CN113651881B

CN113651881B - Cell climbing tablet for detecting acetylcholine receptor autoantibody in human body fluid and preparation method and application thereof

Info

Publication number: CN113651881B
Application number: CN202111021985.2A
Authority: CN
Inventors: 闫亚平; 赵子越; 郝文斌; 封雪; 李科
Original assignee: Shaanxi Mybiotech Co ltd
Current assignee: Shaanxi Mybiotech Co ltd
Priority date: 2021-09-01
Filing date: 2021-09-01
Publication date: 2022-11-25
Anticipated expiration: 2041-09-01
Also published as: CN113651881A

Abstract

The invention discloses a cell climbing sheet for detecting an acetylcholine receptor autoantibody in human body fluid and a preparation method and application thereof, belonging to the technical field of biology. The adopted scheme comprises the following steps: and performing cotransfection on each subunit of the acetylcholine receptor and the unloaded plasmid according to a certain proportion, and performing fixation, washing, termination, washing and drying treatment to prepare the cell slide for detecting the acetylcholine receptor autoantibody in the human body fluid. Compared with the radioimmunoassay and the enzyme-linked immunosorbent assay, the method has higher sensitivity; compared with the immunofluorescence method reported in the prior art document, the method reduces the background and improves the specificity of detection. Therefore, the invention has good application prospect from the research perspective or as an auxiliary diagnosis means of myasthenia gravis.

Description

Cell climbing tablet for detecting acetylcholine receptor autoantibody in human body fluid and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a cell climbing tablet for detecting an acetylcholine receptor autoantibody, and a preparation method and application thereof.

Background

Myasthenia Gravis (MG) is an autoimmune disease with dysfunction of transmission at the neuro-muscular junction (NMJ) mediated by acetylcholine receptor antibodies (AchR-Ab), and the pathology mainly involves the membrane acetylcholine receptors (AchR) behind the NMJ synapses. At present, methods for detecting myasthenia gravis autoantibodies mainly include an radioimmunoassay, an enzyme linked immunosorbent assay, and the like. The prior evidence indicates that the result of the radioimmunoassay detection of a part of patient samples with severe myasthenia symptoms is negative, and the enzyme-linked immunosorbent assay and the radioimmunoassay have the same problems. The immunofluorescence method (CBA, cell Based Assay) using cells as substrates overcomes the defects of the two methods, and can detect the patient with myasthenia gravis with negative or low titer after the detection of an immunization method or an enzyme-linked immunosorbent Assay by expressing complete and correct AchR antigen on the Cell surface, thereby improving the positive detection rate of the serum of the patient with myasthenia gravis. Therefore, the wide application of the AchR immunofluorescence method can provide effective help for early diagnosis and subsequent treatment of diseases.

Rapsyn is a postsynaptic membrane acetylcholine receptor-associated protein, expressed by muscle cells, believed to be a "bionectin," which ensures neurotransmitter transmission by interacting with acetylcholine receptors to catalyze their aggregation (M K, ramarao.j B, cohen. Mechanism of nicotinic acetylcholine receptor clustering by Rapsyn. Proc. Natl. Acad. Sci. Usa.1998, 95. The CBA method of AchR autoantibodies is that Rapsyn and alpha, beta, delta, epsilon and/or gamma subunits of AchR are transiently or stably transfected according to a certain proportion, each subunit of AchR is expressed on the surface of HEK293 cells through Rapsyn aggregation, and then samples with severe myasthenia gravis are detected after the overexpressed cells are fixed (Guang Zhao, xiaoq Wang, xiao owen Yu, et al. Clinical application of blocked AchR for the detection of SNMG. Scientific reports.2015,5 10193. European medical laboratory diagnostics GmbH. A. Cell model for stable expression of acetylcholine receptor cluster and application method are applied. However, transient transfection of Rapsyn as an autoantigen with the various subunits of AchR to obtain AchR-overexpressing antigenic cells all expressed Rapsyn antigen, and thus there is a possibility that Rapsyn could not be distinguished from AchR antibody positivity when tested in a sample (Agius MA, zhu S, kirvan CA, et. Rapsyn antibodies in myseteria gravis. Ann N Y Acad Sci.1998,841: 516-21). Data show that the positive rate of the Rapsyn autoantibody in the serum of the myasthenia gravis patient is up to 15%. However, there is no evidence to date that rapsyn antibody positivity is associated with the severity or a subtype of myasthenia gravis, while rapsyn antibodies have been detected in some other types of autoimmune disease, such as systemic lupus erythematosus. Therefore, there is a need to provide a cell slide containing only the subunits (α β δ ∈ and/or γ subunits) of an overexpressed AchR that, based on the prior art, increases the specificity of detection of AchR autoantibodies.

