CN107522780A - A kind of preparation method and applications method of human prostate Immunodominant Antigenic - Google Patents
A kind of preparation method and applications method of human prostate Immunodominant Antigenic Download PDFInfo
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Abstract
Application the invention discloses a kind of preparation method of human prostate Immunodominant Antigenic and its in the detection of human prostate autoantibody.The human prostate Immunodominant Antigenic method provided by the invention for preparing includes：(1) preparation of human prostate tissue protein purification liquid；(2) the sero-fast preparation of anti-human prostate tissue antigen and antibody titer detection；(3) identification of human prostate Immunodominant Antigenic；(4) purifying of human prostate Immunodominant Antigenic.It is to be based on using normal human prostate histogenic immunity rabbit, immune response only is occurred to prostate specific antigen using rabbit, and the antigen that rabbit and people share does not occur the principle of immune response, so as to obtains special rabbit anti-serum；After rabbit anti-serum immunoglobulin purification, the human prostate Immunodominant Antigenic of purifying is obtained from human prostate tissue using chromatographic column；Using this Immunodominant Antigenic as envelope antigen, the enzyme linked immunosorbent assay analysis method of detection human prostate autoantibody is established.
Description
Technical field
The invention belongs to Biological Detection technical field, in particular it relates to a kind of system of human prostate Immunodominant Antigenic
Preparation Method and its application in the detection of human prostate autoantibody.
Background technology
Prostatitis is the common disease of urological department, and a main medical problem, it is estimated that influences 30% man
Property, chronic prostatitis/chronic Pelvic Pain Syndrome (CP/CPPS) accounts for all prostatitic 90%, is the right side of fifty
The most common urological diagnostic of male.Lack perineum, rectum, prostate, penis, testis and the abdominal pain and inflammation under infection
Disease is the mark of CP/CPPS patient.The CP/CPPS cause of disease is unclear, and therapeutic effect is limited.It is generally believed that CP/CPPS disease
Shape is probably interactional result between psychological factor, immune, neural and internal system dysfunction, and it may be with itself
Immunologic process is relevant.
In Past 30 Years, nibble different with qualitative experiment autoimmune prostatitis is being established in substantial amounts of work always
Tooth class animal model, to study CPPS possibility pathomechanism.These model validations prostate extract immune rat or small
Mouse can induce the T cell and antibody response to prostate antigen, and it is related to the Histological Evidence of prostatitis.But select
The rat in different mouse ages is possible to produce different results；Moreover, the difference of rat or mouse mouse system, to itself prostate antigen
Cell and humoral response ability and intensity it is also different.Therefore, animal model experiment is possible to underestimate prostatitic body fluid
LADA response.The evidence about autoimmune prostatitis is few in male.Most of studies have shown that CP/
Have the related cell factor of high-caliber inflammation in CPPS patients serums or refining, as IL-1, IL-1 α, IL-1 β, IL-4, IL6,
IL-8, IL-13, TNF-α, IFN γ, MCP 1 (MCP-1), platelet derived growth factor BB (PDGF-BB)
Deng.Material-specific such as PAP (PAP), the prostate steroids combination egg in small part research prostate source
(PSBP), PSA immune rats in vain, successfully induction generates significant prostatitis, it is thus regarded that CPPS is related to autoimmunity.
But diagnosis shortage specificity of the rise of cell factor to autoimmune prostatitis, and known several prostate-specific eggs
The presence for confirming clinically autoimmune prostatitis patient, and prostate patient itself are difficult to the successful immunization of animal in vain
The species of antigen is more.But if the presence for having the autoantibody of anti-human prostate Immunodominant Antigenic is confirmed in patients serum
It then can determine that male may occur from body immunity prostatitis
The content of the invention
Goal of the invention：To achieve these goals, the invention provides a kind of preparation of human prostate Immunodominant Antigenic
Method and its application in the detection of human prostate autoantibody.
