and HEK293/pCI-neo-M23-AQP4 cell discharges cell cytosol content, Be then centrifuged for, abandon supernatant plasmosin, be added hypertonic solution into obtained precipitating, 4 DEG C act on 15~30 minutes, again from The heart, abandons supernatant karyon albumen, and finally obtained precipitating is the AQP4- cell membrane complexes of insolubility.
The hypertonic solution is by Tris-HCl, NaCl, MgCl2It is formulated with EDTA, Tris-HCl in hypertonic solution Concentration is that the concentration of 20mmol/L, NaCl are 300mmol/L, MgCl2Concentration be 2mmol/L, EDTA concentration be 2~ 5mmol/L, the pH=8 of hypertonic solution.
The specific steps of the step 2) are as follows: first pass through physical grinding, ultrasound or low temperature freeze thawing makes HEK293/pCI- respectively The cellular membrane disruption of neo-M1-AQP4 cell and HEK293/pCI-neo-M23-AQP4 cell discharges cell cytosol content, It is then centrifuged for, abandons supernatant plasmosin, Dextran-PEG-Na is added into obtained precipitating3PO4Liquid two-phase mixed liquor, then Secondary centrifugation, is then allowed to stand, and forms two liquid phases, contains AQP4- cell membrane complexes in the liquid phase of top.
The Dextran-PEG-Na3PO4Liquid two-phase mixed liquor is by Dextran, PEG and Na3PO4It is formulated, Dextran-PEG-Na3PO4It is 1.03g/mL that the volume fraction of Dextran, which is the concentration of 20%, PEG, in liquid two-phase mixed liquor, Na3PO4Concentration be 0.2mol/L, Dextran-PEG-Na3PO4The pH=6.5 of liquid two-phase mixed liquor.
The carrier is nitrocellulose filter, pvdf membrane, nylon membrane, the slide with protein adsorption ability, plastic material Tissue Culture Dish or culture plate.
The specific steps of the step 3) are as follows: dilute AQP4- cell membrane complexes with 1 × TBS buffer, be configured to The dilution of AQP4- cell membrane complexes, the concentration of AQP4 is 0.1~1mg/mL in the dilution of AQP4- cell membrane complexes, Then the dilution of AQP4- cell membrane complexes is pressed into round or linear trace on carrier with stroke film instrument, is then placed in 37 DEG C Drying in oven reuses fixer and fixes 10~20 minutes, then washes away fixer with PBS buffer solution, finally with quality point Number was closed 16~20 hours for 1% BSA confining liquid in closing 1 hour or 4 DEG C under room temperature, is drained confining liquid and is steamed with double Water cleans up, and is dried at 20~28 DEG C to get to the anti-AQP4 autoantibody detection material of human body, is sealed in -20 DEG C For use；It is the ethyl alcohol that 4% paraformaldehyde, ice methanol or volume fraction are 95% that wherein fixer, which is mass fraction,.
The anti-AQP4 autoantibody inspection of human body made from the preparation method of the anti-AQP4 autoantibody detection material of the human body It measures and monitor the growth of standing timber material, which is the AQP4- cell membrane complexes being solidificated on carrier, and wherein AQP4- cell membrane complexes are by table Up to the HEK293/pCI-neo-M1-AQP4 cell of M1 hypotype AQP4 gene and the HEK293/ of expression M23 hypotype AQP4 gene PCI-neo-M23-AQP4 cell extraction obtains.
The main component of AQP4- cell membrane complexes is with bioactivity, keeps the HEK293/ of its original space conformation The cell membrane of pCI-neo-M1-AQP4 and HEK293/pCI-neo-M23-AQP4 cell, while may be comprising some thin in separation The substances such as indivisible organelle during after birth, but wherein play the role of confrontation AQP4 autoantibody detection is The cell membrane of HEK293/pCI-neo-M1-AQP4 and HEK293/pCI-neo-M23-AQP4 cell.
