CN104897900B - A kind of Prostasomes leak proteantigen and its antibody and application - Google Patents
A kind of Prostasomes leak proteantigen and its antibody and application Download PDFInfo
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Abstract
The present invention relates to a kind of Prostasomes leak proteantigen, with its special corresponding antibody and application, and further obtain based on this based on Prostasomes leak protein quantification Immunofluorescent antibody kit, with effective for detection and diagnosing chronic prostatitis.The precision detection of the reacted system of urine detection kit based on the reaction of PSEP antibody antigens, stability test, its reagent quality of destructive test all show that height can be mass.Its application is real Noninvasive, easily operated, easy, and has high sensitivity and specificity.It will not only mitigate because the patient of chronic pelvic pain syndrome/chronic prostatitis is painful, and early stage diagnosis and treatment will further strengthen the preventing and treating to prostate cancer, be with a wide range of applications.
Description
Technical field
Leaked proteantigen the present invention relates to a kind of Prostasomes, and in particular to a kind of Prostasomes leak albumen
Antigen and preparation method and application.
Background technology
Chronic prostatitis (ChronicProstatitis, CP) be the most common disease of Urology Surgery Lipid on Young-middle Male it
One, it was reported that it accounts for the 30% of Number of Outpatients.According to epidemiology survey, in China adult human male crowd, more than 10% has prostatitis
Sample symptom, 50% male's different times suffered from prostatitis.Illustrate potential chronic prostatitis or chronic pelvic pain
（CPPS) patient is numerous.Meanwhile the hair of inflammatory process or human cancer as caused by chemicals, physical factor or biological agent
Important factor in interpretation of the cause, onset and process of an illness system.In prostate cancer, regeneration is considered as pernicious after the presence and cell epithelia tissue damage of inflammation
The key factor (DeMarzoetal., 2007) of conversion.Prostata tissue is infiltrated by inflammatory cell, various so as to discharge
Active material, the release of these materials also with bacterium and virus infection, that uric acid increases and eaten prostate carcinogenic substance is relevant.In mistake
Go in 10 years, the contact between inflammation and cancer, and prostatitis has flowed as the hypothesis of prostate cancer high risk factor
Sufficiently studied and be confirmed on row disease, clinic, morphology, aetology, cell and molecular level.It is in fact, literary
Pointed out in offering, about more than 12% prostatitis, such as treatment can be converted into prostate cancer not in time.So prostatitis is serious
Influence men's health (Chen Zhi refined etal., 2009；That man of virtue and ability group etal., 2011).
Prostatitis clinical manifestation lacks specificity because of patient individual difference, therefore clinically therapeutic effect is not always
Satisfied (Kriegeretal., 1999;Engeleretal.,2012；That man of virtue and ability group etal., 2011).According to US National in 1997
Institutes of Health Research (NIH) to prostatitic classification, i.e., NIH classification (Kriegeretal., 1999;The refined etal. of Chen Zhi,
, including I types 2009):Acute bacterial prostatitis (ABP);II types:Chronic bacterial prostatitis (CBP);Type III:Divide again
For IIIA, chronic non-bacterial prostatitis (CAP) and IIIB, chronic pelvic pain syndrome (CPPS);IV types:It is asymptomatic
Inflammatory prostatitis (AIP).It is generally common with III type chronic prostatitis in clinical practice, 90% or so is accounted for, so slowly
Property prostatitis generally refers to type III chronic prostatitis.In clinical practice work, uropoiesis andrology doctor is examined head
First 3 of the inspection method (multiselect) of CAP and CPPS patients selection is routine urinalysis (80.3%), massage of prostate fluid inspection
(71.0%), physical examination (including digital rectal examination) (63.2%).Massage of prostate fluid inspection and digital rectal examination have certain intrusion to patient
Property, and most of diagnosis still rely on Clinical signs and doctors experience, therefore be highly desirable to easy without invasive while accurate
True reliable molecular diagnosis method.
China application CN102998455A disclose it is a kind of detect or diagnosis of prostate cancer kit, its include δ-
Catenin absorption antibody, coating buffer solution, lavation buffer solution, Block buffer, δ-catenin detections antibody, enzyme labelled antibody.
Or comprising δ-catenin absorption antibody, coating buffer solution, lavation buffer solution, Block buffer, δ-catenin detection antibody, receive
Rice is quantum dot-labeled.The kit can from patient urine with ELISA method detection with prostate cancer into high correlation
δ-catenin albumen, and ELISA method is ripe, and Sensitivity and Specificity is higher, is Non-Invasive to patient, can
Repetition is taken, and patient is willing to accept.Nano-quantum point (QD) labelling method sampling amount is small, by antibody used in ELISA method with
QD is combined, and can more delicately detect the change of δ-catenin albumen in urine, so as to be used for diagnosis of prostate cancer.But the examination
Agent box is only applicable to prostate cancer detection, it is impossible to chronic prostatitis syndrome patient and normal human sample are distinguished very well, thus it is inapplicable
Detected in prostatitis.
