CN110606885B

CN110606885B - Detection material for anti-AMPA 1 autoantibody in human body fluid, preparation method and application

Info

Publication number: CN110606885B
Application number: CN201910738476.8A
Authority: CN
Inventors: 闫亚平; 李怡婷; 李科
Original assignee: Shaanxi Mybiotech Co ltd
Current assignee: Shaanxi Mybiotech Co ltd
Priority date: 2019-08-12
Filing date: 2019-08-12
Publication date: 2021-03-23
Anticipated expiration: 2039-08-12
Also published as: CN110606885A

Abstract

The invention discloses a detection material for anti-AMPA 1 autoantibody in human body fluid, a preparation method and an application, wherein AMPA1 antigen is coated on an NC membrane to prepare the anti-AMPA 1 receptor detection material, specific AMPA1 antibody in human serum and cerebrospinal fluid can be combined with the antigen, alkaline phosphatase substrate-ligand reaction is utilized for color development, the sealing material is added in the color development reaction, and whether the detection sample contains the AMPA1 antibody or not can be directly judged by visual observation. The method has the advantages of high sensitivity, simple operation, rapid detection and the like, and is favorable for identifying and diagnosing the anti-AMPA 1 receptor encephalitis.

Description

Detection material for anti-AMPA 1 autoantibody in human body fluid, preparation method and application

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a detection material for an anti-AMPA 1 autoantibody in human body fluid, a preparation method and an application.

Background

Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) belongs to the family of ionic glutamate receptor proteins, is divided into GluR1 subtypes and GluR2 subtypes (namely AMPA1 and AMPA1), is mainly expressed in hippocampal neuropil and mediates rapid excitatory synaptic transmission in brain. anti-AMPAR antibodies have been found to cause a reduction in the number of pre-synaptic or postsynaptic GluR1/2 receptors on hippocampal neurons, often manifested as limbic encephalitis with symptoms of memory loss, confusion, and behavioral abnormalities. A novel autoimmune cell surface antigen, AMPAR, was discovered in 2009 by Meizan Lai et al, who detected 7 anti-AMPA 1 antibodies in 109 patients with limbic encephalitis using immunoprecipitation and immunoblotting. Romana et al used immunohistochemical and immunoblotting methods to detect 22 AMPAR-containing antibodies in blood or cerebrospinal fluid of 10573 patients suspected of being free from encephalitis and patients with neuro-tumor syndrome, and the 22 patients showed limbic encephalitis with occasional mental symptoms, which were easily misdiagnosed, and about 64% of the patients had side-effects related to tumors.

The incidence of AMPAR encephalitis is low, and a large amount of blood or cerebrospinal fluid samples of suspected patients need to be screened. The conventional detection means comprise methods such as immunofluorescence, ELISA, immunoblotting, immunohistochemistry and the like, and the methods generally have the defects of long detection process time, complex steps, large antibody demand, high cost, low sensitivity and the like, and are difficult to detect a large number of samples simultaneously or repeatedly for a certain sample at one time.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a detection material for detecting an AMPA1 autoantibody, a preparation method and an application of the material, and solves the problems of long detection time consumption, complex steps, large antibody demand, high cost, low sensitivity and the like of the existing detection method.

In order to achieve the purpose, the invention adopts the following technical scheme:

the preparation method of the detection material for the anti-AMPA 1 autoantibody in human body fluid comprises the following steps:

step 1, acquiring a CDS sequence of AMPA1 as a target gene, and inserting the target gene with a restriction enzyme site into a 17T2A plasmid vector to obtain a recombinant plasmid vector 17T2A-AMPA 1;

wherein, the 17T2A plasmid vector comprises an AmpR sequence, a prokaryotic replicon pUC Ori sequence, a virus replicon SV40 Ori sequence, a RSV promoter, a lentivirus 5 'LTR, a lentivirus 3' LTR, a Gag cis-element, an RRE cis-element, an env cis-element, a cPPT cis-element, an eWPRE enhanced woodchuck hepatitis B virus post-transcriptional regulatory element, a CMV promoter, a MCS multiple cloning site and a T2A element;

step 2, transfecting the recombinant plasmid vector 17T2A-AMPA1 into 293T cells to obtain 17T2A-AMPA1-293T cells;

step 3, cracking the 17T2A-AMPA1-293T cells, removing nucleoprotein, DNA and cytoplasmic protein of the 17T2A-AMPA1-293T cells, adding the composite extracting solution, and standing for 15-20min at room temperature;

