CN112305226A - Anti-human GFAP autoantibody detection kit and preparation method thereof - Google Patents
Anti-human GFAP autoantibody detection kit and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a preparation method of a GFAP high-expression detection component for astrocyte aggregation activation required by a human GFAP autoantibody detection kit, an indirect immunofluorescence kit and application thereof. The preparation method of the GFAP high expression detection assembly comprises the following steps: obtaining GFAP high-expression mouse brain tissue activated by astrocyte aggregation; preparing a tissue slice of a specific brain area and preparing a detection assembly. The kit comprises a GFAP high-expression detection assembly, a blocking solution, PBST, a blocking tablet, a positive control antibody and an anti-human fluorescent secondary antibody. The kit can be used for detecting the anti-GFAP autoantibody in clinical serum and cerebrospinal fluid samples. The kit provided by the invention is simple to operate, high in sensitivity, strong in dyeing form specificity, simple in result interpretation, accurate and reliable, and capable of meeting the detection requirement of the anti-GFAP autoantibody for assisting clinical diagnosis.
Description
Technical Field
The invention relates to the field of disease molecule detection in the field of biomedicine, in particular to an indirect immunofluorescence detection technology for detecting a human GFAP autoantibody, which comprises the preparation of a detection kit and the detection of the GFAP antibody in clinical blood samples and cerebrospinal fluid samples, is a method for quickly and effectively detecting the human GFAP autoantibody, and can meet the requirements of differential diagnosis of clinical meningitis-myelitis and other nervous system diseases.
Background
As an important biomarker of autoimmune diseases, the detection of human autoantibodies has important application value for differential diagnosis of autoimmune diseases, establishment of targeted diagnosis and treatment schemes, improvement of diagnosis and treatment levels and improvement of prognosis. The detection method based on different detection matrixes has influence on the detection rate of the antibody and the determination of the target position of the antibody, and the diagnosis and treatment of diseases.
Glial Fibrillary Acidic Protein (GFAP) is an acidic protein with the molecular weight of 50-52 KDa, belongs to cytoskeletal protein, is a specific protein in astrocyte cytoplasm, is a marker protein, and is abundantly and uniquely expressed in astrocyte. The mouse brain detection matrix based on the high expression of GFAP can specifically detect GFAP antibodies in clinical serum and cerebrospinal fluid samples. The research proves that the meningitis-myelitis is a central nervous system immune disease which is mediated by the GFAP antibody and takes meningitis, encephalitis, myelitis and the like as main manifestations. Encephalopathy, epilepsy, mental symptoms, tremor, meninges stimulation signs, myelopathy, blurred vision and other similar clinical manifestations of neurological diseases bring difficulties to diagnosis and treatment of the diseases. The establishment of the human GFAP autoantibody detection project has important significance for differential diagnosis of meningococcygeus-myelitis and other nervous system diseases, such as hypertrophic meningitis, myelitis, neuromyelitis optica and the like, a targeted diagnosis and treatment scheme is formulated, and diagnosis and treatment level improvement prognosis is improved.
Currently, methods for detecting GFAP autoantibodies in clinical serum and cerebrospinal fluid samples include the following: 1. autoantibodies to GFAP were detected using Indirect Immunofluorescence analysis (IIF) using sections of brain tissue at positions such as pia mater, midbrain, periventricular region, etc. expressing GFAP antigen. The method has the defects that the preparation of the nerve tissue section of the selected area is complex, the difference between batches is large, and the quality control is difficult; and the GFAP antigen is not subjected to high expression treatment, the morphological specificity is insufficient, the requirement on inspectors is high, and the inspectors are required to have rich neurophysiological knowledge to accurately identify the position and the significance of the positive signal in the slice, so that the commercialization difficulty is high, and the clinical detection requirement is difficult to meet. 2. Immunofluorescence analysis (Cell based Assay, CBA) was performed using cells that highly express the GFAP antigen. However, the main defect of the detection method is the background coloring problem caused by GFAP non-membrane expression protein and the possibility of combining multiple proteins in cells with a sample to be detected. In the whole experiment system, although the air-transfected cells are used as a control, when the expression level of the exogenous antigen is low, the partial detection result is still difficult to be clearly read.
Currently, two detection methods of anti-GFAP autoantibodies, IIF and CBA, have certain limitations. The sensitivity and specificity of the IIF method are relatively low due to intergeneric differences in nerve tissue sheets and the expression level of antigens. The transient transformation technology of the CBA method is good in GFAP autoantibody detection specificity, but the transfection efficiency is low, and the transfection efficiency is improved through stable transformation, but the specificity is deficient. GFAP changes its expression in response to stimulation of astrocytes. The astrocyte specially prepared by the project is aggregated and activated with brain tissue slices, the GFAP is high in expression, and the detection sensitivity is high; the dyeing form has strong specificity, is easy to judge results and has low requirement on detection personnel. On the other hand, the detection material is relatively easy to obtain and is convenient to popularize.