Technical scheme is：(1) preparation of human prostate tissue protein purification liquid：By the human prostate hyperplasia of prostate group of acquisition
Knit and be placed in glacial phosphoric acid salt buffer (PBS) (0.01mmol/L, pH7.4), trim the unnecessary tissue such as fat, bladder, then
Cleaning 5 times, shreds prostata tissue, adds the physiological saline containing 0.5%TritonX-100, ice bath homogenate, 12000r/min 4
After DEG C centrifugation 30min, remove upper-layer fat tissue, the supernatant of acquisition is human prostate tissue protein purification liquid, with it is complete from
Packing preserves (- 80 DEG C) after its total protein concentration of dynamicization analysis-e/or determining.
(2) the sero-fast preparation of anti-human prostate tissue antigen and antibody titer detection：Back is passed through to experimental rabbit first
The human prostate tissue protein purification liquid (albumen that the step of intracutaneous and subcutaneous multi-point injection Freund's complete adjuvant emulsification (1) obtains
Concentration is 0.5mg/ml), every experimental rabbit 1ml；Compare the physiological saline of rabbit injection Freund's complete adjuvant emulsification, every 1ml.Family
Rabbit was injected again at the 10th, 20,30 day, is put to death rabbit, and is collected the whole blood of rabbit within 7 days after last time is injected,
Serum is centrifuged, serum freezes standby in -80 DEG C.The anti-prostata tissue protein antibodies potency of rabbit anteserum is examined by ELISA method
Survey, its potency is 1:More than 10000.
(3) identification of human prostate Immunodominant Antigenic：The human prostate tissue protein purification that step (1) is obtained first
Liquid carries out two dimensional electrophoresis, and the experiment rabbit anti-serum and control rabbit anteserum then obtained respectively with step (2) carries out Western blotting, then
Mass spectral analysis is carried out to both differential protein spots, identifies 16 kinds of prostate Immunodominant Antigenics altogether, wherein 3 kinds of albumen ---
Semaphorin-7A, heat shock protein beta-1 and the class antigen of MHC 2 have been found and prostate disease
It is sick related.Western blot determines that these antigens are primarily present on the cell membrane and cytoplasm of prostate gland epithelial cell.
(4) purifying of human prostate Immunodominant Antigenic：Saturated ammonium sulfate method and Protein A-Agarose layers are used successively
Analysis post is purified into rabbit immunoglobulin from rabbit anti-serum, on the Sepharose chromatographic columns that then cross-linking to CNBr activates,
Human prostate tissue protein purification liquid is crossed into this chromatographic column, by combine and elute can obtain purifying human prostate be immunized it is excellent
Gesture antigen.
(5) ELISA method of detection human prostate autoantibody is established：The human prostate for the purifying that step (4) is obtained is exempted from
Epidemic disease dominant antigen coating polystyrene ELISA Plate (lath), coating human prostate Immunodominant Antigenic 50ng, serum sample per hole
With 1:It is loaded after 10 dilutions, the goat anti-human igg of horseradish peroxidase (HRP) mark is secondary antibody.Developed the color with TMB nitrite ions
10min, 2mol/L sulfuric acid terminating reaction.Using the judged result of P/N >=2.1 as the positive, so as to establish human prostate autoantibody
ELISA detection methods.
Wherein, if standard positive serum is positive control, if normal healthy male serum is negative control, phosphate-buffered
Liquid is blank control.
P/N=(sample OD values-blank control OD values)/(negative control OD value-blank control OD values).P/N >=2.1 are sun
Property, 1.5≤P/N<2.1 be suspicious, P/N<1.5 be feminine gender.
(6) clinical practice of human prostate autoantibody detection method：The human prostate autoantibody established with step (5)
ELISA method, 75 normal healthy controls male serum and 78 chronic prostatitis/chronic Pelvic Pain Syndrome (CP/ are detected respectively
CPPS) patients serum, it is in weakly positive that the former, which has 2 anti-prostate autoantibodies, positive rate 2.7%, and the latter has 18 sun
Property, positive rate 23.1%.Positive patient sera confirms that it can be with human prostate Immunodominant Antigenic shape with western blot
Into obvious reaction zone, it was demonstrated that be implicitly present in CP/CPPS patients serums can be combined with human prostate Immunodominant Antigenic from
Body antibody, it was demonstrated that the reliability of human prostate autoantibody detection method provided by the invention.