wherein the compound extract is prepared by mixing CaMgBuffer, NP40 and glucose, and the CaMgBuffer is prepared by CaCl₂、MgCl₂KCl and NaCl, in the composite extract, CaCl₂The concentration is 2 to 3mmol/L, MgCl₂The concentration is 8-15 mmol/L, KCl, the concentration is 50-70 mmol/L, NaCl, the concentration is 10-20 mmol/L, the adding volume of NP40 is 0.05-0.1% of the volume of the compound extracting solution, and the adding mass of glucose is 0.5-2% of the volume of the compound extracting solution;

and 4, step 4: taking the layered precipitate, adding the mixed solution into the precipitate, uniformly mixing, centrifuging, taking supernatant, adding the resuspension and BSA (bovine serum albumin) into the supernatant, uniformly mixing, and solidifying the obtained supernatant on a carrier membrane to obtain the detection material for resisting the AMPA1 autoantibody in the human body fluid;

the mixed solution is formed by mixing a resuspension and sucrose, and the volume molar concentration of the sucrose is 1.9-5 mol/L; the heavy suspension is formed by mixing 0.6-1% of NaCl, 0.05-0.1% of NP40 and 1 xPI.

Preferably, the step 1 specifically comprises the following steps:

step 1.1: obtaining CDS sequence of AMPA1 as target gene by artificial synthesis or PCR method, and adding SalI/NotI enzyme cutting sites at two ends of the target gene;

step 1.2: inserting a target gene with a restriction enzyme site into a 17T2A plasmid vector, wherein the insertion site is SalI/NotI to obtain a recombinant plasmid, and the recombinant plasmid is named as 17T2A-AMPA 1;

step 1.3: sequencing the recombinant plasmid 17T2A-AMPA1, carrying out amplification culture on strains with target genes with correct sequencing, and then carrying out plasmid extraction to obtain the recombinant plasmid vector 17T2A-AMPA 1.

Preferably, in the step 2, 17T2A-AMPA1 is transfected into 293T cells by using a PEI transfection method, lipofectamin2000, lipofectamin3000, lipA or an electrotransformation method, wherein the electrotransformation conditions are 1500-2000V, 25 muF and 200 omega.

Preferably, the carrier membrane is a nitrocellulose membrane, a PVDF membrane, a nylon membrane, a slide with protein adsorption capacity, a cell culture dish or a culture plate made of plastic.

The invention also discloses a detection material for resisting the AMPA1 autoantibody in human body fluid, which is prepared by the preparation method and comprises a carrier membrane and a membrane protein-cell membrane compound which is solidified on the carrier membrane and extracted from 17T2A-AMPA1-293T cells.

The invention discloses a detection material for anti-AMPA 1 autoantibody in human body fluid, which is used for detecting AMPA1 antibody in a sample.

Specifically, the detection process for detecting the AMPA1 antibody in the sample specifically includes: mixing a sample to be detected and a sealing material, incubating, adding the incubated sample into the detection material for resisting the AMPA1 autoantibody according to any one of claims 1 to 6, incubating again, washing, adding an AP-labeled goat anti-human IgG secondary antibody, incubating, and developing a substrate;

wherein the sealing material comprises a treated carrier membrane and 17T2A-293T cell protein solidified on the treated carrier membrane; the treatment process of the carrier film comprises the following steps: and adding the carrier membrane into a mixed reagent in which sucrose and glucose are mixed according to a volume molar concentration ratio of 1:1, and drying at 100 ℃ for 20-30min or 37 ℃ for 8-16 h.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention selects the modified lentiviral vector 17T2A, utilizes a virus expression system to express the AMPA1 protein on 293T cell membrane, and the vector does not contain fluorescent gene, thereby reducing the size of the vector to a greater extent and being more beneficial to the expression of target gene. Meanwhile, the invention extracts the AMPA1 protein-cell membrane compound by using the compound extracting solution, and obtains the AMPA1 protein-cell membrane compound with a more complete structure. Therefore, the AMPA1 protein-cell membrane compound with more complete structure and higher concentration can be obtained by the preparation method, and the detection material has higher sensitivity and specificity when used for detecting the AMPA1 antibody in body fluid.