Disclosure of Invention
The invention aims to provide a human anti-GFAP autoantibody detection kit and a preparation method thereof, the detection kit is based on a GFAP high-expression nerve tissue slice detection matrix of astrocyte aggregation activation, the staining form specificity is strong, the detection method is simple to operate, the sensitivity is high, the experimental result repeatability is good, the interpretation is simple, and the detection kit can meet the requirement of auxiliary clinical diagnosis on detection of the GFAP autoantibody of a sample
The purpose of the invention is realized by the following technical scheme:
a preparation method of a GFAP high-expression detection component activated by astrocyte aggregation for an indirect immunofluorescence kit for detecting human GFAP autoantibodies comprises the following steps:
1) construction of an animal model for astrocyte aggregation activation: selecting a classical demyelinating animal model, namely a Cuprinone model, and specifically damaging oligodendrocytes surrounding axons to cause activation of astrocytes and aggregation to corpus callosum with more myelinated nerve fibers;
2) preparing GFAP high-expression brain area tissue slices: freezing and slicing the coronal surface of the rat brain, and reserving brain slices with obvious callus structures;
3) manufacturing a detection assembly: the cut rat brain slices are pasted in the grids of the detachable glass slides and stored at low temperature of 4 ℃ for standby.
The Cuprine model in the step 1) of the preparation method of the astrocyte aggregation activated GFAP high-expression detection component for the indirect immunofluorescence kit for detecting the human GFAP autoantibody comprises the following steps:
1.1) 6-week-old male C57 mice are raised in a standard environment in an SPF animal room for one week to adapt to the environment;
1.2) feeding with 0.4% dicyclohexyl oxalyl dihydrazone (Cuprizone) feed for three weeks for molding.
In the preparation method of the astrocyte aggregation activated GFAP high expression detection component for the indirect immunofluorescence kit for detecting the human GFAP autoantibody, 2) the preparation of the GFAP high expression brain region tissue slice comprises the following steps:
2.1) after anesthesia, perfusing the heart of the mouse, emptying blood by normal saline, perfusing by paraformaldehyde stationary liquid, and taking the brain after fixation;
2.2) freezing the section after OCT embedding, cutting the section along the midline into left and right half brains, and selecting the brain slice from 1.10mm in front of fontanel to 1.46mm behind fontanel in the coronal section, wherein the thickness is 10-50 μm.
An indirect immunofluorescence kit for detecting human GFAP autoantibody, the kit comprises a GFAP high-expression brain tissue detection assembly activated by astrocyte aggregation, a blocking solution, PBST, a blocking tablet, a positive control and an anti-human fluorescent secondary antibody.
The GFAP high-expression brain tissue detection assembly for detecting astrocyte aggregation activation in the indirect immunofluorescence kit for detecting the human GFAP autoantibody is prepared from a brain area tissue slice with high GFAP expression and a glass slide with grids, wherein the glass slide with the grids is a detachable glass slide with a plurality of grids.
Further, the blocking solution is 5% BSA; the PBST is prepared from 3 per mill triton X-100 and PBS.
A method for using a kit for detecting human GFAP autoantibodies, comprising the steps of:
1) placing the prepared GFAP high expression detection assembly activated by astrocyte aggregation at room temperature for balancing for 20min, and adding 100 μ l of PSBT blocking solution containing 5% BSA in each square for blocking at 37 ℃ for 20 min;
2) discarding the confining liquid; adding 100 mul of a positive control and a diluted serum or cerebrospinal fluid sample to be detected, setting a square grid as a negative control, adding equivalent PBST, and incubating for 1H at 37 ℃; diluting a serum sample to be detected by PBST 1: 10; the cerebrospinal fluid sample to be detected is not diluted;
3) discarding the liquid; PBST is washed for 3 times, 5min each time;
4) diluting an Alexa Fluor 488-labeled anti-human fluorescent secondary antibody with PBST 1:400, and incubating for 30min at 37 ℃ in the dark;
5) and (3) repeating PBST for 3 multiplied by 5min in the step 3), discarding liquid, dropwise adding a small amount of sealing agent for sealing, observing under a fluorescence microscope, and photographing and recording results.
Compared with the prior art, the invention has the beneficial effects that:
the indirect immunofluorescence kit for detecting the human GFAP antibody, provided by the invention, is characterized in that firstly, a planetary glial cell aggregation activation animal model is constructed, a classical demyelinating animal model, namely a Cuprizone model, is selected, oligodendrocytes wrapping axons are specifically damaged, and the astrocytes are activated and aggregated to a corpus callosum with more myelinated nerve fibers; preparing GFAP high expression brain area tissue slices, perfusing and taking brains, cutting coronal planes of mouse brains, and reserving brain slices with obvious callus structures; finally, a detection component and a detection kit are prepared. The rat brain detection matrix based on GFAP high expression can specifically detect GFAP antibodies in clinical serum and cerebrospinal fluid samples, has high detection sensitivity and strong specificity, and can meet the requirement of auxiliary clinical diagnosis and detection of GFAP autoantibodies.