The content of the invention
The first object of the present invention is to be to provide a kind of Prostasomes available for prostatitis detection to leak egg
Bai Kangyuan.
Prostasomes of the present invention leak proteantigen, are prepared with the following method：
500 milliliters of chronic prostatitis syndrome patient's urina sanguinis midstream urines are taken, add equivalent pH7.3 HEPES buffer solution and egg
White enzyme inhibitor, uses filtered through gauze；Filtered fluid is taken to be respectively placed in 10 50 milliliters of centrifuge tubes, the 400g centrifugations 10 under 4 DEG C of low temperature
Minute, go to precipitate, take supernatant 10,000g under 4 DEG C of low temperature to centrifuge 20 minutes, go to precipitate, take supernatant to be respectively placed in 200
3 milliliters of ultracentrifugation pipes, 100,000g is centrifuged 120 minutes under 4 DEG C of low temperature；Supernatant is removed, takes precipitation to add 2.5 milliliters
PH7.8 TBS buffer solutions and protease inhibitors, mix；100,000g is centrifuged 120 minutes under 4 DEG C of low temperature, removes supernatant
Liquid, take precipitation to be dissolved in 2.5 milliliters of 0.5%NP-40,80mMNaCl, 0.2%Deoxycholate respectively, 50mMTris, pH8.0 and
Protease inhibitors；100,000g is centrifuged 20 minutes under 4 DEG C of low temperature；Take supernatant, loading TSKgelG3000PW, 280 nanometers
Reading, take in 40kDa plots peak albumen, -80 DEG C of freezen protectives, produce.
Meanwhile the present invention further protects above-mentioned Prostasomes to leak the preparation method of proteantigen：
500 milliliters of chronic prostatitis syndrome patient's urina sanguinis midstream urines are taken, add equivalent pH7.3 HEPES buffer solution and egg
White enzyme inhibitor, uses filtered through gauze；Filtered fluid is taken to be respectively placed in 10 50 milliliters of centrifuge tubes, the 400g centrifugations 10 under 4 DEG C of low temperature
Minute, go to precipitate, take supernatant 10,000g under 4 DEG C of low temperature to centrifuge 20 minutes, go to precipitate, take supernatant to be respectively placed in 200
3 milliliters of ultracentrifugation pipes, 100,000g is centrifuged 120 minutes under 4 DEG C of low temperature；Supernatant is removed, takes precipitation to add 2.5 milliliters
PH7.8 TBS buffer solutions and protease inhibitors, mix；100,000g is centrifuged 120 minutes under 4 DEG C of low temperature, removes supernatant
Liquid, take precipitation to be dissolved in 2.5 milliliters of 0.5%NP-40,80mMNaCl, 0.2%Deoxycholate respectively, 50mMTris, pH8.0 and
Protease inhibitors；100,000g is centrifuged 20 minutes under 4 DEG C of low temperature；Take supernatant, loading TSKgelG3000PW, 280 nanometers
Reading, take in 40kDa plots peak albumen, -80 DEG C of freezen protectives, produce.
Above-mentioned preparation method can be prepared described Prostasomes albumen with rapid batch and be leaked proteantigen, methods described
It is stable reproducible, it is easy to utilize.
Another object of the present invention is to protect antibody corresponding with above-mentioned antigen-specific.
Another object of the present invention is to protect above-mentioned antigen and antibody preparing the reagent of detection or diagnosis of prostate cancer
Application in box.
Mentioned reagent box can be prepared using a variety of preparation methods disclosed in prior art, skilled person will appreciate that
Dawn, leaked obtaining Prostasomes of the present invention in the case of proteantigen and its antibody, based on this special antigen and
Antibody leaks to Prostasomes, and accurately detection acts on albumen, no matter using which kind of preparation method, can obtain can be used for examining
Survey or the kit of diagnosis of prostate cancer.
Preferably described kit of the invention is prepared using ELISA method or colloidal gold method.
Kit of the present invention include for Prostasomes leak proteantigen monoclonal detection antibody, coating
Buffer solution, lavation buffer solution, Block buffer, enzyme labelled antibody.Specifically comprise those skilled in the art to be understood, the present invention is right
This is not particularly limited.
Wherein, Prostasomes of the present invention leak proteantigen detection antibody be monoclonal mouse anti-human's Prostasomes
Leak the antibody of proteantigen.