In 1995, A.Vincent et al found that P3A + -containing alpha subunits could not bind to MIR mAbs and alpha-Butx, and thus considered that P3A + -containing alpha subunits could not assemble into functional AchR (C.F.Newland, D.Beeseon, A.Vincent et al.functional and non-functional entities of the human muscle acetic ether receptor. Journal of physiology.1995,489: 767-778). In 2002, the Shigeaki Suzuki et al research team stimulated T cells from the sera of MG patients and healthy persons using the alpha subunit of recombinant P3A +, which indicated the presence of T cells that specifically recognized the P3A + sequence and that these T cells could be detected in the sera of myasthenia gravis patients with thymoma and secondary disease (Shigeaki Suzukia, kortano Tanaka, hidekata Yasuoka, et al. Thus, there are inconsistent conclusions about whether the AchR α subunit P3A + is functional based on different approaches.

Thus, the drawbacks and disadvantages of the prior art are summarized as follows:

1. the existing research results do not need to be clearly determined whether the AchR alpha subunit P3A sequence is necessary to detect a positive sample;

2. rapsyn is an autoantigen which is used as an auxiliary protein to promote correct folding of each subunit of AchR, transient transfection is carried out on the rapyn and each subunit of AchR to obtain antigen cells over expressing AchR, the expression of Rapsyn antigens can be realized, and when sample detection is carried out, rapsyn and AchR antibody positivity can not be distinguished, so that a false positive result can be generated;

3. as an available auxiliary detection means, the immunofluorescence method has not been commercialized at present.

Disclosure of Invention

In order to overcome the disadvantages of the prior art, the present invention aims to provide a cell-climbing sheet for detecting acetylcholine receptor autoantibodies in human body fluid, and a preparation method and application thereof.

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

the invention discloses a preparation method of a cell climbing sheet for detecting an acetylcholine receptor autoantibody, which comprises the following steps: the recombinant eukaryotic expression vector of each subunit of the acetylcholine receptor and an eukaryotic expression vector without exogenous gene are mixed according to a certain proportion, and then are subjected to cotransfection, and then are subjected to fixation, washing, termination, washing and drying treatment, so as to prepare the cell climbing tablet for detecting the acetylcholine receptor autoantibody.

Further, in use, the sample to be detected is a body fluid comprising antibodies, preferably selected from the group consisting of whole blood, serum, cerebrospinal fluid and saliva.

Preferably, the recombinant eukaryotic expression vector of each subunit of the acetylcholine receptor is mixed with a eukaryotic expression vector without exogenous gene linkage, and co-transformation is carried out according to a certain proportion, specifically comprising the following steps:

respectively connecting each subunit gene alpha 1 (P3A-), beta 1, delta 1, epsilon and gamma of the acetylcholine receptor to a no-load plasmid by a molecular cloning method, and respectively constructing recombinant vectors of each subunit gene alpha 1 (P3A-), beta 1, delta 1, epsilon and gamma;

then mixing the recombinant vectors of the subunit genes alpha 1 (P3A-), beta 1, delta 1, epsilon and gamma with a eukaryotic expression vector without exogenous gene connection to obtain a cotransformation plasmid, and transfecting the cotransformation plasmid onto a cell-paved creep.

Further preferably, the transfection mass ratio of the recombinant vector of each subunit gene alpha 1 (P3A-), beta 1, delta 1, epsilon and gamma to a eukaryotic expression vector without exogenous gene ligation is 2.

It is further preferred that the total mass of the co-transfected plasmid is between 1ug and 10ug, corresponding to a mass of transfection reagent used for transfection of between 2ug and 20ug.

It is further preferred that the density of the plated cells on the cell-plated crawlers is 30% to 40%.

Further preferably, the empty plasmid is selected from pCDNA3.1 or other commonly used eukaryotic expression vectors overexpressing foreign genes, and the eukaryotic expression vector without exogenous gene ligation is selected from 17-T2A (plasmid map is shown in figure 3) or other commonly used eukaryotic expression vectors overexpressing foreign genes.

Preferably, the fixing is carried out by treating the cell fixing solution on the slide after the cotransfection treatment, and the washing is carried out by washing treatment with PBS.

Preferably, the termination is the application of (NH) to the slide after the first wash ₄ ) ₂ SO ₄ The solution is processed.

The invention also discloses a cell climbing sheet for detecting the acetylcholine receptor autoantibody, which is prepared by the preparation method.

The invention also discloses application of the cell slide for detecting the acetylcholine receptor autoantibody in preparing a kit for detecting the myasthenia gravis. Compared with the prior art, the invention has the following beneficial effects:

the invention discloses a preparation method of a cell climbing tablet, which is characterized in that recombinant eukaryotic expression vectors of various subunits of AchR and an eukaryotic expression vector without exogenous gene linkage are mixed according to a certain proportion, co-transformation is carried out, and the cell climbing tablet for AchR detection is prepared by fixing, washing, stopping, washing and drying. The invention mixes the recombinant eukaryotic expression vector of each subunit of AchR with 17-T2A empty vector and co-transfers them to obtain functional polymer expressed on the surface of eukaryotic cell, and the polymer can correctly identify the antibody in positive sample by verification. Therefore, the method disclosed by the invention is a preparation method of the cell slide for detecting the AchR receptor antibody without using Rapsyn as an auxiliary protein. Compared with the radioimmunoassay and the enzyme-linked immunosorbent assay, the method has higher sensitivity; compared with the immunofluorescence method reported in the prior art document, the method reduces the background and improves the specificity of detection. Therefore, the invention has good application prospect from the research perspective or as an auxiliary diagnosis means of myasthenia gravis.