(2) The existing detection method of the AMPA1 antibody mainly comprises an indirect immunofluorescence method, flow cytometry, an enzyme-linked immunosorbent assay and a protein immunoblotting method, wherein both the indirect immunofluorescence method and the protein immunoblotting method have the disadvantages of complex operation, strong operation skill (needing to be operated by professionals or experienced persons), and long detection time (needing to be two days); the ELISA and the flow cytometry have high detection cost, and simultaneously special equipment and experienced people are required for operation. The detection method provided by the invention is simple to operate, does not need professional equipment assistance, is short in detection period, and only needs about 1 hour in the whole experimental process.

(3) The invention independently prepares the sealing material in the detection process, the sealing material is from the control cell which is homologous with the detection material, the sealing material is applied to the detection of the AMPA1 antibody, the pollution caused by impurity protein can be effectively reduced, the detection sensitivity is increased, strong positive serum is diluted by more than thousand times, medium strength positive serum is diluted by more than hundred times, weak positive serum is diluted by more than 10 times, and the antibody can still be detected.

Drawings

FIG. 1 shows a process of detecting a color reaction when the detection material prepared according to the present invention is used for detecting a sample.

FIG. 2 is a map of the 17T2A plasmid vector of the present invention.

FIG. 3 is a graph showing the results of measurement of the extract of example 1.

FIG. 4 is the result of the test of example 3, in which FIG. 4a is a schematic view of a test material in which region A is coated with AMPA1 cell membrane complex and region B is coated with control cell membrane complex; FIG. 4b is the positive interpretation result; FIG. 4c is the negative interpretation result; FIG. 4d is the negative interpretation result.

Fig. 5 is the test results obtained in example 3 and comparative example 1.

Fig. 6 is the test results obtained in example 3 and comparative example 2.

Fig. 7 is the test results obtained in example 3 and comparative example 3.

Detailed Description

The following embodiments of the present invention are provided, and it should be noted that the following embodiments are only preferred embodiments of the present invention, and the present invention is not limited to the following embodiments, and all equivalent changes based on the technical solutions of the present invention fall into the protection scope of the present invention.

The pCDH-CMV-MCS-EF1-copGFP vector of the following examples of the present invention was purchased from SBI, 293T cells were purchased from ATCC, PLP1 plasmid, PLP2 plasmid and PMDG plasmid were purchased from SBI, and the carrier membranes used, such as NC membrane, PVDF membrane, nylon membrane, nitrocellulose membrane and glass fiber mat, were purchased from MILLIPORE.

Example 1

Step 1, plasmid construction: the method comprises the steps of obtaining a CDS sequence of AMPA1 as a target gene, inserting the target gene with a restriction enzyme site into a 17T2A plasmid vector to obtain a recombinant plasmid vector 17T2A-AMPA1, extracting a plasmid after correct sequencing, and performing subsequent experiments, wherein the plasmid vector construction of the embodiment specifically comprises the following steps:

step 1.1: obtaining CDS sequence of AMPA1 as target gene by PCR method (or artificial synthesis method), and adding SalI/NotI enzyme cutting sites at two ends of the target gene;

step 1.2: inserting a target gene with a restriction enzyme site into a 17T2A plasmid vector, wherein the insertion site is SalI/NotI to obtain a recombinant vector, and the recombinant vector is named as 17T2A-AMPA 1;

wherein, the 17T2A plasmid vector is obtained by deleting the copGFP element on the basis of the pCDH-CMV-MCS-EF1-copGFP vector, and replacing the FE1 promoter with the T2A element, and the map of the modified plasmid vector is shown in figure 2. Specifically, the 17T2A plasmid vector comprises an ampR sequence containing ampicillin resistance gene, a prokaryotic replicon pUC Ori sequence, a virus replicon SV 40O ri sequence, an RSV promoter, a lentivirus 5 'LTR, a lentivirus 3' LTR, a Gag cis-element, an RRE cis-element, an env cis-element, a cPPT cis-element, an eWPRE enhanced woodchuck hepatitis B virus post-transcriptional regulatory element, a CMV promoter, a MCS multiple cloning site and a T2A element;

the preparation method of the 17T2A plasmid vector comprises the following steps:

(1) synthesis of primers NotI-T2A-puromycin-SalI fragment was amplified from pCDH-CMV-MCS-T2A-puromycin vector, and PCR product was double digested with NotI and SALI enzymes.

(2) The pCDH-CMV-MCS-EF1a-copGFP vector was subjected to double digestion with NotI and SalI to remove EF1 a-copGFP. (3) NotI-T2A-puromycin-SalI is ligated into the digested pCDH-CMV-MCS-EF1a-copGFP vector to obtain 17-T2A.

