CN108845134A - Cellular assay detects 4 antibody of LDH receptor related protein - Google Patents
Cellular assay detects 4 antibody of LDH receptor related protein Download PDFInfo
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- CN108845134A CN108845134A CN201810403072.9A CN201810403072A CN108845134A CN 108845134 A CN108845134 A CN 108845134A CN 201810403072 A CN201810403072 A CN 201810403072A CN 108845134 A CN108845134 A CN 108845134A
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- related protein
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
Abstract
The present invention relates to a kind of cellular assays to detect 4 antibody of LDH receptor related protein, including step：Using pReceiver-M98 plasmid, the carrier for expression of eukaryon of the complete open reading frame with EGFP label L RP4 is constructed, recombinant plasmid is obtained；By the cultured HEK293T cell of Transfected Recombinant Plasmid, the HEK293T cell for carrying recombinant plasmid is obtained；The expression cell for carrying recombinant plasmid is successively cultivated, cell is fixed, is closed and is incubated for, then observed using fluorescence microscope, detect LDH receptor related protein.It is provided by the invention that 4 antibody of LDH receptor related protein is detected using cellular assay, because antigen-antibody corresponds, specificity almost 100%, necessary experimental basis is provided for the diagnosis and treatment of Patients With Myasthenia Gravis, the diagnosis and treatment of Patients With Myasthenia Gravis are provided with new direction, is had great importance.
Description
Technical field
The present invention relates to medicine technology fields, and in particular to a kind of cellular assay detection LDL receptor is related
4 antibody of albumen.
Background technique
From 1672 myasthenia gravis (MG) be found since, pathogenesis be typically considered to mainly and acetylcholine by
The humoral immunity that body antibody (AChR-Ab) mediates is closely bound up, but as numerous scientists are to MG Immunological Pathogenesis
Further investigation, generally believe now a variety of muscle antigens (such as：MuSK-Ab), complement system, antigenspecific T lymphocyte,
Cell factor and thymus gland etc. play important function to the morbidity of MG.
Serology antibody test is carried out to MG patient and its research of immunology pathogenic mechanism has been increasingly becoming world's model
The hot issue that the disease discusses in enclosing, there are about can detect that AChR-Ab in 85% MG patients serum；Remaining MG patient there are about
It can detecte out muscle specific tyrosine-kinase enzyme antibody (MuSK-Ab) in 70% serum.It is experimentally confirmed that more in Guizhou High aititude
Amount in 116 MG patients collected by national gathering area, the mono- positive rate of AChR-Ab is 50.0%.Wherein women accounts for mostly
Number；Parting is based on ocular type and slight whole body type.Only 6 people in 56 MG patients for not detecting AChR-Ab antibody
(10.7%) the MuSK-Ab positive is detected.This ratio is far below the data of American-European ethnic group, but close with domestic related data.
Although most MG patient can detect that AChR-Ab and MuSK-Ab, but still (10% is left by some patient
It is right) above two antibody can not be detected in vivo, they whether there is unknown pathogenic antibody in vivo and are still not clear, this type
The appearance of type Patients With Myasthenia Gravis brings certain difficulty to clinical diagnosis and treatment.2011, Higuchi etc. was for the first time in dSN-MG
It detected LRP4-Ab in patients serum, therefore think that newfound LRP4 is the target of dSN-MG patient's body autoantibody.
These antibody belong to IgG1, it may be by inhibiting the combination of the Agrin and extracellular LRP4 of neuron release, mediating NMJ transmitting
Obstacle and cause a disease.Meanwhile LRP4-Ab activating complement system leads to that the reaction of autoantigen antibody mediated immunity, Agrin- occur in vivo
LRP4-MuSK signal path is destroyed and is caused a disease.9 are shared in experiment detection 300 dSN-MG patients of discovery of Higuchi
The LRP4-Ab positive (3%).Pevzner etc. detects 13 dSN-MG altogether, and positive (positive rate is about 7 LRP4-Ab in obvious
53.8%)；Tzartos's studies have shown that there are about detect LRP4-Ab in 9.2% dSN-MG patients serum；Georgios etc.