Aiming at the defects of the prior detection technology, the invention selects the rat brain tissue aggregated and activated by the astrocytes as the detection matrix, so that the GFAP is highly expressed, the detection sensitivity is improved, the dyeing form specificity is strong, the result interpretation is simple, and the requirement on detection personnel is low; on the other hand, the detection material is relatively easy to obtain and is convenient to popularize.
In order to optimize the kit, the prepared GFAP high-expression nerve tissue slice with activated astrocyte aggregation is pasted on a detachable glass slide with grids, so that the subsequent experimental operation is facilitated, and the operation burden of detection personnel is reduced. The kit is also provided with a negative control and a positive control, and compared with the dyeing result of the sample to be detected, the accuracy and the scientificity of experimental judgment can be obviously improved. The experiment proves that the prepared kit is convenient to store and transport, and does not influence the detection effect.
Drawings
FIG. 1 shows the result of the negative serum test and the positive control chart according to the present invention.
FIG. 2 is a flow chart of the detection steps of the detection kit according to the present invention.
Detailed Description
The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Example 1
This example provides a method for preparing a GFAP high expression detection module activated by astrocyte aggregation for an indirect immunofluorescence kit for detecting human GFAP autoantibodies, comprising the following steps.
1) Construction of an animal model for astrocyte aggregation activation: a classical demyelinating animal model, the Cuprine model, was selected which specifically injured oligodendrocytes surrounding axons, causing astrocytes to activate and aggregate to the corpus callosum where myelinated nerve fibers are more abundant.
The step 1) specifically comprises the following steps:
1.1) breeding male C57 mice of 6 weeks old in standard environment of SPF animal house for one week to adapt to environment;
1.2) feed supplemented with 0.4% bicyclohexanoneoxalyl dihydrazone (purchased from Sigma Aldrich, 14690-100G, USA) was given and fed for three weeks for molding.
2) Preparing GFAP high-expression brain area tissue slices: and (4) taking brains by perfusion, cutting the coronal plane of the brains of the mice, and reserving brain slices with obvious callus structures.
The step 2) specifically comprises the following steps:
2.1) after anesthesia, perfusing the heart of the mouse, emptying blood by normal saline, perfusing by paraformaldehyde stationary liquid, and taking the whole brain after fixation;
2.2) frozen section after OCT embedding, cut into left and right half brains along midline, and select brain slice from 1.10mm anterior to 1.46mm posterior to fontanel in coronal section, with thickness of 25 μm.
3) Manufacturing a detection assembly: the cut rat brain slices are pasted in 8 grids of the detachable glass slide, and are stored at a low temperature of 4 ℃ for standby, wherein the grid size is as follows: 13 mm. times.14 mm.
Example 2
The embodiment provides an indirect immunofluorescence kit for detecting a human GFAP autoantibody, which comprises a GFAP high-expression brain tissue detection component activated by astrocyte aggregation, a blocking solution, PBST, a blocking tablet, a positive control and an anti-human fluorescent secondary antibody.
A GFAP high-expression brain tissue detection assembly for detecting astrocyte aggregation activation in an indirect immunofluorescence kit for detecting human GFAP autoantibodies is prepared from a brain region tissue slice with high GFAP expression and a glass slide with square grids, wherein the glass slide with the square grids is a detachable glass slide with 8 square grids.
Example 3
The present embodiment provides a method for using a kit for detecting human GFAP autoantibody, comprising the steps of:
1) placing the prepared GFAP high expression detection assembly activated by astrocyte aggregation at room temperature for balancing for 20min, adding 100 μ l of PSBT blocking solution containing 5% BSA in a square grid, and blocking for 20min at 37 ℃;
2) discarding the confining liquid; adding 100 mul of a positive control and a diluted serum or cerebrospinal fluid sample to be detected, setting a square grid as a negative control, adding equivalent PBST, and incubating for 1H at 37 ℃; diluting a serum sample to be detected by PBST 1: 10; the cerebrospinal fluid sample to be detected is not diluted;
3) discarding the liquid; PBST is washed for 3 times, 5min each time;
4) diluting an Alexa Fluor 488-labeled anti-human fluorescent secondary antibody with PBST 1:400, and incubating for 30min at 37 ℃ in the dark;
5) and (3) repeating PBST for 3 multiplied by 5min in the step 3), discarding liquid, dropwise adding a small amount of sealing agent for sealing, observing under a fluorescence microscope, and photographing and recording results.