Step 1.3: sequencing the recombinant plasmid 17T2A-AMPA1, carrying out amplification culture on a strain with a target gene and a carrier strain (namely 17T2A carrier) which are correctly sequenced, and then carrying out plasmid extraction to obtain the recombinant carrier 17T2A-AMPA 1.

In this example, a glycerol strain of 17T2A plasmid was inoculated into ampicillin-resistant 3mLLB liquid medium, incubated at 37 ℃ for 220r/min and shaken overnight. On the second day, 500. mu.L of the bacterial liquid is sucked for storing strains, and the remaining 2.5mL of the bacterial liquid is extracted by a plasmid miniextraction kit and the concentration is measured.

17T2A vector digestion: mu.L of the digestion system, 3. mu.g of the vector, 1. mu.L of each of SalI and NotI enzymes, 5. mu.L of Buffer, ddH, 10 Xdigestion₂Supplementing O to 50 mu L, and carrying out water bath digestion at 37 ℃ for 2 h.

And (3) recovering the enzyme digestion product, separating the enzyme digestion product by 0.8% agarose gel electrophoresis, and recovering the vector fragment according to the instruction of the gel recovery kit.

Connecting the target gene with a vector fragment: 20 μ L ligation system, 50ng 17T2A vector, 200ng AMPA1 gene fragment, T4 ligase 1 μ L, ddH₂The O was replenished to 20. mu.L and ligated overnight at 16 ℃.

The above-mentioned ligation product plasmids were transformed into E.coli DH 5. alpha. respectively for homologous recombination, spread on ampicillin solid LB plates, incubated overnight at 37 ℃ and 5 strains were picked the next day, inoculated into ampicillin (25ug/ml) liquid LB medium, and incubated overnight at 37 ℃. The next day, each tube sucks 500 μ L of bacterial liquid, the bacterial liquid is stored at the temperature of minus 20 ℃, the plasmid of the residual bacterial liquid is extracted slightly, and the bacterial strain with correct enzyme digestion identification is sent to sequencing for re-identification. The strains with correct sequencing and identification are reserved and stored at-80 ℃.

And (4) carrying out amplification culture on strains with the target genes and the carrier strains with correct sequencing to carry out large plasmid extraction, and measuring the concentration. Finally, the recombinant plasmid vector 17T2A-AMPA1 is obtained.

Step 2, amplification and infection of lentivirus:

transfecting a recombinant plasmid vector 17T2A-AMPA1 into 293T cells to obtain lentivirus 17T2A-AMPA1-293T cells, and simultaneously, amplifying and infecting a control group 17T2A Plasmid (PS) in the step, wherein the method specifically comprises the following steps:

step 2.1, 293T cell culture: preparing 10% FBS-DMEM high-sugar medium by DMEM high-sugar medium and FBS according to the ratio of 9:1, adding 293T cells into the high-sugar medium, culturing until the cells are fused to 90% and passaged according to the ratio of 1:5, placing at 37 ℃, and placing 5% CO₂Cultured in a cell culture box.

And (3) slow virus packaging: and (2) after 293 cells in the T-25 culture bottle grow to 70-80% of the bottom area of the culture dish, replacing a serum-free DMEM high-sugar culture medium to continue culturing for 2h, taking 15 mug of the total amount of the 17T2A-AMPA1 recombinant vector and the 17T2A vector (PS) obtained in the step 1, mixing the 15 mug of the recombinant vector and the 17T2A vector with an auxiliary plasmid PLP1 plasmid, a PLP2 plasmid and a PMDG plasmid in a vortex mode, wherein the mass ratio of the PLP1 plasmid to the PLP2 plasmid to the PMDG plasmid is 6.5:2.5:3.5, and 82.5 mug of PEI, transfecting the 17T2A-AMPA1 recombinant vector and the 17T2A vector respectively according to a transfection method, replacing a fresh culture solution after 6h, and replacing PEI again after 12.

The transfection method can also use lipofectamin2000, lipofectamin3000, lipA or electric transfer (the electric transfer adopts an electric transfer cup with low field intensity of 0.4cm, and the electric transfer condition is 1500V-2000V, 25 muF and 200 omega).

2.3, lentivirus infection: and collecting cell culture solution after 24h of transfection, adding the cell culture solution into 293T cells (the cell density is about 50-60%) in another dish, and adding puromycin after 24h of culture for antibiotic pressure screening until the final concentration is 6-8 mug/mL. After 24 hours of screening, cell passage is carried out, puromycin with the concentration of 3 mu g/mL is added into a culture medium, a cell line (hereinafter referred to as AMPA1 cell) for stably expressing AMPA1 protein and a control cell line (hereinafter referred to as 17T2A cell and PS) for stably expressing 17T2A are obtained after 3-5 passages, and protein extraction in the next step is prepared.