Detect and analyze demography feature, clinical manifestation and the curative effect of 3 LRP4-Ab positive patients with ethnic group.These discoveries are equal
Show that LRP4-Ab may be the new diagnosis marker of dSN-MG patient.Therefore the positive rate of the antibody is how many in Chinese, and
Detection method is the target that we study.
Summary of the invention
For the defects in the prior art, it is an object of that present invention to provide a kind of cellular assays to detect low-density lipoprotein
4 antibody of receptor-related proteins provides necessary reality to improve the specificity of detection method for the diagnosis and treatment of Patients With Myasthenia Gravis
Foundation is tested, the diagnosis and treatment of Patients With Myasthenia Gravis are provided with new direction, is had great importance.
To achieve the above object, technical solution provided by the invention is：
The present invention provides a kind of methods for detecting LDH receptor related protein, including step：Building recombination
Plasmid；By the cultured HEK293T cell of Transfected Recombinant Plasmid, the HEK293T cell for carrying recombinant plasmid is obtained；Weight will be carried
The expression cell (HEK293T cell) of group plasmid is successively cultivated, cell is fixed, is closed and is incubated for, then aobvious using fluorescence
Micro mirror is observed, and detects LDH receptor related protein.
Preferably, recombinant plasmid is to construct completely opening with EGFP label L RP4 using pReceiver-M98 plasmid
Put the carrier for expression of eukaryon of reading frame.Green fluorescent protein (Green fluorescent protein, GFP) why can
It is enough shine be because containing color base, the color base of GFP be by serine, tyrosine, that glycine (Ser/Tyr/Gly) is formed is special
Structure " cyclisation tripeptides ", the structure can absorb blue light (maximum excitation wavelength is 395nm, the peak dot of launch wavelength be
509nm), it can be activated when being irradiated with ultraviolet radiation, emit green (or yellow green) fluorescence.The color base of EGFP and having for CFP
Institute is different, and the substitution of amino acid occurs in special construction " cyclisation tripeptides ", near maximum excitation wavelength shift to 490nm.Therefore,
The fluorescence signal ratio GFP that EGFP is obtained is more bright, launches stronger green fluorescence.By the gene of EGFP and mesh in test
It is gene constructed at so-called " mosaic gene ", cause its expression product recombinant protein with fluorescent marker.It is recombinated with plasmid
Method easily research purpose gene, especially expression, the process for positioning and operating.PReceiver-M98 recombinant plasmid was both
It can be replicated in mammalian cells again in Escherichia coli, so producer can complete fusion in Escherichia coli
Building process, after buying, transfection cell can be expressed in cell, can also under screening pressure complete stablize duplication.
The target gene of pReceiver-M98 recombinant plasmid insertion, i.e. the region ORF of LRP4 is located at EGFP gene upstream.
Preferably, culture specifically includes step：The cell climbing sheet handled through poly-D-lysine is placed in 24 hole cell culture
In plate, the HEK293T cell that carrying recombinant plasmid is then added is cultivated.
Preferably, cell fixation is used when carrying the density of HEK293T cell of recombinant plasmid is 70%~80%
4% paraformaldehyde fixer is fixed.
Preferably, cell regular time is 16~20min.
Preferably, close the confining liquid used be containing mass fraction for 5% sheep blood serum albumin PBS solution.
Preferably, the closed time is 2.5~3.0h.
Preferably, be incubated for includes that primary antibody is incubated for and the incubation of fluorescence secondary antibody.
Preferably, primary antibody incubation is incubated overnight using diluted test serum；The temperature of incubation is 4 DEG C；Dilution is root
It is carried out according to confining liquid multiplication dilution method.