Beneficial effect：The human prostate Immunodominant Antigenic method provided by the invention for preparing is to be based on using normal human prostate
Histogenic immunity rabbit, immune response only is occurred to prostate specific antigen using rabbit, and the antigen shared to rabbit and people is simultaneously
The principle of immune response does not occur, so as to obtain special rabbit anti-serum；After rabbit anti-serum immunoglobulin purification, layer is utilized
Analysis post obtains the human prostate Immunodominant Antigenic of purifying from human prostate tissue；It is anti-using this Immunodominant Antigenic as coating
Original, establishes the enzyme linked immunosorbent assay analysis method (ELISA method) of detection human prostate autoantibody, and is applied to the inspection of clinic
Survey.
Compared with prior art, the present invention has advantages below：
(1) people and rabbit common antigen present in most prostata tissue homogenate can be removed by immunizing rabbit,
And caused antibody is mainly for human prostate Immunodominant Antigenic.
(2) by two dimensional electrophoresis and the differential disply of Western blotting, 16 kinds of human prostate Immunodominant Antigenics are identified altogether,
Find that these Immunodominant Antigenics are primarily present in the cell membrane and cytoplasm of prostate gland epithelial cell by SABC
In.
(3) human prostate Immunodominant Antigenic is obtained by the method purifying of affinity chromatography, and it is anti-in this, as coating
Original establishes the ELISA detection methods of human prostate autoantibody.
(4) detect anti-prostate certainly in CP/CPPS patients serums by human prostate autoantibody ELISA detection methods
Body antibody, and confirmed by western blot, so as to the diagnosis for clinically male's autoimmune prostatitis and treatment prison
Survey provides new means.
Brief description of the drawings
Accompanying drawing 1 is the two dimensional electrophoresis and differential protein spot schematic diagram of human prostate tissue protein purification liquid, and wherein A is two dimension
The silver staining picture of running gel, B and C are respectively the western blot result tested rabbit anti-serum and compare rabbit anteserum；
Accompanying drawing 2 is the immunohistochemical analysis of human prostate tissue Immunodominant Antigenic, and wherein A and B are respectively to test rabbit-anti
The ImmunohistochemistryResults Results of serum and control rabbit anteserum；
Accompanying drawing 3 is the purifying of human prostate Immunodominant Antigenic and SDS-PAGE checkings, and wherein A is experiment rabbit anti-serum
IgG elution curve, B are the elution curve of human prostate Immunodominant Antigenic, and C is that SDS-PAGE verifies purification effect；
Accompanying drawing 4 be western blot confirm CP/CPPS patients serums in exist anti-human prostate Immunodominant Antigenic from
Body antibody, left figure and right figure are respectively the anti-prostate autoantibody positive sera of CP/CPPS patient and healthy male serum
Western blot result.
Embodiment
Below will be by several specific embodiments, the present invention is furture elucidated, these embodiments simply to illustrate that problem,
It is not a kind of limitation.
Embodiment 1
The preparation of human prostate tissue protein purification liquid：Human prostate hyperplasia of prostate tissue is by Nanjing General Hospital, Nanjing Military Area Command, PLA
Urology Surgery provides, from 5 Benign Prostatic Hypertrophies.This research obtains Hospital Ethical Committee's approval, and obtains patient
Informed consent.The prostata tissue of acquisition is placed in ice PBS (0.01mmol/L, pH7.4), it is more to trim fat, bladder etc.
Remaining tissue, then clean 5 times, prostata tissue is shredded, adds the physiological saline containing 0.5%TritonX-100, ice bath is homogenized,
After 4 DEG C of centrifugation 30min of 12000r/min, the adipose tissue on upper strata is removed, it is that human prostate tissue albumen carries to leave and take supernatant
Pure liquid, packing preservation (- 80 DEG C) after determining its total protein concentration with automatic clinical chemistry analyzer.