And step 3: the method for extracting the membrane protein-cell membrane compound of the 17T2A-AMPA1-293T cell specifically comprises the following steps:

3.1, abandoning the cell culture solution, washing the cells 1-2 times by using CaMg Buffer (CaCl 2mM, MgCl 10mM, KCl 60mM, NaCl 15mM), adding 1.5ml of hypotonic lysis solution (ddH2O, 1xPI, 0.25-0.5x CaMgBuffer), and placing on a shaking bed for about 5-10 min. Scraping all cells with a cell scraper, collecting cells and suspension into a 2mL EP tube, blowing and beating the cells and suspension with a gun head for 20-30 times at 300g and 4 ℃ for 3min, and collecting supernatant into a new 2mL EP tube. The supernatant was centrifuged at 19000g for 5min, the precipitate was retained, and again, 19000g was centrifuged for 3min, the residual liquid was discarded, and the precipitate was retained. Adding 80-100 ul of the composite extracting solution into the precipitate, and standing at room temperature for 15-20 min. In this example, the complex extract was prepared by mixing 1xCaMgBuffer, 0.05% NP40, 1% glucose, and 1 xPI.

Step 3.2: centrifuging 19000g for 5min, retaining the precipitate (membrane protein-cell membrane complex), adding 10ul of mixed solution into the precipitate, blowing, stirring uniformly, transferring to a 1.5ml EP tube, centrifuging 21380g for 10-15 min, and collecting 40ul of supernatant; wherein the mixed solution consists of 10ul of resuspension (0.8% NaCl, 0.05% NP40,1xPI) and 40ul of sucrose with concentration more than or equal to 1.9M.

Then 40-80 ul of resuspension (0.8% NaCl, 0.05% NP40,1xPI) and 0.6ul of 0.125% BSA were added to the supernatant, mixed well and stored at-20 ℃ for further use. As shown in FIG. 3, a clear band at 100kD by Western Blot using the AMPAR1 antibody indicates that the extracted sample contains AMPAR1 protein.

And 4, taking 0.8 mu L of the membrane protein-cell membrane complex diluted to a proper concentration, using a pipette to dot the membrane protein-cell membrane complex on a carrier membrane (the carrier is generally an NC membrane), baking the membrane protein-cell membrane complex in an oven at 37 ℃ for 10min to obtain the detection material for the anti-AMPA 1 autoantibody in the human body fluid, and sealing and storing the detection material at 4 ℃.

When the anti-AMPA 1 autoantibody detection material prepared in the above examples is used for detecting AMPA1 antibodies in a sample, the present invention first prepares a novel blocking material. As some proteins existing in human serum and cerebrospinal fluid can be subjected to nonspecific binding interference detection results with antigens in the detection process, the invention uses a sealing material to seal a detection sample, and the implementation effect finds that the background of the sample can be obviously reduced, and the specific embodiment of the preparation of the sealing material is given below.

Example 2

The embodiment discloses a preparation method of a sealing material, which specifically comprises the following steps:

1) cutting the carrier film (glass fiber mat selected in this example) into small square blocks of 0.5cm × 0.5cm, dropping 15 μ L of a mixture of 1.9M sucrose and 1.9M glucose on each small glass fiber mat, baking at 100 deg.C for 20min, and standing at room temperature for use;

2) extraction of 17T2A cell (PS) protein: the 17T2A cells obtained in step 2.3 of the above example were scraped off with a spatula, 17T2A cells were collected in a 15ml centrifuge tube at 1000rpm for 5min at room temperature, and the supernatant was discarded. The precipitate was added to 1mL of 1 XPBS (or physiological saline), the solution was pipetted into a 1.5mL EP tube at 700g for 5min at room temperature, and the supernatant was discarded. 1/2 cell pellet volume of blocking protein extract (1.25% sodium deoxycholate, 0.25% Triton X-100, 0.75% CHAPS, 20mM NaCl, 2 XPI) was added, the mixture was pipetted into a 2ml EP tube, vortexed for several seconds, and allowed to stand for several minutes until significant pellet formation occurred in the tube. Adding 1/2 NaCl with the cell mass volume of 0.8%, beating by blowing, 4000g for 5min, collecting the supernatant, and discarding the precipitate. The supernatant was mixed with 1/4 vol 25% BSA to give a 17T2A cell protein solution.