Further, in fixing cell slide, by using (NH) ₄ ) ₂ SO ₄ The solution is terminated, so that the cell slide can be stably stored for a long time, and the positive signals and the background are well maintainedAnd (4) horizontal.

Drawings

FIG. 1 is a graph showing the results of combinations 5, 6, 7 and 8 of plasmids of example 1 of the present invention;

FIG. 2 is a graph showing the results of combinations 1, 2, 5 and 6 of plasmids in example 2 of the present invention.

FIG. 3 is a plasmid map of 17-T2A.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, shall fall within the protection scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in other sequences than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the accompanying drawings:

example 1

Step 1, construction of recombinant vector

The genes alpha 1 (P3A +), alpha 1 (P3A-), beta 1, delta 2, delta 3, delta 4, epsilon, gamma and Rapsyn (ringer) of each subunit of AchR are connected to pCDNA3.1 by a PCR or artificial synthesis method, wherein, the sequences of alpha 1 (P3A +), alpha 1 (P3A-), delta 1, delta 2, delta 3 and delta 4 are shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, and the sequence numbers of the rest sequences on NCBI are NM-000747.3, NM-000080.4, NM-005199.5 and NM-055.5, and the constructed recombinant vector is connected to pCDNA3.1 by a method of molecule cloning, and is used for great improvement after the sequencing is correct;

step 2, cell transfection

(1) 293T cell culture: the DMEM high-glucose medium and FBS were prepared into a 10-% FBS-DMEM high-glucose medium at a ratio of 9, passaged at a ratio of 1 to 5 to 1;

(2) When the cell density is 30% -40%, the genes are transfected according to the combination of table 1:

taking the recombinant vectors pCDNA3.1-alpha 1 (P3A +), pCDNA3.1-alpha 1 (P3A-), pCDNA3.1-beta 1, pCDNA3.1-delta 2, pCDNA3.1-delta 3, pCDNA3.1-delta 4, pCDNA3.1-epsilon, pCDNA3.1-gamma, pCDNA3.1-rapsyn (ringer) obtained in step 1, wherein the mass ratio of the pCDNA3.1-alpha 1 (P3A +)/pCDNA3.1-alpha 1 (P3A-), pCDNA3.1-beta 1, pCDNA3.1-delta 1/pCDNA3.1-delta 2/pCDNA3.1-delta 3/pCDNA3.1-delta 4, pCDNA3.1-epsilon, pCDNA3.1-gamma and pCDNA3.1-delta 2/pCDNA3.1-delta 3.1-delta 4 is that the total mass ratio of the transfected plasmid is 1: after 7ug,4h, the fresh culture medium was replaced, and after 48h, the cells were fixed.

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

Table 1 trial of the combination conditions of different species of plasmids.

TABLE 1

Step 3 climbing film fixing

(1) Washing: cells grown for 48h were washed 2 times with PBS,

(2) Fixing: adding 1% formaldehyde for fixing for 5min;

(3) Washing: the formaldehyde-fixed slide was washed 2 times with PBS.

4. Results of the experiment

(1) Transfection was performed according to the plasmid combination of table 1, and it was found that combination 5 gave a significant positive signal, while the other combinations gave no or a weak signal, corresponding to the immunofluorescence results shown in fig. 1 (only the results of plasmid combination 5 are presented). In FIG. 1,1 positive blood is taken as an example, and the results of experiments with plasmid combinations 5, 6, 7 and 8 are described, but the plasmid combinations 1, 2, 3 and 4 are not shown because they have no positive signal.

5. Conclusion

(1) Under the experimental condition, alpha 1 (P3A +) and alpha 1 (P3A-) are respectively transfected together with the rest beta 1, delta 1, epsilon, gamma and rapsyn, the alpha 1 (P3A-) can make a positive signal, and the alpha 1 (P3A +) can not make the positive signal, so that the invention confirms that the sequence of the alpha subunit needs to be P3A-;

(2) The delta 2, the delta 3 and the delta 4 are delta 1 subunits with different deletion lengths, only when the delta 1 and other subunits rotate together, positive signals are obvious, and the subunit signals with the deletion of other sequences are weaker or have no signals, so that the invention confirms that the sequence of the delta subunit needs to be 1554bp when the cell slide is prepared by using the method.

Different splice variants of the α 1 subunit:

in the nucleotide sequence table, SEQ.ID.NO.1 and SEQ.ID.NO.2 are both AchR alpha 1 subunit, and the correct expression of AchR is seriously influenced because the 233-307bp of the SEQ.ID.NO.2 is deleted from the SEQ.ID.NO.1, and the excessive 233-307bp expression can make the AchR lose the binding capacity on the antibody.