Further, the kit also includes standard items and negative control；The standard items are that Prostasomes are leaked
The human prostate corpusculum that Protein Detection antibody produces immune response leaks proteantigen.
Wherein, the kit coating buffer solution is carbonate or dPBS；The lavation buffer solution is containing detergent
dPBS。
The detection antibody identification Prostasomes leak proteantigen.
Kit of the present invention, in use, specific flow is as follows：
1st, prepare：
1. taking out kit from 2-8 DEG C of cold storage environment, BSA pulvis is poured into premade closure liquid in PBS bottle（Must
PBS is being sucked in BSA pulvis pipes with pipettor again after emptying pulvis when wanting, is pouring into PBS bottle after shaking up again
In, in case pulvis is adhered on tube wall）, fully mix.
2. by 20X concentrated cleaning solutions 50ml（Two bottles）Poured into completely in the bottle for handling liquid toilet or cosmetic substance of board-washing machine after shaking up（It is brilliant if any precipitation
Body dissolves under the conditions of being placed in 37 DEG C）, then pour into purified water（Resistivity is more than 15M Ω）, 1000ml is settled to, it is fully mixed
It is even.
2nd, number：Microwell plate is taken out from aluminium foil bag.A1 is blank well, and A2-A6 holes have been coated with 5 works of positive criteria product
Make concentration：0.1ng;1ng;2ng;5ng;10ng.Remaining each hole serial number as required.
3rd, it is loaded：In addition to positive criteria hole and blank well, remaining adds urine sample in corresponding hole respectively by number（Pat
Microwell plate edge is to have prevented bubble）.90 person-portions/box is individual by the inspection μ l of people's urine sample 100 per Kong Jiayi（Blank control wells are without adding
Urine sample, closing plus primary antibody, add the operation such as secondary antibody, remaining each step operation is identical）.Microwell plate is put into 37 with after shrouding film shrouding
DEG C insulating box（Or water bath）One hour.
4th, clean：Washed 5 times with cleaning fluid on board-washing machine（Optionally setting is each per hole μ l of cleaning fluid 300 or so,
Soak time 5 seconds）, washed plate is taken out, patted if necessary on paper handkerchief, to remove raffinate in hole（Including owning below
Clappers plate bottom can not be all propped up with finger, to prevent from influenceing last measure effect）.
5th, close：In addition to blank well, in each hole（Containing positive gauge orifice, without blank control wells）100 μ l of middle addition are prefabricated
Good confining liquid, 37 DEG C of insulating boxs are put into after shrouding film shrouding by microwell plate（Or water bath）40 minutes.
6th, clean：Washed 5 times with cleaning fluid on board-washing machine（Optionally setting is each per hole μ l of cleaning fluid 300 or so,
Soak time 5 seconds）, washed plate is taken out, patted if necessary on paper handkerchief, to remove raffinate in hole.
7th, PSEP antibody is added：To each hole（Containing positive gauge orifice, without blank control wells）The middle μ l of addition PSEP antibody 50（Gently
Microwell plate edge is clapped to have prevented stomata）.Microwell plate is put into 37 DEG C of insulating boxs with after shrouding film shrouding（Or water bath）40 points
Clock.
8th, clean：Washed 5 times with cleaning fluid on board-washing machine（Optionally setting is each per hole μ l of cleaning fluid 300 or so,
Soak time 5 seconds）, washed plate is taken out, patted if necessary on paper handkerchief, to remove raffinate in hole.
9th, enzyme-added labeling antibody：To each hole（Containing positive gauge orifice, without blank control wells）Middle addition enzyme labelled antibody 100 μ l
（Microwell plate edge is patted to have prevented stomata）.Microwell plate is put into 37 DEG C of insulating boxs with after shrouding film shrouding（Or water bath）20
Minute.
10th, clean：Washed 5 times with cleaning fluid on board-washing machine（Optionally setting is left with the μ l of cleaning fluid 300 per hole every time
The right side, soak time 5 seconds）, washed plate is taken out, patted if necessary on paper handkerchief, to remove raffinate in hole.
11st, develop the color：To every hole（Containing positive gauge orifice and blank well）Inject 50 μ l nitrite ions A and 50 μ l nitrite ions B（Pat
Microwell plate edge mixes it and has prevented stomata）, by its normal temperature avoid light place 20 minutes.
12nd, terminate：To every hole（Containing positive gauge orifice and blank well）Inject 50 μ l terminate liquids（Patting microwell plate edge makes it
Mix and prevented stomata）.
13rd, determine：Microwell plate is put into enzyme non-analysis meter, each hole OD values is determined using dual wavelength 450nm/630nm and remembers
Record result.