3) Dropping 12 μ L of 17T2A cell protein solution on each piece of glass fiber mat treated by 1), baking at 75 deg.C for 20min to obtain sealing material, and standing at 4 deg.C for use.

The sealing material is glass fiber, the protein of the control cell is fixed on the sealing material through high-temperature drying, and when the sealing material is used, the sealing material is added into serum or cerebrospinal fluid diluent, and the protein can be released into the liquid to react with an interference antigen in a detection sample, so that the sealing effect is achieved.

Example 3

The detection material for the anti-AMPA 1 autoantibody in the human body fluid prepared in example 1 was used for detecting the AMPA1 antibody in a sample, and specifically, the detection process for detecting the AMPA1 antibody in the sample included:

1) the membrane protein-cell membrane complex detection material obtained in step 4 of the above example was placed in a 24-well plate with the side coated with the membrane protein antigen facing upward.

2) Serum blocking: a sample to be detected is diluted by working solution (1 XPBS, 0.5% Triton X-100 and 0.04% EDTA) according to a ratio of 1:250, one piece of the sealing material prepared in example 4 is added into every 250 microliter of diluted serum, and after standing for 2min at room temperature, vortex and shake for a plurality of seconds, and then standing for 5min at room temperature.

3) And (3) serum incubation: the blocked serum was added to a 24-well plate containing a membrane protein-cell membrane complex detection material at 250. mu.L/well, and the 24-well plate was placed in a horizontal shaker (frequency about 20 times/min) and incubated at room temperature for 25 min.

4) Washing: removing the serum incubation solution from the wells, adding the working solution at a rate of 500. mu.L/well, placing in a shaker (frequency about 40 times/min), standing at room temperature for 3min, discarding the working solution from the wells, and repeatedly washing 3 times.

5) And (3) secondary antibody incubation: the working solution in the wells was removed, and 300. mu.L/well of AP-labeled goat anti-human IgG antibody diluted 1:1000 was added. The cells were incubated in a shaker (frequency about 20 times/min) at room temperature for 25 min.

6) Washing: the wells were cleared of the secondary antibody incubation solution, and 500. mu.L/well of washing solution (0.8% NaCl, 0.5% Triton X-100) was added and placed in a shaker (frequency about 40 times/min) at room temperature for 3min, and the washing solution in the wells was discarded. The washing was repeated a total of 3 times.

7) Color development: after removing the washing solution in the wells, 300. mu.L/well of a substrate diluent (substrate: reaction buffer: 1:49) was added, and the mixture was left standing at room temperature in the dark for 2 to 5 minutes to obtain a color development result.

8) And (3) stopping color development: and discarding the substrate diluent in the hole, rinsing the detection material for 1-2 times by using distilled water, taking out and airing the detection material or drying the detection material at 37-45 ℃, and analyzing the result.

Wherein, the schematic diagram of the color reaction detection process is shown in FIG. 1. FIG. 4 shows the results of the measurement in this example, in which FIG. 4a is a schematic diagram of a human anti-AMPA 1 autoantibody measurement material prepared according to the present invention, in which region A is coated with an AMPA1 cell membrane complex; the B area is coated with a control cell membrane complex and used as a negative control; FIG. 4b is the positive interpretation result; FIG. 4c is the negative interpretation result; FIG. 4d is the negative interpretation result.

Positive result interpretation: the membrane protein antigen coating area of the membrane protein-cell membrane complex detection material prepared by the invention presents obvious color spots, the shape is similar to a circle, the color is from light blue gray to dark blue black, and the color is darker than the peripheral area and the negative control area (as shown in figure 4 b).

Negative result interpretation: the membrane protein antigen coating area of the membrane protein-cell membrane complex detection material prepared by the invention is lighter in color than the negative control area (shown in figure 4 c) or equal to the negative control area (shown in figure 4 d) and the like, and the negative result is judged.

Comparative example 1

This comparative example differs from example 1 in that: the extract used in step 3.1 was CHAPS 10 mmol/L.

The detection process was the same as example 3, and three sera (nos. 1, 2, and 3) which were determined to be positive to AMPA1 were detected, and the results are shown in fig. 5. The group of example 3 could correctly detect the positive result, while the group of comparative example 1 failed to detect the positive result in all three positive sera. The composite extract in the method can obtain better detection effect.