Compared with the existing technology, the invention has the benefit that
The preparation method of the anti-AQP4 autoantibody detection material of human body provided by the invention, first obtains M1, M23 hypotype Target gene is transfected into HEK293 cell by pCI-neo plasmid vector, constructs expression by AQP4 overall length target gene The HEK293/pCI-neo-M1-AQP4 cell and HEK293/pCI-neo-M23-AQP4 cell of M1, M23 hypotype AQP4 gene； The AQP4- cell membrane in HEK293/pCI-neo-M1-AQP4 cell and HEK293/pCI-neo-M23-AQP4 cell is extracted again Compound；Finally the AQP4- cell membrane complexes of extraction are solidificated on carrier and are detected to get to the anti-AQP4 autoantibody of human body Material.The present invention using the AQP4- cell membrane complexes extracted as the detection material for detecting the anti-AQP4 autoantibody of human body, The intrinsic protein amount for causing background coloration can be lowered, more current CBA method is the immunofluorescence for detecting material and carrying out using cell The method of detection further increases detection sensitivity and specificity, can further decrease nonspecific signals, make testing result Interpretation is more easier clear.And the application method of the anti-AQP4 autoantibody detection material of human body prepared by the present invention is also simpler It is single, reviewer is required low, entire testing process expends that the time is short, and required instrument is simple, can simply and rapidly be used to examine Survey patient AQP4 autoantibody.
The anti-AQP4 autoantibody of human body produced by the present invention detects material, is that the AQP4 cell membrane of space conformation will be kept to answer It closes object and is solidificated in and obtained on carrier, it, can be further based on cell detection method high sensitivity and under the premise of specificity Nonspecific signals are reduced, is more easier testing result interpretation clear, simply and rapidly can be used to detect patient AQP4 itself Antibody.
Further, in the preparation method of the anti-AQP4 autoantibody detection material of human body provided by the invention, key point It is to obtain the AQP4 cell membrane complexes for keeping space conformation.The entire mistake of AQP4- cell membrane complexes is extracted in the present invention Cheng Zhong is able to maintain membrane protein configuration and is not destroyed, and loses Membrane protein antigen not.Abandon cell cytosol and karyon at Point, the AQP4- cell membrane complexes of extraction can lower the intrinsic protein amount for causing background coloration, increase as detection material Detection sensitivity and specificity.In addition the present invention in AQP4- cell membrane complexes simple and convenient extraction, do not need special instrument Device relatively purifies the cost of memebrane protein and technical requirements substantially reduces.
Detailed description of the invention
Fig. 1 is the schematic diagram that the anti-AQP4 autoantibody of human body prepared by the present invention detects material, and wherein a-quadrant is coated with M23- AQP4 cell membrane complexes；B area is coated with pCI-neo cell membrane complexes.
Fig. 2 is that the anti-AQP4 autoantibody of human body prepared by the present invention detects in material tests NMO patient and normal human serum Anti- AQP4 Antibody Results, it is known NMO patients serum that wherein a, which is detection serum, and b is that detection serum is known normal blood Clearly.
Specific embodiment
Below with reference to specific embodiment, the present invention is described in further detail.
The preparation method of the anti-AQP4 autoantibody detection material of human body provided by the invention specifically includes the following steps:
One, AQP4 overall length target gene that is artificial synthesized or being obtained by PCR method, building stabilization or transient expression M1, M23 HEK293/pCI-neo-M1-AQP4, HEK293/pCI-neo-M23-AQP4 cell of hypotype, and the HEK293/pCI-neo that dallies Cell is as a control group.The specific steps of which are as follows:
1. plasmid construction
1.1 target gene obtain: the AQP4 overall length purpose base of M1, M23 hypotype is obtained by artificial synthesized or PCR method Cause, both ends add the restriction enzyme site of NotI/NheI.
Obtain two kinds of target gene are inserted into carrier pCI-neo plasmid by 1.2 respectively, and insertion point NotI/NheI will Two kinds of obtained plasmids are respectively designated as pCI-neo-M1-AQP4 and pCI-neo-M23-AQP4, and correct rear extraction plasmid is sequenced Carry out subsequent experimental.The specific method is as follows:
A. the glycerol strain of pCI-neo plasmid is taken to be inoculated in the 3mL of ammonia benzyl resistance, LB liquid medium sets 37 DEG C of constant temperature Incubator, 220r/min shake bacterium overnight.
B. second day draws 500 μ L of bacterium solution and saves strain, and the remaining small extraction reagent kit of 2.5mL bacterium solution plasmid is extracted, surveyed dense Degree.
C.pCI-neo carrier digestion: 40 μ L digestion systems, 2 μ g carriers, NotI, each 1 μ L, 10x digestion Buffer of NheI enzyme 4 μ L, ddH2O polishing is to 40 μ L, 37 DEG C of insulating box digestion 2h.