14th, result judgement:According to PSEP antibody test features, PSEP positive findingses：Tested sample is assessed with standard curve,
Concentration value ＞ 1.20ng/ml；
PSEP negative findingses：Tested sample, concentration value≤1.20ng/ml are assessed with standard curve；OD values are less than 0.10 nothing
Effect.
The invention provides a kind of Prostasomes leak proteantigen and with its special corresponding antibody, and can be further
Obtain based on Prostasomes leak protein quantification Immunofluorescent antibody kit, with effective for detecting and diagnosing
Chronic prostatitis.Prostasomes (prostasomes) are the super of about 30~150 nanometers of the diameter being present in prostatic fluid
Micro-structural, by prostatic epithelium or interstitial cell etc. it is outer tell (exocytosis) or leak (excretion) formed, with other groups
Outer in knitting tells corpusculum (exosome) structure, and size, composition is similar but differs.
Although outer at present tell approach, signal transduction mechanism understands very few, Recent study discovery, deposited in Prostasomes
In nucleic acid, albumen, carbohydrate, fat, small molecule metabolites etc..Prostasomes leak, and (it is named as albumen
Prostateexosomal protein, or abbreviation PSEP) it is very more, not yet identify (Lietal2008 completely at present;
Luetal.,2009;).The Prostasomes that the application separates from Patients with Chronic Prostatitis are antigen, are screened, and are obtained special
Different antibody, it is albumen (PSEP) humanized murine antibodies of being leaked for anti-Prostasomes, for further studying it for chronic
The sensitivity and specificity of prostatitis detection.
EPS methods used in current diagnosis prostatitis, with massage of prostate liquid, there is the very big property invaded and not to patient
It is suitable, and the present invention based on special Prostasomes leak proteantigen offer detection method it is easy to be reliable, sensitivity reaches
85%, it is a kind of a kind of novel Noninvasive available for Diagnosis of Chronic Prostatitis, painless molecule measuring means.
Brief description of the drawings
Fig. 1 is the evaluation to PSEP kits Test of accuracy (ROC curve analysis), and wherein abscissa is specificity, is indulged
Coordinate is sensitivity.
Embodiment
The description that gives an actual example is carried out to the principle of the invention and feature below, the given examples are served only to explain the present invention, not uses
In restriction the scope of the present invention.
Embodiment 1
Prostasomes leak proteantigen（PSEP preparation):Take in 500 milliliters of chronic prostatitis syndrome patient's urina sanguinis
Section urine, adds equivalent HEPES buffer solution (pH7.3) and protease inhibitors (Sigma Co., USA), use filtered through gauze.Took
Filtrate, 10 50 milliliters of centrifuge tubes are respectively placed in, 400g is centrifuged 10 minutes under 4 DEG C of low temperature.Go to precipitate, take supernatant.At 4 DEG C
10,000g is centrifuged 20 minutes under low temperature.Go to precipitate, take supernatant.200 3 milliliters of ultracentrifugation pipes are respectively placed in, it is low at 4 DEG C
Lower 100, the 000g of temperature is centrifuged 120 minutes.Supernatant is removed, takes precipitation.Add 2.5 milliliters of TBS buffer solutions (pH7.8) and protease
Inhibitor (Sigma Co., USA), mix.100,000g is centrifuged 120 minutes under 4 DEG C of low temperature.Supernatant is removed, takes precipitation.
2.5 milliliters of 0.5%NP-40,80mMNaCl, 0.2%Deoxycholate, 50mMTris, pH8.0 and albumen enzyme level are dissolved in respectively
Agent (Sigma Co., USA).100,000g is centrifuged 20 minutes under 4 DEG C of low temperature.Take supernatant, loading TSKgelG3000PW (U.S.s
Sigma companies of state), 280 nanometers of readings, take in 40kDa plots peak albumen.- 80 DEG C of freezen protectives.
Embodiment 2
Kit forms are as follows described in the present embodiment：The mud-stream urine of prostatitis patient；Confining liquid；Prostasomes
Leak proteantigen（PSEP)；PSEP detects antibody；Cleaning fluid；Developer；Terminate liquid.
It is as follows to the specific method of prostatitic detection or diagnosis using mentioned reagent box：
1. Specimen origin：According to prostatitic diagnostic criteria in " Chinese urological diseases diagnoses and treatment companion "
(that a man of virtue and ability group etal., 2011) collects 213 parts of the mud-stream urine of prostatitis patient, and selection complies fully with Comprehensive Clinical diagnosis
Chronic prostatitis 176.Age 16-72 year, average age 34 years old.100 parts of normal person's physical examination urine, age 20-68 year.
Average age 34 years old.Routine urinalysis is normal.Urine detects immediately after collecting, or puts -20 DEG C of refrigerators and preserve.