Comparative example 2

The preparation of the test material of this comparative example is the same as that of example 1, and the difference between the test process of serum and that of example 3 is that: the blocking material prepared in example 2 was not added during the detection, and the "2) serum blocking" step in example 3 was not performed.

Three sera (nos. 1, 2, 3) which had been determined to be positive for AMPA1 were tested by two different preparative methods, and the results are shown in fig. 6. The group of example 3 can correctly detect the positive result, while the group of comparative example 2 only detects the positive result in the No. 3 serum in the three positive sera, and the positive signal is weaker. The application of the sealing material in the method can obtain better detection effect and improve the detection sensitivity.

Comparative example 3

The comparative example is used for preparing a detection material and detecting a serum sample according to the method of application No. 201710175642.9, and comprises the following specific steps:

1) obtaining CDS sequence of AMPA1 by artificial synthesis, and adding NotI/NheI enzyme cutting sites at two ends of a target gene;

2) respectively inserting two target genes with enzyme cutting sites into a pCI-neo plasmid vector, wherein the insertion sites are NotI/NheI, and obtaining a plasmid with the target genes, which is named as AMPA 1; and (3) transfecting the cultured HEK293 cells according to a PEI transfection reagent transfection method to obtain HEK293/pCI-neo-AMPA1 cells expressing the AMPA1 gene.

3) Removing cell nucleuses of HEK293/pCI-neo-AMPA1 cells by centrifugation, adding 5-20 mmol/L CHAPS detergent, acting for 30-60 minutes at 4 ℃, and removing undissolved parts by centrifugation to obtain supernatant which is the soluble AMPA 1-cell membrane compound.

4) Diluting the AMPA 1-cell membrane compound by using a1 xTBS buffer solution to prepare a diluent of the AMPA 1-cell membrane compound, wherein the concentration of AMPA1 in the diluent of the AMPA 1-cell membrane compound is 0.1-1 mg/mL, then, using a membrane scribing instrument to print the diluent of the AMPA 1-cell membrane compound on a carrier in a round or linear mode, then, putting the carrier into a drying oven at 37 ℃ for drying, and then, using a fixing solution for fixing for 10-20 minutes, wherein the fixing solution is paraformaldehyde with the mass fraction of 4%, glacial methanol or ethanol with the volume fraction of 95%; washing away the fixing solution by using PBS buffer solution, finally sealing for 1 hour at room temperature or sealing for 16-20 hours at 4 ℃ by using BSA sealing solution with the mass fraction of 1%, draining the sealing solution, cleaning by using double distilled water, drying at 20-28 ℃ to obtain the human anti-AMPA 1 autoantibody detection material, and sealing and storing at-20 ℃ for later use;

5) the method is used for detecting the AMPA1 antibody in a sample, and comprises the following specific detection processes:

the prepared human anti-AMPA 1 autoantibody detection material is placed in a 12-hole plate, and one surface coated with AMPA1 antigen faces upwards;

and (3) serum incubation: the test sample was diluted at 1:500 and added to the well at 1 mL/well. The 12-well plate was placed on a horizontal shaker (frequency about 30 times/min) and incubated at room temperature for 40 min.

Washing: remove the serum incubation solution from the wells, add wash solution 1 mL/well, place in a shaker (frequency about 30 times/min), room temperature for 3-5 min, and discard the wash solution from the wells. The washing was repeated a total of 3 times.

And (3) secondary antibody incubation: the wash solution in the wells was removed and 1 mL/well of AP-labeled goat anti-human IgG antibody diluted 1:100 was added. The cells were incubated on a shaker (frequency about 30 times/min) at room temperature for 30 min.

Washing: removing the secondary antibody incubation liquid in the holes, adding washing liquid with the concentration of 1 mL/hole, placing the secondary antibody incubation liquid in a shaking table (the frequency is about 30 times/minute), keeping the temperature at room temperature for 3-5 minutes, and removing the washing liquid in the holes. The washing was repeated a total of 3 times.

Color development: after removing the washing solution from the wells, a diluted substrate solution (substrate: reaction buffer: 1:49) was added at 1 mL/well. Standing the mixture at room temperature in a dark place for 2-5 minutes, and checking the mixture once every 1 minute.

And (3) stopping color development: and discarding the substrate diluent in the hole, rinsing the detection material for 1-2 times by using distilled water, taking out and airing the detection material or drying the detection material at 37-45 ℃, and analyzing the result.

The results of the two different preparation methods are shown in figure 7, and the group of example 3 can correctly detect the positive results, while only the group of comparative example 3 detects the positive results from the three positive serums of No. 3, and the positive signals are weaker.