Preferably, fluorescence secondary antibody be incubated for be using Alex568 label Goat anti-Human IgG be protected from light incubation at room temperature 2.5~
3.0h。
Technical solution provided by the invention has following beneficial effect：It is provided by the invention to be examined using cellular assay
4 antibody of LDH receptor related protein is surveyed, because antigen-antibody corresponds, specificity almost 100%, is severe
The diagnosis and treatment of myasthenia gravis patients provide necessary experimental basis, and the diagnosis and treatment of Patients With Myasthenia Gravis are provided with new direction, tool
There is important meaning.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Detailed description of the invention
Fig. 1 is the process signal of cellular assay detection 4 antibody of LDH receptor related protein in the present invention
Figure.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description.The following examples are only intended to illustrate the technical solution of the present invention more clearly, therefore is intended only as example, without
It can be limited the scope of the invention with this.
Experimental method in following embodiments is unless otherwise specified conventional method.Examination as used in the following examples
Material is tested, is to be commercially available from conventional reagent shop unless otherwise specified.Quantitative test in following embodiment, is all provided with
Three repeated experiments are set, data are the average value or flat of three repeated experiments
The present invention provides a kind of method for detecting LDH receptor related protein, including step：
S1 (construction recombination plasmid)：Using pReceiver-M98 plasmid, the Complete Open with EGFP label L RP4 is constructed
The carrier for expression of eukaryon of reading frame obtains recombination LRP4 overall length and carries the plasmid of EGFP；
S2 (plasmid transfection)：LRP4 overall length will be recombinated and carry the cultured HEK293T cell of plasmid transfection of EGFP, obtained
To the HEK293T cell for carrying recombinant plasmid；
S3 (culture)：The cell climbing sheet handled through poly-D-lysine is placed in 24 porocyte culture plates, is then added and takes
HEK293T cell with recombinant plasmid is cultivated；
S4 (fixation)：When carrying the density of HEK293T cell of recombinant plasmid is 70%~80%, using 4% poly
Formaldehyde fixer room temperature fixes 16~20min；
S5 (closing)：After cell is fixed, 2.5~3.0h is closed using confining liquid room temperature；Wherein, confining liquid is containing matter
Measure the PBS solution for the sheep blood serum albumin that score is 5%；
S6 (primary antibody incubation)：After closing, it is added and is doubled the diluted test serum of dilution method using confining liquid, then 4
DEG C be incubated overnight；
S7 (incubation of fluorescence secondary antibody)：After primary antibody is incubated for, room temperature is protected from light using the Goat anti-Human IgG that Alex568 is marked
It is incubated for 2.5~3.0h；
S8 (mounting fluorescence microscope is taken pictures)：It after fluorescence secondary antibody is incubated for, is observed, is overlapped using fluorescence microscope
Yellow is presented or fluorescent orange is judged as positive, and according to strong and weak 0~4 point of the scoring of fluorescence, to detect low density lipoprotein
Protein receptor GAP-associated protein GAP.
Technical solution provided by the invention is described further combined with specific embodiments below.
Embodiment one：Carry the building of the HEK293T cell of recombinant plasmid
1, wild plasmid and recombinant plasmid are constructed：PReceiver-M98 plasmid is selected, enhanced green fluorescence is constructed
The carrier for expression of eukaryon of albumen (Enhanced green fluorescent protein, EGFP), with EGFP label L RP4's
Complete open reading frame (Open Reading frame, ORF), while allowing destination protein in HEK-293T cell (human embryo kidney (HEK)
It is given full expression in 293T), carries out subsequent detection research as antigen.Green fluorescent protein (Green fluorescent
Protein, GFP) why can to shine be because contain color base, the color base of GFP is by serine, tyrosine, glycine
(Ser/Tyr/Gly) special construction " cyclisation tripeptides " formed, the structure can absorb blue light (maximum excitation wavelength is 395nm,
The peak dot of its launch wavelength is in 509nm), it can be activated when being irradiated with ultraviolet radiation, emit green (or yellow green) fluorescence.