The proteantigen 2. Prostasomes leak（PSEP preparation):Take 500 milliliters of chronic prostatitis syndrome patient's urina sanguinis
Midstream urine, equivalent HEPES buffer solution (pH7.3) and protease inhibitors (Sigma Co., USA) are added, use filtered through gauze.Take
Filtered fluid, 10 50 milliliters of centrifuge tubes are respectively placed in, 400g is centrifuged 10 minutes under 4 DEG C of low temperature.Go to precipitate, take supernatant.4
10,000g is centrifuged 20 minutes under DEG C low temperature.Go to precipitate, take supernatant.200 3 milliliters of ultracentrifugation pipes are respectively placed in, at 4 DEG C
100,000g is centrifuged 120 minutes under low temperature.Supernatant is removed, takes precipitation.Add 2.5 milliliters of TBS buffer solutions (pH7.8) and albumen
Enzyme inhibitor (Sigma Co., USA), mix.100,000g is centrifuged 120 minutes under 4 DEG C of low temperature.Supernatant is removed, it is heavy to take
Form sediment.2.5 milliliters of 0.5%NP-40,80mMNaCl, 0.2%Deoxycholate, 50mMTris, pH8.0 and protease are dissolved in respectively
Inhibitor (Sigma Co., USA).100,000g is centrifuged 20 minutes under 4 DEG C of low temperature.Take supernatant, loading
TSKgelG3000PW (Sigma Co., USA), 280 nanometers of readings, take in 40kDa plots peak albumen.- 80 DEG C of freezen protectives.
3.PSEP detects the preparation of antibody:Defrosting PSEP albumen, it is public with BCA (U.S. ThomasFisherScientific)
Take charge of kit measurement concentration.6 BALB/c mouses are taken, every is injected 50 micrograms for the first time.After 2 weeks, inject again.10 days
Afterwards, mouse blood is taken, makees ELISA titer determinations with 96 orifice plates of the nanogram bed board of PSEP albumen 50.Then make third time injection to exempt from
Epidemic disease.Titre reaches 1 after 2 weeks:More than 10,000, do the 4th booster shots.After 10 days, 2 mouse of titre highest are taken
Spleen merged with SP2/0 murine myeloma cells.Culture cell supernatant is taken to screen PSEP protein ELISAs,
Obtain to PSEP specific antibody clone strains.Tested after RPMI1640+ calf serums expand culture with chronic prostatitis syndrome urine
Card positive antibody simultaneously obtains IgG to immunoglobulin parting.Nutrient solution is expanded to 3000 milliliters, takes supernatant, is loaded onto albumen
Purified to obtain PSEP antibody purifications on A/G chromatographic columns.
2. reagent and instrument:96 hole polystyrenes check plate by U.S. Becton-Dickson and U.S. ThomasFisher
Scientific is provided；The sheep anti-mouse antibody IgG of HRP marks is purchased from Sigma companies；Cleaning fluid, developer, terminate liquid are using this
Room preparation, and compared with Shanghai Kehua Bio-technology Co., Ltd product.TMB, carbamide peroxide, polysorbas20, lemon
Lemon acid, sodium acetate, EDTA, thiosulfuric acid etc. are purchased from Chinese medicines group chemical reagent Suzhou Co., Ltd.
The automatic enzyme label washing plates of DEM- III, using Beijing Tuo Pu Analytical Instrument Co., Ltd.ELIASA（680 types）It is purchased from
BIO-RAD companies.WHS type intelligence constant humidity insulating boxs, purchased from Ningbo south of the River instrument plant.Micropipettor, using Eppendorf,
Gilson brands.
2nd, method
1.ELISA is detected：（Indirect ELISA method）
A certain proportion of PSEP protein standard substances will be diluted to and be coated in 96 orifice plates (A2-A6 holes), remaining hole adds urine
Sample, 37 DEG C of insulating boxs are incubated 1 hour, after board-washing machine washing plate 5 times, add 1%BSA to close, per 100 microlitres of hole, 37 DEG C of insulating boxs are incubated
Educate 40 minutes, board-washing 5 times.Specific PSEP detections antibody is added, 37 DEG C of insulating boxs are incubated 40 minutes, after board-washing, add HRP marks
The sheep anti-mouse igg of note, 37 DEG C of insulating boxs are incubated 20 minutes, and lucifuge develops the color 20 minutes after board-washing, adds terminate liquid, terminating reaction.
OD value of each reacting hole at double wave 450nm/630nm is determined with ELIASA.Mark is drawn according to the standard items OD values of various concentrations
Directrix curve, by the numerical value of patient compared with positive criteria hole count value, so as to draw the actual PSEP concentration of testee's urine sample.