D. digestion products recycle, and 0.8% agarose gel electrophoresis separates digestion products, illustrate according to gel reclaims kit Book carries out carrier segments recycling.
E. target gene is connect with carrier segments: 20 μ L linked systems, 50ng pCI-neo carrier, 150ng M23- AQP4, T4 ligase 1 μ L, ddH2O polishing is to 20 μ L, and 4 DEG C of connections are overnight.
F. by the pCI-neo-M23-AQP4 plasmid equipped with M23-AQP4 genetic fragment or equipped with M1-AQP4 genetic fragment PCI-neo-M1-AQP4 plasmid is transformed into bacillus coli DH 5 alpha, applies ammonia benzyl solid LB culture plate, 37 DEG C overnight, and second day is chosen 5 bacterial strains are taken, the LB liquid medium of inoculation and ammonia benzyl resistance, 37 DEG C are incubated overnight.Next day, each pipe draw 500 μ L bacterium solutions ,- 20 DEG C of preservations, remaining bacterium solution plasmid is small to be mentioned, and digestion identifies that correct bacterial strain send sequencing to identify again.Correct bacterial strain is identified in sequencing Leave and take strain, -80 DEG C of preservations.
G. sequencing correctly carries out plasmid and mentions greatly with target gene strain and the expansion culture of carrier strain, side concentration.
2.AQP4 expresses 293T cell construction
2.1HEK293 cell culture, DMEM high glucose medium and FBS are configured to 10%FBS-DMEM high sugar in 9:1 ratio Culture medium is passed on by 1:3 to cell fusion to 90%, continues subculture to the 3rd generation, cover with to the 3rd generation cell to 80-90% When, cell suspension inoculation is made in 10cm Tissue Culture Dish with complete medium in 0.05% trypsin digestion, is inoculated with 1.5x106 A cell is added complete medium to 10mL, sets 37 DEG C, 5%CO2Cell incubator in cultivate.
2.2 is long to culture dish floor space 70-80% to HEK293 cell in 10cm culture dish, replaces fresh culture and continues 4h is cultivated, is transfected by PEI transfection reagent transfection method, takes pCI-neo-M1-AQP4 plasmid or pCI-neo-M23-AQP4 Plasmid 30 μ g, PEI are 45 μ g.Fresh medium is replaced after transfection after 6h, collects cell after transfecting 72h, is stablized or instantaneous Express HEK293/pCI-neo-M1-AQP4, HEK293/pCI-neo-M23-AQP4 cell of M1, M23 hypotype.
2.3, by pCI-neo plasmid transfection HEK293 cell, obtain HEK293/pCI-neo cell and compare.
Two, the acquisition of AQP4- cell membrane complexes.
It collects HEK293/pCI-neo-M1-AQP4 cell and HEK293/pCI-neo-M23-AQP4 cell height expresses purpose AQP4 albumen and the HEK293/pCI-neo cell of idle running extract AQP4- cell membrane complexes, and it is compound to obtain AQP4- cell membrane Object includes but is not limited to following several method:
A: soluble AQP4- cell membrane complexes are obtained with de-sludging agent method.5-20mmol/ can be used in detergent herein L CHAPS, 0.5-1%Triton X-100 etc..Cell low-speed centrifugal is removed into core first, detergent cold service is then added 30-60 minutes, then undissolved part is removed by low-speed centrifugal, supernatant is soluble AQP4- cell membrane complexes.
B: with the cell membrane extracting method for gradually removing nucleus and endochylema, the AQP4- cell membrane for obtaining insolubility is multiple Close object.Cellular membrane disruption is made by physical grinding, ultrasound or low temperature freeze thawing first, discharges cell cytosol content.High speed centrifugation Supernatant plasmosin is abandoned, hypertonic solution is added in precipitating: 20mmol/L Tris-HCl, 300mmol/L NaCl, 2mmol/L MgCl2, 2-5mmol/L EDTA, pH 8, cold service 15-30 minutes, 12000-15000rpm, 10 minutes, abandoning supernatant karyon Albumen, precipitating are the AQP4- cell membrane complexes of insolubility.
C: AQP4- cell membrane complexes are obtained with cell membrane liquid two-phase centrifugal separation.Smudge cells, remove endochylema and Nucleoprotein refers to b method, and some organelles are also contained in the precipitating that high speed centrifugation obtains herein, Dextran- is added in precipitating PEG-Na3PO4Liquid two-phase mixed liquor (20%Dextran, 103g PEG, 0.2mol/L Na3PO4, pH 6.5), 2000- 3000g, 4 DEG C, 10 minutes.It slightly stands, forms two liquid phases, mainly contain cell membrane fragment in the liquid phase of top.