Embodiment 2
The sero-fast preparation of anti-human prostate tissue antigen：Experimental rabbit scheme passes through the animal committee and Ethics Committee
Approval.Experimental rabbit passes through before the people that is obtained in back is intracutaneous and subcutaneous multi-point injection Freund's complete adjuvant emulsifies embodiment 1
Row glandular tissue protein purification liquid (protein concentration 0.5mg/ml), every experimental rabbit 1ml；Compare rabbit injection Freund's complete adjuvant breast
The physiological saline of change, every 1ml.Rabbit received injection at the 10th, 20,30 day again, put to death house within 7 days after last time is injected
Rabbit, and the whole blood of rabbit is collected, serum is centrifuged, serum freezes standby in -80 DEG C.Immunizing rabbit prepare antiserum into
Whether is work(, is assessed by the potency of contained specific antibody in ELISA method detection its rabbit anteserum.
Embodiment 3
The detection of experimental rabbit serum antibody titer：The experiment anti-prostata tissue protein antibodies potency of rabbit anteserum passes through routine
ELISA method detection.Employment prostatein homogenate 96 hole elisa Plates of coating, per hole 50ng albumen.Serum to be checked presses 1:10、1:
100、1:1000、1:10000、1:100000 gradient dilutions.The goat anti-rabbit igg polyclonal antibody of HRP marks is as secondary antibody (1:
1000 dilutions), with TMB nitrite ions colour developing 10min, 2mol/L sulfuric acid terminating reactions.To test absorbance/control of rabbit anteserum
The absorbance of rabbit anteserum >=2.1 are used as positive criterion, as a result show, the antibody titer for testing rabbit anteserum is more than 1:
10000。
Embodiment 4
The identification of human prostate Immunodominant Antigenic：The human prostate tissue protein purification liquid (egg that will be obtained in embodiment 1
Bai Hanliang is 500 μ g) isoelectric focusing (IEF) electricity is carried out on Ettan IPGphor 3Isoelectric Focusing Unit
Swimming, the band (pH3-10) of pH gradient is fixed with 13cm.Sample-loading buffer is IPG buffer solutions (pH3-10), and its composition includes
8mol/L urea, 2% (g/mL) 3- [(3- cholesterol aminopropyl) dimethylamino] -1- propane sulfonic acid, 20mmol dithiothreitol (DTT)s,
0.001% bromophenol blue.Isoelectric focusing voltage uses linear ramp pattern.First to the band after electrophoresis in 10ml balance solutions
Equilibrium at room temperature 15min, then balance 15min in the balance solution containing 2.5% (weight/volume) iodoacetamide.Second to electrophoresis
Carried out on 12.5% sds gel, using Ettan DALRtwelve GEL System electrophoresis apparatuses, 5W is used in preceding 30min
Per the power of clotting glue, the power in rear 6~7h using 12W per clotting glue, until last bromophenol blue line is run to gel bottom.Altogether
Prepare 3 gels and carry out electrophoresis, 1 gel carries out cma staining according to conventional scheme, and another 2 gels print for Western
Mark.
Another 2 gels are used as primary antibody (1 by the use of the experiment rabbit anti-serum and control rabbit anteserum obtained in embodiment 2 respectively:1 is dilute
Release) western blot is carried out, the goat-anti rabbit of horseradish peroxidase-labeled is as secondary antibody (1:1000 dilutions), TMB nitrite ions show
Color.Stained gel is with 300dpi resolution scan (Artix Scan 1010plus), and with image analysis software (Image
MasterTM 2D platinum software, Version 5.0) protein site is detected, quantified, is compared and data point
Analysis, with Flex Analysis, v.2.4 software is carried out for mass spectral analysis, final to obtain 16 kinds of difference immunodominant proteins, that is, is people forefront
Gland Immunodominant Antigenic.Concrete outcome is as shown in accompanying drawing 1 and subordinate list 1.