2. autogamy cleaning fluid, developer, terminate liquid:Cleaning fluid is the PBS solution containing 0.05% Tween-20；Developer A contains
Citric acid, sodium acetate, urea peroxide；Developer B is containing citric acid, EDTA, TMB hydrochloride, 1% hypo solution；Terminate liquid
For 2mol/LH2SO4.
3. pattern detection:Prostatitis and normal person's urine sample are detected with indirect ELISA method.Examined according to clinical sample
True positives a, false positive b, false negative c, true negative d are surveyed, obtains detection sensitivity, specificity, positive desired value, negative desired value
And calculate Cutoff values.
4. methodology is identified:
4.1 analytical precision：Variation within batch：With the sample of two concentration levels（Inflammation and normal person）It is each to repeat detection 10
It is secondary, the average value M and standard deviation SD of 10 measurement concentration results are calculated, draws coefficient of variation CV.
Difference between batch：Same two parts of samples are detected respectively with the kit of 3 lot numbers, are respectively repeated 10 times, and calculate 30 results
Average value M and standard deviation SD, obtain coefficient of variation CV.
The stability experiment of 4.2 each parts：Detect elisa plate, specific antibody, enzyme labelled antibody, developer, the positive
The OD values measured after being preserved 3 days, 5 days, 7 days respectively at 37 DEG C such as standard items.And each part is put into 4 DEG C of refrigerators,
Monthly detect once, observe period of validity.
4.3 orthogonal experiment：Specific binding antibody according to 1:50、1:100 dilutions；Enzyme labelled antibody is according to 1:2000、1:
2500、1:3000、1:5000 dilutions, then first this 37 DEG C of sample-adding is incubated one hour, after board-washing closing, adds the special of various concentrations
Property antibody after, 37 DEG C are incubated 40 minutes, and the enzyme labelled antibody of various concentrations is added after board-washing 5 times, after 37 DEG C are incubated 20 minutes, board-washing
Colour developing.The concentration of optimal specific antibody and enzyme labelled antibody is selected according to OD Value Datas.
The checking of 4.4 sample incubation times：The incubation time of sample will directly affect test effect, therefore it is determined that used
The incubation time of sample is determined on the premise of antibody, reagent.The present embodiment compares different incubation times（2 hours, 1 hour,
40 minutes）Influence to testing result, so as to find out the optimal incubation time to clinical detection.
The comparison of 4.5 different developers：The present embodiment by the use of the developer of China of Shanghai section as control, prepared aobvious by detection
The color developing of toner.
The comparison of 4.6 different detection methods（ELISA double-antibody methods and indirect method）：The determination of detection method is for detection
Effect has vital effect, and antibody and reagent is being determined and detection method must be sieved after the testing time
Choosing, the present embodiment compare the influence of double-antibody method and indirect method to testing result.
5. statistical procedures：Statistical procedures are carried out using SPSS17.0 statistical softwares.Inflammatory disease patients and the sample of normal person
This comparison in difference uses rank test.P ＜ 0.05 are that difference is statistically significant.
Result of the test
First, clinical sample testing result:The indirect ELISA detection method established using this experiment, it is slow to 176 parts respectively
The property urine sample of prostatitis patient and the urine sample of 100 normal persons are detected, as a result as follows：Patients with chronic prostatitis via
Clinical symptoms, prostatitis massage liquid lecithin are reduced, and are reference without other relevant diseases.Normal population reference is that nothing is faced
Bed symptom, routine urinalysis are normal.
PSEP protein contents compare in the patients with chronic prostatitis of table 1 and control crowd
* table is noted:PSEP contents are ng/ml, according to the OD values of detection absorbance, are converted and obtained by standard curve.Meanwhile often
Individual data measure in triplicate, calculating of averaging.
PESP comparision contents in the patient of table 2 and normal population（ng/ml）
| Group | N | Mean | SD | Median |
| Case | 176 | 3.013 | 2.199 | 2.34 |
| Control | 100 | 0.734 | 0.574 | 0.605 |
* table is noted:Z=10.74P<0.05
PSEP content datas analysis display in patient and control crowd, for current data, PSEP contents are in prostate
In scorching patient and it is in Non-Gaussian Distribution in normal person.Two groups of data are compared using rank test, show patient and control
In group there is significant sex differernce in the distribution of PSEP contents, and the PSEP detected in clinical samples concentration is higher than PSEP in control group
Concentration.
According to Fig. 1, drawn from this research, if critical point is arranged to 1.17ng/ml, PSEP kits detect chronic forefront
The sensitivity of adenositis disease is up to 82.39%, and specificity is up to 87%.Area represents PSEP kits up to 0.89. under ROC regions
There is preferable diagnostic value to chronic prostatitis.