Delta subunit different splice variants:

SEQ ID No. 3-SEQ ID No.6 in the nucleotide sequence list are delta subunits of AchR, 4 subunits have different lengths, and compared with SEQ ID No.3, SEQ ID No.4 deletion sequences 199-243bp, SEQ ID No.5 deletion 510-820bp, SEQ ID No.6 deletion 1-270bp,322-355bp and different sequence deletions in the former half section of the delta subunit all influence the correct expression of the AchR, so that the binding capacity of the AchR antibody in a sample is reduced. In the present invention, for the delta subunit, the nucleic acid sequence of the first half of the subunit, 1-270, 199-243, 322-355, 510-820bp, is necessary for the complete expression of AchR or for the ability of AchR to correctly recognize antibodies to AchR in serum.

Delta subunit splice variant 1 (1554 bp) as shown in seq.id No. 3; delta subunit splice variant 2 (1509 bp) as shown in seq.id No.4, lacking bases 199-243 and 45bp less than seq.id No. 3; delta subunit splice variant 3 (972 bp) as shown in seq.id No.5 lacks bases 510-820 and is 310bp less than delta 1 of seq.id No. 3; delta subunit splice variant 4 (1251 bp) as shown in seq id No. 6: lacks bases 1-271 and bases 322-353, and is 303bp less than delta 1 of SEQ ID No. 3.

Example 2

1. Construction of recombinant vectors

The Rapsyn (short, sequence number on NCBI: NM-032645.5) gene is connected to pCDNA3.1 by a molecular cloning method through a PCR or artificial synthesis method, the rest required vectors all use the vector constructed in the embodiment 1, and the constructed recombinant vector is used for large extraction after being correctly sequenced;

2. cell transfection

(2) When the cell density is 30% -40%, the genes are transfected according to the combination of the following tables 2 and 3:

transfection system 1 (table 1):

the recombinant vectors pCDNA3.1-. Alpha.1 (P3A-), pCDNA3.1-. Beta.1, pCDNA3.1-. Delta.1, pCDNA3.1-. Epsilon.1, pCDNA3.1-. Gamma.pCDNA3.1-rapsyn (longer) and pCDNA3.1-rapsyn (short) obtained in step 1 of examples 1 and 2 were used, and the total amount of the transfected plasmid was 7ug. Wherein, pCDNA3.1-alpha 1 (P3A-), pCDNA3.1-beta 1, pCDNA3.1-delta 1, pCDNA3.1-epsilon, pCDNA3.1-gamma and pCDNA3.1-rapsyn (ringer)/pCDNA3.1-rapsyn (short) plasmids are transfected according to the following proportion of 2;

transfection system 2 (table 2):

the recombinant vectors pCDNA3.1-alpha 1 (P3A-), pCDNA3.1-beta 1, pCDNA3.1-delta 1, pCDNA3.1-epsilon, pCDNA3.1-gamma, pCDNA3.1-rapsyn (ringer), pCDNA3.1-rapsyn (short) and 17-T2A obtained in step 1 of example 1 and example 2 were taken, and the total amount of the transfected plasmids was 6ug. Wherein, pCDNA3.1-alpha 1 (P3A-), pCDNA3.1-beta 1, pCDNA3.1-delta 1, pCDNA3.1-epsilon, pCDNA3.1-gamma and 17-T2A/pCDNA3.1-rapsyn (ringer)/pCDNA3.1-rapsyn (short) are transfected according to the proportion of 2.

Wherein, the map of the structure of the 17-T2A plasmid is shown in figure 3, and the length of the plasmid is 6131bp.

The transfection method can also use lipofectamine 2000, lipofectamine 3000 or electrotransfer (the electrotransfer uses an electrotransfer cup with low field strength of 0.4cm, and the electrotransfer conditions are 1500V-2000V, 25 muF and 200 omega) or other common transfection methods to carry out transfection.

To ensure expression of each of the AchR subunits, the AchR subunits described in Table 2 were transfected with higher plasmid amounts.

TABLE 2

After introduction of the empty vector 17-T2A into the transfection system, the AchR subunits described in Table 3 were transfected with lower plasmid amounts.

TABLE 3

3. Fixing of climbing sheet

(1) Washing: cells grown for 48h were washed 2 times with PBS,

(2) Fixing: adding 1% formaldehyde for fixation for 5min;

(3) Washing: the formaldehyde-fixed slide was washed 2 times with PBS.

4. Results of the experiment

Taking the detection of one positive sample and one negative sample as an example, the experimental results are shown in the following table 4 and fig. 2, the plasmid combination 6 has good effect, and the plasmid combinations 3 and 4 do not have positive signals on the positive sample.