The western blot of subordinate list 1 and 16 kinds of Immunodominant Antigenics of mass spectral analysis identification
For further positioning of the clear and definite human prostate Immunodominant Antigenic in prostata tissue, benign prostate is taken to increase
Raw tissue progress formalin is fixed and FFPE, is dewaxed in dimethylbenzene, its is regenerated hyrate, antigen is used at 95 DEG C
Liquid EDTA processing 13min are repaired, and cool down 50min, are closed with 1%BSA solution.Histotomy respectively with experiment rabbit anti-serum and
4 DEG C of overnight incubations of rabbit anteserum are compareed, are cleaned 5 times with the PBS solution containing 0.1% tween, and in horseradish peroxidase-labeled
30min is incubated at room temperature in goat anti-rabbit IgG antibody.BAD chromogenic reagents, haematoxylin redyeing.SABC picture is shown with IX73
Micro mirror is observed.Concrete outcome is as shown in Figure 2.
Embodiment 5
The purifying of human prostate Immunodominant Antigenic：It is purified into embodiment 2 and is obtained with saturated ammonium sulphate method first
Experimental rabbit antiserum globulin, reuse Protein A-Agarose column chromatographies and go out in experimental rabbit antiserum globulin
IgG.IgG is eluted using the pH4.0 of the glycine containing 1.5mol/L PBS solution, and with 1mol/L pH8.5Tris-
HCl buffer solutions prevent antibody from inactivating to neutralize elution buffer.Then, by the Sepharose of IgG after purification and CNBr activation
Crosslinking, then the human prostate tissue protein purification with the Sepharose-IgG after crosslinking in chromatographic column to being obtained in embodiment 1
Immunodominant Antigenic in liquid is purified, you can obtains the human prostate Immunodominant Antigenic of purifying.SDS-PAGE electrophoresis enters
One step confirms the effect of purifying.As a result as shown in Figure 3.
Embodiment 6
The foundation of human prostate autoantibody detection method：The ELISA method of human prostate autoantibody detection is established, will
The purifying human prostate Immunodominant Antigenic coating polystyrene micropore plate obtained in embodiment 5, resists per hole 50ng immunodominances
Original, serum sample is with 1:It is loaded after 10 dilutions, the goat anti-human igg of HRP marks is secondary antibody.With TMB nitrite ions develop the color 10min,
2mol/L sulfuric acid terminating reactions.Using the judged result of P/N >=2.1 as the positive.Wherein, if standard positive serum is positive control, if
Normal healthy male serum is negative control, and phosphate buffer is blank control.
P/N=(sample OD values-blank control OD values)/(negative control OD value-blank control OD values).P/N >=2.1 are sun
Property, 1.5≤P/N<2.1 be suspicious, P/N<1.5 be feminine gender.
Embodiment 7
The clinical practice of human prostate autoantibody detection method：Clinically NIH chronic prostatitis symptom index is collected to comment
Divide the CP/CPPS patient 78 of (NIH-CPSI) >=10 point, 30~45 years old age.Collect simultaneously without prostatitis clinical symptoms
Healthy male volunteers 75,27~37 years old age.In morning, collection venous blood, separation serum are standby on an empty stomach for two groups of research objects
With.Patients serum and healthy male serum use human prostate autoantibody detection method (ELISA method) in embodiment 6 to detect.
It is in weakly positive to have 2 anti-prostate autoantibodies in 75 normal healthy controls male's serum, positive rate 2.7%, and
18 anti-prostate autoantibody sun in 78 chronic prostatitis/chronic Pelvic Pain Syndrome (CP/CPPS) patients serums
Property, positive rate 23.1%.Positive patient sera confirms that it can be with human prostate Immunodominant Antigenic shape with western blot
Into obvious reaction zone, as shown in Figure 4, it was demonstrated that being implicitly present in CP/CPPS patients serums can be with human prostate immunodominance
The autoantibody that antigen reacts.
Described above is only several embodiments of invention, it is noted that for those skilled in the art
For, on the premise of inventive principle is not departed from, some improvement can also be made, these improvement also should be regarded as the protection of the present invention
Scope.
Claims (9)
- A kind of 1. preparation method of human prostate Immunodominant Antigenic, it is characterised in that：Comprise the following steps：(1) preparation of human prostate tissue protein purification liquid；(2) the sero-fast preparation of anti-human prostate tissue antigen and antibody titer detection；(3) identification of human prostate Immunodominant Antigenic；(4) purifying of human prostate Immunodominant Antigenic.