The sensitivity of table 3ROC curve difference point of cut-offs and specificity
| AreaundertheROCcurve(AUC) | 0.890 |
| StandardErrora | 0.0192 |
| 95%ConfidenceIntervalb | 0.847to0.924 |
| zstatistic | 20.276 |
| SignificancelevelP(Area=0.5) | <0.0001 |
Diagnostic criteria of the intermediate value as chronic prostatitis corresponding to selecting the maximum point of Youden indexes, draws PSEP's
Diagnostic points are 1.17ng/ml.
Table 4 is with the sensitivity of PSEP antibody test chronic prostatitis and specificity
| Criterion | Sensitivity | 95%CI | Specificity | 95%CI |
| >1.17 | 82.39 | 75.9-87.7 | 87.00 | 78.8-92.9 |
2nd, PSEP detection methods are identified
1. analytical precision：With the sample of two concentration levels（Chronic prostatitis is positive and negative）It is each to repeat detection 10
It is secondary, the average value M and standard deviation SD of 10 measurement concentration results (OD) are calculated, show that coefficient of variation CV is less than 8%.With 3 lot numbers
Kit detect same two parts of samples respectively,（It is positive and negative）Respectively it is repeated 10 times, calculates the average value M and mark of 30 results
Accurate poor SD, its coefficient of variation CV are 10.1%.It the results are shown in Table 5.From data below it can be seen that the kit reaction system have it is good
Good precision.
The precision detection of the reaction system of table 5
2. kit is in the evaluation of 37 DEG C of condition stability inferiors：The prostatitis PSEP protein detection kits that will be assembled
It is placed in 37 DEG C and carries out the experiment that accelerates the failure, the results are shown in Table 6.It was found that in 37 DEG C of insulating boxs at 7 days, its positive sample OD values are to work as
81%, P/N of its reagent preparation detection>4.Ng values such as are scaled according to each standard curve, the positive sample numerical value after 7 days
It is still the 96% of the positive sample numerical value of 0 day.From experimental result it can be seen that 37 DEG C preserve 7 days after, experimental result keeps steady substantially
It is fixed.The antigen standard being coated on elisa plate is respectively put into 37 DEG C of insulating boxs after 3 days, 5 days, 7 days, the standard of positive criteria product
Curve R2Value both greater than 0.99.There is document to point out that 37 DEG C often preserve equivalent to 4 DEG C preservations in one day 45 days, it is possible to calculate reagent
Box main component can keep stable in more months of Cord blood.
637 DEG C of destructive testing results of table
3. the stability experiment evaluation that kit preserves under the conditions of 4 DEG C:
Kept dry under the conditions of prostatitis PSEPELISA kits are further put 4 DEG C by the present embodiment, monthly periodically takes out
Inspection detected prostatitis positive sample and normal person's sample up to 12 months.It the results are shown in Table 7.The data visible 1-12 months from table,
Positive sample ng values monthly reduce without obvious, negative sample numerical stability, in below 1ng.P/N>4.7.The standard items of each moon
Curve R2Value is all more than 0.99, and very close 1.After preserving 12 months, positive sample ng values still reach normal condition
90%.Illustrate that each composition of kit can keep stable for 12 months in Cord blood.
4 DEG C of 7 kit of table preserves experiment
The selection of 4.PSEP specific antibodies and enzyme labelled antibody optimum dilution degree:
The PSEP specific antibodies and influence of the enzyme labelled antibody to testing result for selecting various concentrations are shown in Table 8.
The reaction result of the various concentrations PSEP specific antibodies of table 8 and enzyme labelled antibody
It will be seen that when enzyme labelled antibody concentration is high from table, negative sample OD values are higher, easily cause false positive
Occur；And when enzyme labelled antibody concentration is 1:During 5000 dilution proportion, positive sample numerical value is relatively low.Consider, select PSEP special
Different binding antibody 1:100 dilutions, enzyme labelled antibody 1:2500 dilutions are preferably.
5. influence of the different incubation times to testing result：
The present embodiment compares influence of three different incubation times to testing result.It was found that for positive sample.37
DEG C being incubated 2h and 1h does not have notable difference, p ＞ 0.05.37 DEG C were incubated 2h compared with 40 minutes, there is significant difference, p ＜ 0.05.It is right
For negative sample, 37 DEG C are incubated 2h compared between 1h and 40 minute, equal indifference, p ＞ 0.05.Therefore the temperature of detection kit
It is 37 DEG C of 1h to educate selection of time.