TABLE 4

Gene	Positive signal	Background (whether a suspected positive signal exists in a negative sample)
			Plasmid combination 1	Is provided with	Is provided with
Plasmid combination 2	Is provided with	Is provided with
			Plasmid combination 3	Is free of	Is composed of
Plasmid combination 4	Is free of	Is free of
			Plasmid combination 5	Is free of	Is free of
Plasmid combination 6	Is provided with	Is free of

In table 4 above:

plasmid combination 1:

pCDNA3.1-α1+pCDNA3.1-β1+pCDNA3.1-δ1+pCDNA3.1-ε+pCDNA3.1-γ+pCDNA3.1-rapsyn(1239bp)

plasmid combination 2:

pCDNA3.1-α1+pCDNA3.1-β1+pCDNA3.1-δ1+pCDNA3.1-ε+pCDNA3.1-γ+pCDNA3.1-rapsyn(1062bp)

plasmid combination 3:

plasmid combination 4:

plasmid combination 5:

pCDNA3.1-α1+pCDNA3.1-β1+pCDNA3.1-δ1+pCDNA3.1-ε+pCDNA3.1-γ

plasmid combination 6:

pCDNA3.1-α1+pCDNA3.1-β1+pCDNA3.1-δ1+pCDNA3.1-ε+pCDNA3.1-γ+17-T2A

and (4) analyzing results:

as can be seen from the result chart of the positive sample in FIG. 2, the signal intensity and the positive signal morphology are substantially consistent in the combination of the four plasmids. Therefore, the following conclusions are drawn:

(1) By introducing the unloaded 17-T2A into a transfection system, the correct expression of AchR can be promoted, and then the antibody in a positive sample can be identified;

(2) In order to control the background of a negative sample, the plasmid quantity is reduced, and meanwhile, no-load 17-T2A is introduced, so that the correct expression of AchR can be promoted, and antibodies in a positive sample can be identified;

(3) By introducing the empty load 17-T2A into a transfection system, the effect same as that of a plasmid combination containing rapsyn plasmid cotransformation is obtained, and the positive signal intensity and the signal quantity are basically consistent.

In a further development of the invention, the application of ammonium sulphate to the cell wash, after termination, is effective to extend the shelf life of the cell slide.

The drying method comprises a drying method such as microwave oven heating drying, metal bath heating drying, oven heating drying and the like.

Example 3

1. 293T cell culture: DMEM high-glucose medium was formulated with FBS at a ratio of 9 to 1 into 10% FBS-DMEM high-glucose medium, passaged at 1;

2. when the cell density is 30-40%, the carrier constructed in the embodiment 1 is used for transfection according to the following three modes, after 4 hours, the fresh culture solution is replaced, and after 48 hours, the cells are fixed.

(1) Adult + fetal: pCDNA3.1-. Alpha.1 (P3A-), pCDNA3.1-. Beta.1, pCDNA3.1-. Gamma.pCDNA3.1-. Delta.1, pCDNA3.1-. Epsilon.17-T2A, the plasmids were transfected in the following proportions of 2: 6ug;

(2) Adult type: pCDNA3.1-. Alpha.1 (P3A-), pCDNA3.1-. Beta.1, pCDNA3.1-. Gamma.pCDNA3.1-. Delta.1, 17-T2A, the plasmids were transfected in the following proportions of 2: 6ug;

(3) Fetal type: pCDNA3.1-. Alpha.1 (P3A-), pCDNA3.1-. Beta.1, pCDNA3.1-. Delta.1, pCDNA3.1-. Epsilon.17-T2A, the plasmids were transfected in the following proportions of 2: 6ug;

3. the transfected cells were treated as described in example 3, slide treatment;

4. performing immunofluorescence detection on 40 AchR patients with positive ELISA results, and using a combination of fetal type and adult type subunits; verifying the fluorescent slide of fetal subunit combination and adult subunit combination;

5. the results are shown in Table 5 below

TABLE 5

And (4) analyzing results:

from the results in table 5, the combined positive rate of the fetal subunit alone was 85%, the combined positive rate of the adult subunit alone was 90%, and the combined detection rate of the fetal subunit and the adult subunit was 92.5%. Thus, in detecting AchR suspected samples, the combination: the detection of pCDNA3.1-alpha 1 (P3A-), pCDNA3.1-beta 1, pCDNA3.1-gamma, pCDNA3.1-delta 1, pCDNA3.1-epsilon and 17-T2A has higher sensitivity.

Example 4

Typing of IgG antibodies was performed on 37 sera examined using the cell slide of the combination of fetal type and adult type subunits obtained in example 3, and the results obtained are shown in table 6 below:

TABLE 6

Antibody typing	IgG1	IgG1、IgG3	IgG1、IgG4	IgG1、IgG2、IgG3、IgG4
					Number of positive cases	22	10	2	3

From the above results, the proportion of patients with IgG1 subtype among AchR-positive patients was the largest. AchR-positive blood, also predominantly of the IgG1 subtype, was screened by Maria Isabel Leite et al in serial myathena gravis patients (Maria Isabel Leite, saiju Jacob, stuart Viegas, et al. IgG1antibodies to acetyl choline receptors in 'serial' myathena gravis. Brain.2008,131, 1940-1952).

Example 5

The cell slide was processed as follows for combination 6 of example 2.

1. Washing: cells grown for 48h were washed 2 times with PBS,

2. fixing: adding 1% formaldehyde for fixing for 5min;

3. washing: washing the formaldehyde-fixed slide with PBS for 2 times;

4. and (4) terminating: stopping the slide for 5min by using an ammonium sulfate solution;

5. washing: the slide after termination was washed 2 times with PBS;

6. drying: drying for 5min using a metal bath.