- 2. the preparation method of human prostate Immunodominant Antigenic according to claim 1, it is characterised in that：The people forefront The preparation of glandular tissue protein purification liquid, comprises the following steps：Human prostate hyperplasia of prostate tissue is placed in glacial phosphoric acid salt buffer, trims unnecessary tissue, the excess tissue Including fat, bladder；Cleaning 5 times, shreds prostata tissue, adds the physiological saline containing 0.5%TritonX-100, ice bath homogenate, 12000r/ After min4 DEG C of centrifugation 30min, upper-layer fat tissue is removed, the supernatant of acquisition is human prostate tissue protein purification liquid, is surveyed Preservation is dispensed after determining total protein concentration.
- 3. the preparation method of human prostate Immunodominant Antigenic according to claim 1, it is characterised in that：It is described it is anti-human before The sero-fast preparation of row glandular tissue antigen, comprises the following steps：The human prostate tissue protein purification liquid that Freund's complete adjuvant emulsifying step (1) obtains, it is intracutaneous by back to experimental rabbit And the human prostate tissue protein purification liquid of subcutaneous multi-point injection Freund's complete adjuvant emulsification, protein concentration 0.5mg/ml, often Experimental rabbit 1ml；Rabbit is compareed by the physiological saline that back is intracutaneous and subcutaneous multi-point injection Freund's complete adjuvant emulsifies, every 1ml；Experimental rabbit and control rabbit received injection at the 10th, 20,30 day again, and last time puts to death experimental rabbits and right for 7 days after injecting According to rabbit, and collect experimental rabbit and compare the whole blood of rabbit, centrifuge serum, serum freezes standby in -80 DEG C.
- 4. the preparation method of human prostate Immunodominant Antigenic according to claim 3, it is characterised in that：The antibody effect Valency detection is detected by enzyme linked immunosorbent assay analysis method, and rabbit anteserum anti-human prostate histone antibody titer is 1:10000 with On.
- 5. the preparation method of human prostate Immunodominant Antigenic according to claim 1, it is characterised in that：The people forefront The identification of gland Immunodominant Antigenic, comprises the following steps：The human prostate tissue protein purification liquid that step (1) is obtained first carries out two dimensional electrophoresis, is then obtained respectively with step (2) The experiment rabbit anti-serum and control rabbit anteserum obtained carries out Western blotting, then carries out mass spectral analysis to both differential protein spots, obtains The human prostate Immunodominant Antigenic.
- 6. the preparation method of human prostate Immunodominant Antigenic according to claim 5, it is characterised in that：The people forefront Gland Immunodominant Antigenic includes Semaphorin-7A, heat shock protein beta-1 and the class of MHC 2 resists It is former.
- 7. the preparation method of human prostate Immunodominant Antigenic according to claim 1, it is characterised in that：The people forefront The purifying of gland Immunodominant Antigenic, comprises the following steps：Saturated ammonium sulphate method and Protein A-Agarose layers are used successively Analysis post is purified into rabbit immunoglobulin from rabbit anti-serum, on the Sepharose chromatographic columns that then cross-linking to CNBr activates, Human prostate tissue protein purification liquid is crossed into this chromatographic column, by combine and elute can obtain purifying human prostate be immunized it is excellent Gesture antigen.
- 8. the application process of the human prostate Immunodominant Antigenic according to claim any one of 1-7, it is characterised in that：Bag Include following steps：The human prostate Immunodominant Antigenic coating polystyrene ELISA Plate of purifying, the coating human prostate Immunodominant Antigenic per hole 50ng, serum sample is with 1:It is loaded after 10 dilutions, the goat anti-human igg of horseradish peroxidase-labeled is secondary antibody；With TMB nitrite ions Develop the color 10min, 2mol/L sulfuric acid terminating reactions.
- 9. the application process of human prostate Immunodominant Antigenic according to claim 8, it is characterised in that：By healthy male Serum and/or chronic prostatitis or chronic Pelvic Pain Syndrome patients serum use the ELISA of human prostate autoantibody Detection method is detected.
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