For obtaining M23-AQP4- cell membrane complexes using b method, below specific steps:
After transfecting 72h, HEK293/pCI-neo-M23-AQP4 cell is collected, cell, glass is resuspended in 500 μ L hypotonic solutions Homogenizer is homogenized 50 times, makes cellular membrane disruption, discharges cell cytosol content.12000rpm, 4 DEG C of centrifugation 10min abandon supernatant born of the same parents Albumen is starched, hypertonic solution is added in precipitating: 20mmol/L Tris-HCl, 300mmol/L NaCl, 2mmol/L MgCl2, 2mmol/L EDTA, pH 8, cold service 30min, 12000rpm, 4 DEG C, 10min abandon supernatant karyon albumen, precipitate as thin Mixing is sufficiently resuspended in after birth compound, 1xTBS.
Three, solidification AQP4- cell membrane complexes detect material as the anti-AQP4 autoantibody of human body.
Used solidified carrier can be nitrocellulose filter, pvdf membrane, nylon membrane, and the glass with protein adsorption ability Piece, the Tissue Culture Dish of plastic material, culture plate etc..By adjusting the coated AQP4- cell membrane complexes quantity of unit area, Uniform the AQP4 antigen protein amount (or density) of experimental group and negative control group.With point film instrument, film instrument is drawn by AQP4- cell On carrier, film is placed in 37 DEG C of incubators 30-60 minutes by round or linear trace for the dilution of membrane complex.Use fixation Liquid is such as: 4% paraformaldehyde, ice methanol, and 95% dehydrated alcohol etc. is 10-20 minutes fixed, with cleaning solution washes clean fixer.Envelope Liquid is closed in closing closing in 1 hour or 4 DEG C 16-20 hours under room temperature, confining liquid is drained and is cleaned with distilled water, at 20-28 DEG C Under the conditions of dry, be sealed in -20 DEG C.
For solidifying M23-AQP4- cell membrane complexes, the specific steps are as follows:
M23-AQP4 antigen protein concentration in the M23-AQP4- cell membrane complexes extracted is measured with BCA, with 1xTBS tune Its whole ultimate density is to 1mg/mL.It is with point film instrument that M23-AQP4 cell membrane complexes dilution and pCI-neo cell membrane is compound Object dilution by round trace on nitrocellulose membrane, slightly dry, and film is placed in 37 DEG C of incubators 30 minutes by room temperature.It is complete to film 10 minutes are fixed using 4% paraformaldehyde after absolutely dry, PBS is washed film 3 times, 3 minutes/time.1%BSA confining liquid is in room temperature item It is closed 1 hour under part, distilled water cleans 3 times, 3 minutes/time.It dries under the conditions of 20-28 DEG C, is sealed in -20 DEG C.
The anti-AQP4 autoantibody detection material of human body prepared by the present invention can be used for color developing detection.It is coated with correct space structure The M1-AQP4- cell membrane complexes of elephant or the detection material of M23-AQP4- cell membrane complexes, in combination with needle in dilute sample To the autoantibody IgG of AQP4 linear epitope or comformational epitope, the antibody on detection material is integrated to by enzyme mark anti-human igg secondary antibody Identification carrys out in judgement sample whether there is the autoantibody of anti-AQP4 finally by chromogenic reaction.
Specific detecting step is as follows:
A) the anti-AQP4 autoantibody detection material of human body prepared by the present invention is placed in 12 orifice plates, is coated with the one of AQP4 antigen Up.
B) serum is incubated for: test sample adds in hole after diluting by 1:500, the hole 1mL/.12 orifice plates are placed in horizontal shaker (frequency is about 30 beats/min) is incubated at room temperature 40 minutes.
C) wash: the serum Incubating Solution in removal hole is added cleaning solution, the hole 1mL/, be placed in shaking table (frequency is about 30 times/ Minute), room temperature 3-5 minutes, discard the cleaning solution in hole.Repeated washing totally 3 times.
D) secondary antibody is incubated for: the cleaning solution in removal hole, and the diluted AP of 1:100 is added and marks goat anti-human igg antibody, 1mL/ Hole.It is placed in shaking table (frequency is about 30 beats/min), is incubated at room temperature 30 minutes.