Shelf life data are shown in table 7 below:

TABLE 7

Slide treatment	Without using ammonium sulfate treatment	Ammonium sulfate termination
			Shelf life (Tian) at 4 DEG C	7	180 days or longer
Preservation at 37 ℃ (Tian)	3	21 days

As can be seen from the above results, the shelf life of the cell slide treated by ammonium sulfate is obviously prolonged.

The above contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention should not be limited thereby, and any modification made on the basis of the technical idea proposed by the present invention falls within the protection scope of the claims of the present invention.

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ccagtggaag accaccgcca ggtcgtggag gtcaccgtgg gcctgcagct gatacagctc 180

atcaatgtgg atgaagtaaa tcagatcgtg acaaccaatg tgcgtctgaa acagggtgac 240

atggtagatc tgccacgccc cagctgcgtg actttgggag ttcctttgtt ttctcatctg 300

cagaatgagc aatgggtgga ttacaaccta aaatggaatc cagatgacta tggcggtgtg 360

aaaaaaattc acattccttc agaaaagatc tggcgcccag accttgttct ctataacaat 420

gcagatggtg actttgctat tgtcaagttc accaaagtgc tcctgcagta cactggccac 480

atcacgtgga cacctccagc catctttaaa agctactgtg agatcatcgt cacccacttt 540

ccctttgatg aacagaactg cagcatgaag ctgggcacct ggacctacga cggctctgtc 600

gtggccatca acccggaaag cgaccagcca gacctgagca acttcatgga gagcggggag 660

tgggtgatca aggagtcccg gggctggaag cactccgtga cctattcctg ctgccccgac 720

accccctacc tggacatcac ctaccacttc gtcatgcagc gcctgcccct ctacttcatc 780

gtcaacgtca tcatcccctg cctgctcttc tccttcttaa ctggcctggt attctacctg 840

cccacagact caggggagaa gatgactctg agcatctctg tcttactgtc tttgactgtg 900

ttccttctgg tcatcgtgga gctgatcccc tccacgtcca gtgctgtgcc cttgattgga 960

aaatacatgc tgttcaccat ggtgttcgtc attgcctcca tcatcatcac tgtcatcgtc 1020

atcaacacac accaccgctc acccagcacc catgtcatgc ccaactgggt gcggaaggtt 1080

tttatcgaca ctatcccaaa tatcatgttt ttctccacaa tgaaaagacc atccagagaa 1140

aagcaagaca aaaagatttt tacagaagac attgatatct ctgacatttc tggaaagcca 1200

gggcctccac ccatgggctt ccactctccc ctgatcaaac accccgaggt gaaaagtgcc 1260

atcgagggca tcaagtacat cgcagagacc atgaagtcag accaggagtc taacaatgcg 1320

gcggcagagt ggaagtacgt tgcaatggtg atggaccaca tactcctcgg agtcttcatg 1380

cttgtttgca tcatcggaac cctagccgtg tttgcaggtc gactcattga attaaatcag 1440

caaggatga 1449

<210> 3

<211> 1554

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggaggggc cagtgctgac actggggctg ctggctgccc tggcggtgtg tggcagctgg 60