The steps of using the human GFAP antibody detection kit are shown in FIG. 2.
Example 4
The experiment was carried out according to the method of the previous example, and the results of the negative serum test and the results of the fluorescence microscope photograph at different magnifications of the positive control staining are shown in FIG. 1.
The 6-week-old male C57BL mice used in the present invention were purchased from experimental animal technology ltd, viton, beijing. Alexa Fluor 488-labeled anti-human fluorescent secondary antibody was purchased from Thermo fisher.
In the present invention, the detachable glass slide having a plurality of cells is a glass slide on which prepared neural tissue slices can be attached, as shown in fig. 2; the slide with the grid can be replaced by other similar devices as long as the function of the invention can be realized.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Various modifications and alterations to this invention will become apparent to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the technical spirit of the present invention should be included in the scope of protection of the present invention.
Claims (6)
1. A method for preparing a GFAP high-expression detection component for detecting the aggregation and activation of astrocytes required by an indirect immunofluorescence kit for detecting a human GFAP autoantibody is characterized in that the expression level of the GFAP of the activated astrocytes is high, and the method comprises the following steps:
1) construction of an animal model for astrocyte aggregation activation: selecting a classical demyelinating animal model, namely a Cuprinone model, and specifically damaging oligodendrocytes surrounding axons to cause activation of astrocytes and aggregation to corpus callosum with more myelinated nerve fibers;
2) preparing GFAP high-expression brain area tissue slices: freezing and slicing coronal section of mouse brain, and reserving brain slice with obvious corpus callosum structure, wherein the brain slice positioning region is from 1.10mm before fontanel to 1.46mm after fontanel;
3) manufacturing a detection assembly: the cut rat brain slices are pasted in the grids of the detachable glass slides and stored at low temperature of 4 ℃ for standby.
2. The method for preparing the GFAP high-expression detection module for astrocyte aggregation activation required in the indirect immunofluorescence kit for detecting human GFAP autoantibody according to claim 1, wherein the model construction of step 1) comprises the following steps:
1.1) 6-week-old male C57 mice are raised in a standard environment in an SPF animal room for one week to adapt to the environment;
1.2) feeding with a feed supplemented with 0.4% bicyclohexanoneoxalyl dihydrazone for three weeks for molding.
3. The method for preparing a GFAP high-expression detection module activated by astrocyte aggregation for use in an indirect immunofluorescence kit for detecting human GFAP autoantibody according to claim 1, wherein the step 2) of preparing a tissue section of the GFAP high-expression brain region comprises the steps of:
2.1) after anesthesia, perfusing the heart of the mouse, emptying blood by normal saline, perfusing by paraformaldehyde stationary liquid, and taking the brain after fixation;
2.2) freezing the section after OCT embedding, cutting the section along the midline into left and right half brains, and selecting the brain slice from 1.10mm in front of fontanel to 1.46mm behind fontanel in the coronal section, wherein the thickness is 10-50 μm.
4. An indirect immunofluorescence kit for detecting human GFAP autoantibody, characterized in that: the kit comprises an astrocyte aggregation activated GFAP high-expression brain tissue detection component prepared according to any one of the preceding claims, a blocking solution, PBST, a blocking tablet, a positive control and an anti-human fluorescent secondary antibody.
5. The indirect immunofluorescence kit for detecting human GFAP autoantibody according to claim 4, wherein: the GFAP high-expression brain tissue detection assembly for astrocyte aggregation activation is prepared from a brain region tissue slice with high expression of GFAP and a glass slide with square grids, wherein the glass slide with the square grids is a detachable glass slide with a plurality of square grids; the blocking solution is 5% BSA; the PBST is prepared from 3 per mill triton X-100 and PBS.
6. A method of using a kit for detecting human GFAP autoantibodies, comprising the steps of:
1) the GFAP high expression detection module activated by astrocyte aggregation prepared according to claim 1 is left to stand at room temperature for 20min for equilibration, and 100. mu.l of PSBT blocking solution containing 5% BSA is added to each square for blocking at 37 ℃ for 20 min;
2) discarding the confining liquid; adding 100 mul of a positive control and a diluted serum or cerebrospinal fluid sample to be detected, setting a square grid as a negative control, adding equivalent PBST, and incubating for 1H at 37 ℃; diluting a serum sample to be detected by PBST 1: 10; the cerebrospinal fluid sample to be detected is not diluted;
3) discarding the liquid; PBST is washed for 3 times, 5min each time;
4) diluting an Alexa Fluor 488-labeled anti-human fluorescent secondary antibody with PBST 1:400, and incubating for 30min at 37 ℃ in the dark;
5) and (3) repeating PBST for 3 multiplied by 5min in the step 3), discarding liquid, dropwise adding a small amount of sealing agent for sealing, observing under a fluorescence microscope, and photographing and recording results.
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