E) wash: the secondary antibody Incubating Solution in removal hole is added cleaning solution, the hole 1mL/, be placed in shaking table (frequency is about 30 times/ Minute), room temperature 3-5 minutes, discard cleaning solution in hole.Repeated washing totally 3 times.
F) it develops the color.After removing the cleaning solution in hole, the dilution (substrate: reaction buffer=1:49) of substrate is added, The hole 1mL/.Room temperature is protected from light standing 2-5 minutes, can check every 1 minute primary.
G) color development stopping.The substrate dilution in hole is discarded, and rinses detection material of the invention 1-2 times with distilled water, It is drawn off drying or 37 DEG C -45 DEG C dries, analyze result.
Testing result interpretation is as follows:
Positive findings interpretation: the AQP4 antigen coat area of the anti-AQP4 autoantibody detection material of human body prepared by the present invention is in Now apparent color spot, shape is approximate circle, light slate gray to deep blue-black, and color is deep compared with its neighboring area and negative control area (as shown in Figure 2 a).
Negative findings interpretation: the AQP4 antigen coat area face of the anti-AQP4 autoantibody detection material of human body prepared by the present invention Color is shallower than negative control area, or be equal to negative control area (as shown in Figure 2 b) etc., it is determined as negative findings.
Fig. 1 is the schematic diagram that the anti-AQP4 autoantibody of human body prepared by the present invention detects material, and wherein a-quadrant is coated with M23- AQP4 cell membrane complexes；B area is coated with pCI-neo cell membrane complexes, as negative control.
Fig. 2 is to detect material tests NMO patient and normal human serum with the anti-AQP4 autoantibody of human body prepared by the present invention In anti-AQP4 Antibody Results, it is known NMO patients serum that wherein Fig. 2 a, which is detection serum, through chromogenic reaction, a-quadrant AQP4- Cell membrane complexes are coated with area and more apparent color occur compared with B area negative control group, and such situation regards as detection serum In contain anti-AQP4 antibody；Fig. 2 b is that detection serum is known normal serum, and through chromogenic reaction, a-quadrant AQP4- cell membrane is multiple It closes object coating area and does not occur more apparent color compared with B area negative control group, the two is presented identical color, such situation, It regards as in detection serum without anti-AQP4 antibody.
The above is only presently preferred embodiments of the present invention, is not intended to limit the invention in any way, it is all according to the present invention Technical spirit any simple modification, change and equivalent structure transformation to the above embodiments, still fall within skill of the present invention In the protection scope of art scheme.

Claims (5)

1. a kind of preparation method of the anti-AQP4 autoantibody detection material of human body, which comprises the following steps:
1) the AQP4 overall length target gene for obtaining M1, M23 hypotype, is transfected target gene by pCI-neo plasmid vector respectively Enter in HEK293 cell, constructs the HEK293/pCI-neo-M1-AQP4 cell and expression M23 of expression M1 hypotype AQP4 gene The HEK293/pCI-neo-M23-AQP4 cell of hypotype AQP4 gene；
2) the AQP4- cell of HEK293/pCI-neo-M1-AQP4 cell and HEK293/pCI-neo-M23-AQP4 cell is extracted Membrane complex；
3) the AQP4- cell membrane complexes of extraction are solidificated on carrier, obtain the anti-AQP4 autoantibody detection material of human body；
The specific steps of step 2) are as follows: first by centrifugation respectively removal HEK293/pCI-neo-M1-AQP4 cell and Then detergent is added in the nucleus of HEK293/pCI-neo-M23-AQP4 cell, 4 DEG C act on 30~60 minutes, then are centrifuged Except undissolved part, supernatant is soluble AQP4- cell membrane complexes；Wherein detergent is the CHAPS of 5~20mmol/L Or the Triton X-100 that volume fraction is 0.5~1%；