The color base of EGFP and CFP's is different, and the substitution of amino acid occurs in special construction " cyclisation tripeptides ", and maximum excitation wavelength is inclined
It moves near 490nm.Therefore, the fluorescence signal ratio GFP that EGFP is obtained is more bright, launches stronger green fluorescence.Examination
It tests the middle gene by EGFP and target gene is built into so-called " mosaic gene ", cause its expression product recombinant protein with glimmering
Signal.Method easily research purpose gene, the especially expression, the process for positioning and operating recombinated with plasmid.
PReceiver-M98 recombinant plasmid can not only be replicated in Escherichia coli but also in mammalian cells, so producer can be with
The building process that fusion is completed in Escherichia coli, after buying, transfection cell can express in cell, can also screen
It completes to stablize duplication under pressure.The target gene of pReceiver-M98 recombinant plasmid insertion, i.e. the region ORF of LRP4 is located at
EGFP gene upstream.
2, the culture of HEK293T cell：Using conventional experimental method culture HEK293T cell.
3, plasmid transfection：First according to lipofectamine2000 transfection reagent specification grope DNA with
Lipofectamine2000 optium concentration ratio, to obtain best transfection.We are groped dense by 24 porocyte culture plates
Degree, finally found that DNA and lipofectamine2000 optium concentration ratio is 1:2.5, the concentration for the wild plasmid that we obtain
For 613 μ g/mL, blank group plasmid concentration is 7.08 μ g/ μ L, respectively by two kinds of plasmid transfections to 2 12 porocyte culture plates into
Row preliminary experiment.
Embodiment two：Cellular immunofluorescence method detects anti-LRP4 antibody
1, it cultivates：The cell climbing sheet handled through poly-D-lysine is placed in 24 porocyte culture plates, is then added and carries
The HEK293T cell of recombinant plasmid is cultivated.
2, fixed：When carrying the density of HEK293T cell of recombinant plasmid is 75%, fixed using 4% paraformaldehyde
Liquid chamber temperature fixes 18min.
3, it closes：After cell is fixed, 2.8h is closed using confining liquid room temperature；Wherein, confining liquid is containing mass fraction
For the PBS solution of 5% sheep blood serum albumin.
4, primary antibody is incubated for：After closing, it is added using the confining liquid multiplication diluted test serum (1 of dilution method:20), so
4 DEG C of overnight incubations afterwards.
5, fluorescence secondary antibody is incubated for：After primary antibody is incubated for, room temperature is protected from light using the Goat anti-Human IgG that Alex568 is marked and is incubated
Educate 2.8h.
6, mounting fluorescence microscope is taken pictures：Every part of sample is repeated twice the operation of 1-5.After fluorescence secondary antibody is incubated for, adopt
It is observed with fluorescence microscope.Fluorescence microscope (Olympus-IX51) excitation wavelength is respectively blue light (420~490nm)
With green light (535~550nm), green fluorescence and red fluorescence are generated, they, which are overlapped, is presented yellow (or orange) fluorescence i.e. judgement
For the positive, scored by the observation of two researchers independently in 1 point or more person according to strong and weak 0~4 point of the scoring of fluorescence
For the positive.The serum of healthy premenopausal volunteers is as negative control.Meanwhile it need to be to transfect the HEK293T cell of empty plasmid as control group.
Preferred steps, the positive serum detected need to be detected again, primary antibody dilution, i.e., from 1:20 are diluted to 1:
40,1:100,1:200 etc., it may determine that the antibody titer of positive patient with this.
The present invention is all made of cellular assay (CBA) to all 116 MG patients and 80 control group serum and carries out
The detection of LRP4-Ab, as a result, it has been found that 2 dSN-MG (4%) patient LRP4-Ab are the positive.