Claims

1. A detection material for anti-AMPA 1 autoantibody in human body fluid is characterized by comprising a sample sealing material and a sample detection material;

the sample detection material is prepared by the following preparation method:

the step 1 specifically comprises the following steps:

step 1.3: sequencing the recombinant plasmid 17T2A-AMPA1, carrying out amplification culture on a strain with a target gene with correct sequencing, and then carrying out plasmid extraction to obtain a recombinant plasmid vector 17T2A-AMPA 1;

step 3, cracking the 17T2A-AMPA1-293T cells, removing nucleoprotein, DNA and cytoplasmic protein of the 17T2A-AMPA1-293T cells, adding the composite extracting solution, and standing for 15-20min at room temperature; the method specifically comprises the following steps:

step 3.1, discarding the cell culture solution, washing the cells for 1-2 times by using CaMgBuffer, adding 1.5ml of hypotonic lysis solution, and placing on a shaking bed for 5-10 min; the hypotonic lysis solution is prepared from ddH₂O, 1xPI and 0.25-0.5 xCaMgBuffer;

scraping all cells by using a cell scraper, collecting the cells and suspension into a 2mL EP tube, blowing and beating the cell and suspension by using a gun head for 20-30 times at 4 ℃ for 3min, collecting supernatant into a new 2mL EP tube, centrifuging the supernatant for 5min, keeping precipitates, centrifuging the supernatant again for 3min, discarding residual liquid, and keeping the precipitates;

adding 80-100 ul of the composite extracting solution into the precipitate, and standing at room temperature for 15-20 min; wherein the compound extract is prepared by mixing CaMgBuffer, NP40 and glucose, the CaMgBuffer is CaCl₂、MgCl₂KCl and NaCl, wherein CaCl₂The concentration is 2 to 3mmol/L, MgCl₂The concentration is 8-15 mmol/L, KCl, the concentration is 50-70 mmol/L, NaCl, the concentration is 10-20 mmol/L, the adding volume of NP40 is 0.05-0.1% of the volume of the compound extracting solution, and the adding mass of glucose is 0.5-2% of the volume of the compound extracting solution;

step 3.2: centrifuging for 5min, keeping the precipitate, adding 10ul of mixed solution into the precipitate, blowing, uniformly mixing, completely transferring to a 1.5ml EP tube, centrifuging for 10-15 min, and collecting 40ul of supernatant; the mixed solution consists of 10ul of heavy suspension and 40ul of sucrose, the volume molar concentration of the sucrose is 1.9-5 mol/L, and the heavy suspension is formed by mixing 0.6-1% of NaCl, 0.05-0.1% of NP40 and 1 xPI;

adding 40-80 ul of the resuspension and 0.6ul of 0.125% BSA into the supernatant, mixing uniformly, and storing at-20 ℃ for later use; the resuspension consists of 0.8% NaCl, 0.05% NP40,1 xPI;

and 4, step 4: taking the layered precipitate, adding the mixed solution into the precipitate, uniformly mixing, centrifuging, taking supernatant, adding the resuspension and BSA into the supernatant, uniformly mixing, and solidifying the obtained supernatant on a carrier membrane to obtain a sample detection material;

the sample sealing material comprises a processed carrier membrane and 17T2A-293T cell protein solidified on the processed carrier membrane; the treatment process of the carrier film comprises the following steps: adding the carrier membrane into a mixed reagent mixed with sucrose and glucose according to the volume molar concentration ratio of 1:1, and drying at 100 ℃ for 20-30min or 37 ℃ for 8-16 h;

the use process of the sample sealing material comprises the following steps:

and mixing the sample to be detected and the sealing material, incubating, adding the incubated sample into the sample detection material, incubating again, washing, adding the AP-labeled goat anti-human IgG secondary antibody, incubating and developing the substrate.

2. The material for detecting anti-AMPA 1 autoantibody in human body fluid, according to claim 1, wherein 17T2A-AMPA1 is transfected into 293T cells by PEI transfection method, lipofectamin2000, lipofectamin3000, lipA or electroporation method in step 2, wherein electroporation conditions are 1500V-2000V, 25 μ F, 200 Ω.

3. The material for detecting anti-AMPA 1 autoantibody in human body fluid according to claim 1, wherein the carrier membrane is a nitrocellulose membrane, a PVDF membrane, a nylon membrane, a glass slide having protein adsorption ability, a cell culture dish or a culture plate made of plastic.