gggctgaacg aggaggagcg gctgatccgg cacctgtttc aagagaaggg ctacaacaag 120

gagctccggc ccgtggcaca caaagaggag agtgtggacg ttgccctggc cctcacactc 180

tccaacctca tctccctgaa agaagttgag gagaccctca ctaccaatgt gtggatagag 240

cacggctgga cagacaaccg gctgaagtgg aatgctgaag aatttggaaa catcagtgtc 300

ctgcgcctcc ccccggacat ggtgtggctc ccagagattg tgctggagaa caacaatgac 360

ggctccttcc agatctccta ctcctgcaac gtgcttgtct accactacgg cttcgtgtac 420

tggctgccac ctgccatctt ccgctcctcc tgccccatct ctgtcaccta tttccccttc 480

gactggcaga actgctccct caagttcagt tccctcaagt atacggccaa agagatcacc 540

ctgagcctga aacaggatgc caaggagaac cgcacctacc ccgtggagtg gatcatcatt 600

gatcctgaag gcttcacaga gaacggggag tgggagatag tccaccggcc ggccagggtc 660

aacgtggacc ccagagcccc tctggacagc cccagccgcc aggacatcac cttctacctc 720

atcatccgcc gcaagcccct cttctacatc atcaacatcc tggtgccctg cgtgctcatc 780

tccttcatgg tcaacctggt cttctaccta ccggctgaca gtggtgagaa gacatcagtg 840

gccatctcgg tgctcctggc tcagtctgtc ttcctgctgc tcatctccaa gcgtctgcct 900

gccacatcca tggccatccc ccttatcggc aagttcctgc tcttcggcat ggtgctggtc 960

accatggttg tggtgatctg tgtcatcgtg ctcaacatcc acttccgaac acccagcacc 1020

catgtgctgt ctgagggggt caagaagctc ttcctggaga ccctgccgga gctcctgcac 1080

atgtcccgcc cagcagagga tggacccagc cctggggccc tggtgcggag gagcagctcc 1140

ctgggataca tctccaaggc cgaggagtac ttcctgctca agtcccgcag tgacctcatg 1200

ttcgagaagc agtcagagcg gcatgggctg gccaggcgcc tcaccactgc acgccggccc 1260

ccagcaagct ctgagcaggc ccagcaggaa ctcttcaatg agctgaagcc agctgtggat 1320

ggggcaaact tcattgttaa ccacatgagg gaccagaaca attacaatga ggagaaagac 1380

agctggaacc gagtggcccg cacagtggac cgcctctgcc tgtttgtggt gacgcctgtc 1440

atggtggtgg gcacagcctg gatcttcctg cagggcgttt acaaccagcc accaccccag 1500

ccttttcctg gggaccccta ctcctacaac gtgcaggaca agcgcttcat ctag 1554

<210> 4

<211> 1509

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atggaggggc cagtgctgac actggggctg ctggctgccc tggcggtgtg tggcagctgg 60

gggctgaacg aggaggagcg gctgatccgg cacctgtttc aagagaaggg ctacaacaag 120

gagctccggc ccgtggcaca caaagaggag agtgtggacg ttgccctggc cctcacactc 180

tccaacctca tctccctggg ctggacagac aaccggctga agtggaatgc tgaagaattt 240

ggaaacatca gtgtcctgcg cctccccccg gacatggtgt ggctcccaga gattgtgctg 300

gagaacaaca atgacggctc cttccagatc tcctactcct gcaacgtgct tgtctaccac 360

tacggcttcg tgtactggct gccacctgcc atcttccgct cctcctgccc catctctgtc 420

acctatttcc ccttcgactg gcagaactgc tccctcaagt tcagttccct caagtatacg 480

gccaaagaga tcaccctgag cctgaaacag gatgccaagg agaaccgcac ctaccccgtg 540

gagtggatca tcattgatcc tgaaggcttc acagagaacg gggagtggga gatagtccac 600

cggccggcca gggtcaacgt ggaccccaga gcccctctgg acagccccag ccgccaggac 660

atcaccttct acctcatcat ccgccgcaag cccctcttct acatcatcaa catcctggtg 720

ccctgcgtgc tcatctcctt catggtcaac ctggtcttct acctaccggc tgacagtggt 780

gagaagacat cagtggccat ctcggtgctc ctggctcagt ctgtcttcct gctgctcatc 840

tccaagcgtc tgcctgccac atccatggcc atccccctta tcggcaagtt cctgctcttc 900

ggcatggtgc tggtcaccat ggttgtggtg atctgtgtca tcgtgctcaa catccacttc 960

cgaacaccca gcacccatgt gctgtctgag ggggtcaaga agctcttcct ggagaccctg 1020

ccggagctcc tgcacatgtc ccgcccagca gaggatggac ccagccctgg ggccctggtg 1080

cggaggagca gctccctggg atacatctcc aaggccgagg agtacttcct gctcaagtcc 1140

cgcagtgacc tcatgttcga gaagcagtca gagcggcatg ggctggccag gcgcctcacc 1200

actgcacgcc ggcccccagc aagctctgag caggcccagc aggaactctt caatgagctg 1260

aagccagctg tggatggggc aaacttcatt gttaaccaca tgagggacca gaacaattac 1320

aatgaggaga aagacagctg gaaccgagtg gcccgcacag tggaccgcct ctgcctgttt 1380

gtggtgacgc ctgtcatggt ggtgggcaca gcctggatct tcctgcaggg cgtttacaac 1440

cagccaccac cccagccttt tcctggggac ccctactcct acaacgtgca ggacaagcgc 1500

ttcatctag 1509

<210> 5

<211> 972

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgctgaaga atttggaaac atcagtgtcc tgcgcctccc cccggacatg gtgtggctcc 60

cagagattgt gctggagaac aacaatgacg gctccttcca gatctcctac tcctgcaacg 120

tgcttgtcta ccactacggc ttcgtgtact ggctgccacc tgccatcttc cgctcctcct 180

gccccatctc tgtcacctat ttccccttcg actggcagaa ctgctccctc aagttcaggt 240

ggtgagaaga catcagtggc catctcggtg ctcctggctc agtctgtctt cctgctgctc 300

atctccaagc gtctgcctgc cacatccatg gccatccccc ttatcggcaa gttcctgctc 360

ttcggcatgg tgctggtcac catggttgtg gtgatctgtg tcatcgtgct caacatccac 420

ttccgaacac ccagcaccca tgtgctgtct gagggggtca agaagctctt cctggagacc 480

ctgccggagc tcctgcacat gtcccgccca gcagaggatg gacccagccc tggggccctg 540

gtgcggagga gcagctccct gggatacatc tccaaggccg aggagtactt cctgctcaag 600

tcccgcagtg acctcatgtt cgagaagcag tcagagcggc atgggctggc caggcgcctc 660

accactgcac gccggccccc agcaagctct gagcaggccc agcaggaact cttcaatgag 720

ctgaagccag ctgtggatgg ggcaaacttc attgttaacc acatgaggga ccagaacaat 780

tacaatgagg agaaagacag ctggaaccga gtggcccgca cagtggaccg cctctgcctg 840

tttgtggtga cgcctgtcat ggtggtgggc acagcctgga tcttcctgca gggcgtttac 900

aaccagccac caccccagcc ttttcctggg gacccctact cctacaacgt gcaggacaag 960

cgcttcatct ag 972

<210> 6

<211> 1251

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgctgaaga atttggaaac atcagtgtcc tgcgcctccc cccggacatg caatgacggc 60