Or the specific steps of step 2) are as follows: first pass through physical grinding, ultrasound or low temperature freeze thawing makes HEK293/pCI-neo- respectively The cellular membrane disruption of M1-AQP4 cell and HEK293/pCI-neo-M23-AQP4 cell discharges cell cytosol content, then Supernatant plasmosin is abandoned in centrifugation, and hypertonic solution is added into obtained precipitating, and 4 DEG C act on 15~30 minutes, are centrifuged again, is abandoned Supernatant karyon albumen, finally obtained precipitating are the AQP4- cell membrane complexes of insolubility；The hypertonic solution by Tris-HCl、NaCl、MgCl2It is formulated with EDTA, the concentration of Tris-HCl is the dense of 20mmol/L, NaCl in hypertonic solution Degree is 300mmol/L, MgCl2Concentration be the concentration of 2mmol/L, EDTA be 2~5mmol/L, the pH=8 of hypertonic solution；
Or the specific steps of step 2) are as follows: first pass through physical grinding, ultrasound or low temperature freeze thawing makes HEK293/pCI-neo- respectively The cellular membrane disruption of M1-AQP4 cell and HEK293/pCI-neo-M23-AQP4 cell discharges cell cytosol content, then Centrifugation abandons supernatant plasmosin, Dextran-PEG-Na is added into obtained precipitating3PO4Liquid two-phase mixed liquor, again from The heart is then allowed to stand, and is formed two liquid phases, is contained AQP4- cell membrane complexes in the liquid phase of top；The Dextran- PEG-Na3PO4Liquid two-phase mixed liquor is by Dextran, PEG and Na3PO4It is formulated, Dextran-PEG-Na3PO4Liquid two The concentration that the volume fraction for mixing Dextran in liquid is 20%, PEG is 1.03g/mL, Na3PO4Concentration be 0.2mol/L, Dextran-PEG-Na3PO4The pH=6.5 of liquid two-phase mixed liquor.
2. the preparation method of the anti-AQP4 autoantibody detection material of human body according to claim 1, which is characterized in that described Step 1) specifically includes the following steps:
1.1) the AQP4 overall length target gene of M1, M23 hypotype is obtained by artificial synthesized or PCR method, and in target gene two End adds NotI/NheI restriction enzyme site；
1.2) two kinds of target gene with restriction enzyme site are inserted into respectively in pCI-neo plasmid vector, insertion point NotI/ NheI obtains two kinds of plasmids for having target gene, is respectively designated as pCI-neo-M1-AQP4 and pCI-neo-M23-AQP4；
1.3) pCI-neo-M1-AQP4 plasmid and pCI-neo-M23-AQP4 plasmid are taken respectively, by PEI transfection reagent transfection method Cultured HEK293 cell is transfected, the HEK293/pCI-neo-M1-AQP4 cell and table of expression M1 hypotype AQP4 gene are obtained Up to the HEK293/pCI-neo-M23-AQP4 cell of M23 hypotype AQP4 gene.
3. the preparation method of the anti-AQP4 autoantibody detection material of human body according to claim 1, which is characterized in that described Carrier is the Tissue Culture Dish of nitrocellulose filter, pvdf membrane, nylon membrane, the slide with protein adsorption ability, plastic material Or culture plate.
4. the preparation method of the anti-AQP4 autoantibody detection material of human body according to claim 1, which is characterized in that described The specific steps of step 3) are as follows: dilute AQP4- cell membrane complexes with 1 × TBS buffer, it is compound to be configured to AQP4- cell membrane The dilution of object, the concentration of AQP4 is 0.1~1mg/mL in the dilution of AQP4- cell membrane complexes, then with draw film instrument will The dilution of AQP4- cell membrane complexes is then placed in 37 DEG C of drying in oven on carrier by round or linear trace, then makes 10~20 minutes are fixed with fixer, then washes away fixer with PBS buffer solution, is finally closed with the BSA that mass fraction is 1% Liquid drains confining liquid and is cleaned up with distilled water, 20 in closing closing in 1 hour or 4 DEG C 16~20 hours under room temperature It is dried at~28 DEG C to get to the anti-AQP4 autoantibody detection material of human body, is sealed in -20 DEG C stand-by；Wherein fixer It is the ethyl alcohol that 4% paraformaldehyde, ice methanol or volume fraction are 95% for mass fraction.
5. people made from the preparation method of the anti-AQP4 autoantibody detection material of human body described in any one of claim 1-4 The anti-AQP4 autoantibody of body detects material, which is characterized in that the detection material is that the AQP4- cell membrane that is solidificated on carrier is compound Object, wherein AQP4- cell membrane complexes by express M1 hypotype AQP4 gene HEK293/pCI-neo-M1-AQP4 cell and table HEK293/pCI-neo-M23-AQP4 cell extraction up to M23 hypotype AQP4 gene obtains.
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