Detection method specificity provided by the invention nearly reaches 100% (because antigen-antibody corresponds), for this kind
The diagnosis and treatment of patient provide necessary laboratory foundation.
It should be noted that unless otherwise indicated, technical term or scientific term used in this application should be this hair
The ordinary meaning that bright one of ordinary skill in the art are understood.Unless specifically stated otherwise, it otherwise illustrates in these embodiments
Component and opposite step, numerical expression and the numerical value of step are not limit the scope of the invention.It is illustrated and described herein
In all examples, unless otherwise prescribed, any occurrence should be construed as merely illustratively, not as limitation, because
This, other examples of exemplary embodiment can have different values.
In the description of the present invention, it is to be understood that, term " first ", " second " are used for description purposes only, and cannot
It is interpreted as indication or suggestion relative importance or implicitly indicates the quantity of indicated technical characteristic.Define as a result, " the
One ", the feature of " second " can explicitly or implicitly include one or more of the features.In the description of the present invention,
The meaning of " plurality " is two or more, unless otherwise specifically defined.
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, those skilled in the art should understand that：Its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme should all cover in protection scope of the present invention.
Claims (10)
1. a kind of method for detecting LDH receptor related protein, which is characterized in that including step：
Construction recombination plasmid；
By the cultured HEK293T cell of the Transfected Recombinant Plasmid, the HEK293T cell for carrying recombinant plasmid is obtained；
By the HEK293T cell for carrying recombinant plasmid is successively cultivated, cell is fixed, is closed and is incubated for, then use
Fluorescence microscope is observed, and detects LDH receptor related protein.
2. the method for detection LDH receptor related protein according to claim 1, it is characterised in that：
The recombinant plasmid is to construct the entire open reading frame with EGFP label L RP4 using pReceiver-M98 plasmid
The carrier for expression of eukaryon of frame.
3. the method for detection LDH receptor related protein according to claim 1, which is characterized in that
The culture specifically includes step：The cell climbing sheet handled through poly-D-lysine is placed in 24 porocyte culture plates, so
The HEK293T cell for carrying recombinant plasmid is added afterwards to be cultivated.
4. the method for detection LDH receptor related protein according to claim 1, it is characterised in that：
The cell fixation is when carrying the density of HEK293T cell of recombinant plasmid is 70%~80%, using 4% poly
Formaldehyde fixer is fixed.
5. the method for detection LDH receptor related protein according to claim 4, it is characterised in that：
The cell regular time is 16~20min.
6. the method for detection LDH receptor related protein according to claim 1, it is characterised in that：
It is described close the confining liquid that uses be containing mass fraction for 5% sheep blood serum albumin PBS solution.
7. the method for detection LDH receptor related protein according to claim 1, it is characterised in that：
The closed time is 2.5~3.0h.
8. the method for detection LDH receptor related protein according to claim 1, it is characterised in that：
Described be incubated for includes that primary antibody is incubated for and the incubation of fluorescence secondary antibody.
9. the method for detection LDH receptor related protein according to claim 8, it is characterised in that：
The primary antibody incubation is incubated overnight using diluted test serum；The temperature of the incubation is 4 DEG C；The dilution is root
It is carried out according to confining liquid multiplication dilution method.
10. the method for detection LDH receptor related protein according to claim 8, it is characterised in that：
It is to be protected from light 2.5~3.0h of incubation at room temperature using the Goat anti-Human IgG of Alex568 label that the fluorescence secondary antibody, which is incubated for,.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111060695A (en) * | 2019-12-23 | 2020-04-24 | 江苏三联生物工程有限公司 | Electrochemiluminescence kit for detecting anti-LRP 4 antibody and preparation method thereof |
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| CN111060695A (en) * | 2019-12-23 | 2020-04-24 | 江苏三联生物工程有限公司 | Electrochemiluminescence kit for detecting anti-LRP 4 antibody and preparation method thereof |
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