tccttccaga tctcctactc ctgcaacgtg cttgtctacc actacggctt cgtgtactgg 120

ctgccacctg ccatcttccg ctcctcctgc cccatctctg tcacctattt ccccttcgac 180

tggcagaact gctccctcaa gttcagttcc ctcaagtata cggccaaaga gatcaccctg 240

agcctgaaac aggatgccaa ggagaaccgc acctaccccg tggagtggat catcattgat 300

cctgaaggct tcacagagaa cggggagtgg gagatagtcc accggccggc cagggtcaac 360

gtggacccca gagcccctct ggacagcccc agccgccagg acatcacctt ctacctcatc 420

atccgccgca agcccctctt ctacatcatc aacatcctgg tgccctgcgt gctcatctcc 480

ttcatggtca acctggtctt ctacctaccg gctgacagtg gtgagaagac atcagtggcc 540

atctcggtgc tcctggctca gtctgtcttc ctgctgctca tctccaagcg tctgcctgcc 600

acatccatgg ccatccccct tatcggcaag ttcctgctct tcggcatggt gctggtcacc 660

atggttgtgg tgatctgtgt catcgtgctc aacatccact tccgaacacc cagcacccat 720

gtgctgtctg agggggtcaa gaagctcttc ctggagaccc tgccggagct cctgcacatg 780

tcccgcccag cagaggatgg acccagccct ggggccctgg tgcggaggag cagctccctg 840

ggatacatct ccaaggccga ggagtacttc ctgctcaagt cccgcagtga cctcatgttc 900

gagaagcagt cagagcggca tgggctggcc aggcgcctca ccactgcacg ccggccccca 960

gcaagctctg agcaggccca gcaggaactc ttcaatgagc tgaagccagc tgtggatggg 1020

gcaaacttca ttgttaacca catgagggac cagaacaatt acaatgagga gaaagacagc 1080

tggaaccgag tggcccgcac agtggaccgc ctctgcctgt ttgtggtgac gcctgtcatg 1140

gtggtgggca cagcctggat cttcctgcag ggcgtttaca accagccacc accccagcct 1200

tttcctgggg acccctactc ctacaacgtg caggacaagc gcttcatcta g 1251

Claims

1. A method for preparing a cell slide for detecting acetylcholine receptor autoantibodies in human body fluids, comprising: mixing the recombinant eukaryotic expression vector of each subunit of the acetylcholine receptor with an eukaryotic expression vector without exogenous gene linkage according to a certain proportion, carrying out cotransfection, and carrying out fixation, washing, termination, washing and drying treatment to prepare a cell climbing sheet for detecting the acetylcholine receptor autoantibody;

the recombinant eukaryotic expression vector of each subunit of the acetylcholine receptor is mixed with an eukaryotic expression vector without exogenous gene linkage, and cotransformation is carried out according to a certain proportion, and the specific operation is as follows:

respectively connecting each subunit gene alpha 1 (P3A-), beta 1, delta 1, epsilon and gamma of the acetylcholine receptor to a no-load plasmid pCDNA3.1 by a molecular cloning method, and respectively constructing recombinant vectors of each subunit gene alpha 1 (P3A-), beta 1, delta 1, epsilon and gamma;

mixing the recombinant vectors of the subunit genes alpha 1 (P3A-), beta 1, delta 1, epsilon and gamma with a eukaryotic expression vector 17-T2A without exogenous gene connection to obtain a cotransformation plasmid, and transfecting the cotransformation plasmid onto a creel with laid cells;

the transfection mass ratio of the recombinant vector of each subunit gene alpha 1 (P3A-), beta 1, delta 1, epsilon and gamma to a eukaryotic expression vector 17-T2A without exogenous gene ligation is 2.

2. The method of claim 1, wherein the total mass of the co-transmissible plasmid is between 1ug and 10ug, and the mass of the transfection reagent used for transfection is between 2ug and 20ug.

3. The method of claim 1, wherein the density of the cells spread on the cell-seeded slide is 30% to 40%.

4. The method for preparing a cell slide for detecting acetylcholine receptor autoantibodies of claim 1, wherein the fixing comprises treating the slide after the co-transfection with a cell fixative, and the washing comprises washing with a PBS buffer solution.

5. The method for preparing a cell slide for detecting acetylcholine receptor autoantibodies of claim 1, wherein the stopping is (NH) applied to the slide washed after the fixation is completed ₄ ) ₂ SO ₄ The solution is processed.

6. A cell slide for detecting an acetylcholine receptor autoantibody, produced by the production method according to any one of claims 1 to 5.

7. Use of the cell slide for detecting acetylcholine receptor autoantibodies as defined in claim 6 for the preparation of a kit for detecting myasthenia gravis autoantibodies.