Influence of the 9 different incubation times of table to result
Incubation time
| Sample | 2h(OD) | 1h(OD) | 40’(OD) |
| P1 | 1.397 | 1.278 | 0.803 |
| P2 | 0.519 | 0.503 | 0.468 |
| P3 | 0.746 | 0.721 | 0.588 |
| P4 | 0.702 | 0.690 | 0.519 |
| P5 | 1.397 | 1.283 | 1.07 |
| P6 | 0.689 | 0.674 | 0.548 |
| N1 | 0.225 | 0.233 | 0.201 |
| N2 | 0.281 | 0.250 | 0.241 |
| N3 | 0.176 | 0.163 | 0.170 |
| N4 | 0.195 | 0.189 | 0.191 |
P：Positive (positive)；N:Negative (feminine gender)
6. the comparison of autogamy developer and Shanghai Xiamen Kehua developer：Patient that we are measured with autogamy developer and just
The OD values of ordinary person's sample are above the OD values that the Shanghai Xiamen Kehua nitrite ion of outsourcing is measured, but no difference of science of statistics between the two, P
>0.05, illustrating the nitrite ion of autogamy has good color developing effect.
The comparison (OD) of the autogamy developer of table 10 and Shanghai Xiamen Kehua developer
The distribution of PSEP contents is present in prostatitis patient and the normal human urine of control group as can be seen from the above results
Significant sex differernce, the concentration of the PSEP detected in clinical samples concentration apparently higher than PSEP in control group.PSEP excretion eggs
White detection can provide a reliable basis for the scorching diagnosis of clinical prostate.The kit is ruined by strict 37 DEG C simultaneously
Preservation for a long time is tested under the conditions of damaging experiment and 4 DEG C, testing result stabilization, meets the in-vitro diagnosis examination of state food drug surveilance office
Agent appraisal standards.
Chronic pelvic pain syndrome/chronic prostatitis (CPPS/CP) is one of medical principal disease of urological department male,
Even more influence the common disease of Lipid on Young-middle Male health.They are a kind of syndromes of inhomogeneity, are defined as " past 6 months
In at least 3 months pelvis areas are uncomfortable or pain, subsidiary uropoiesis/reproductive system functional disturbance " and differentiate any
Cancer, infection and anatomic abnormalities pathology exist.They also frequently result in passive cognition, behavior, property, or Hypoemotivity.Separately
Outside, the research of cancer gene group landscape convincingly proves that promotion sensitivity gene mutation has begun to class set from one's youth.It is chronic
Pelvic pain syndrome/chronic prostatitis is common in 20~50 years old male, can just embody carcinoma of prostate symptom later for many years.
According in prostate cancer, after the presence and cell epithelia tissue damage of inflammation regeneration be considered as vicious transformation it is crucial because
Element.Obviously, early stage diagnosis and treatment chronic pelvic pain syndrome/chronic prostatitis has great meaning to reducing or preventing and treating prostate cancer
Justice.
At present, chronic pelvic pain syndrome/Diagnosis of Chronic Prostatitis is based primarily upon clinical experience and symptom is rejected.Face
There is no any simple and feasible molecule or immunization method to be used for guidance or auxiliary diagnosis in bed detection, cause clinical diagnosis master
See, inaccurate and mistaken diagnosis.This not only further results in treatment difficulty, also unfavorable searching treatment new drug development.So present invention
Urine detection kit based on PSEP is that the pioneering chronic pelvic pain syndrome/chronic prostatitis molecular immune in the whole world is examined
Disconnected kit, the precision detection of reacted system, stability test, its reagent quality of destructive test all show that height can be criticized
Amount production.Its application is real Noninvasive, easily operated, easy, and has high sensitivity and specificity.It will not only
Mitigate patient's pain due to chronic pelvic pain syndrome/chronic prostatitis, and early stage diagnosis and treatment will further be strengthened
Preventing and treating to prostate cancer, is with a wide range of applications.
Although the present invention is described in detail with most preferred embodiment above, those skilled in the art are not it is to be understood that inclined
On the premise of from inventive concept and spirit, any improvement and modification made to the present invention, still fall within claimed
Within the scope of.
Claims (10)
- The proteantigen 1. a kind of Prostasomes leak, it is characterised in that be prepared with the following method：500 milliliters of chronic prostatitis syndrome patient's urina sanguinis midstream urines are taken, add equivalent pH7.3 HEPES buffer solution and protease Inhibitor, use filtered through gauze；Filtered fluid is taken to be respectively placed in 10 50 milliliters of centrifuge tubes, 400g centrifuges 10 points under 4 °C of low temperature Clock, go to precipitate, take supernatant 10,000g under 4 °C of low temperature to centrifuge 20 minutes, go to precipitate, take supernatant to be respectively placed in 200 3 Milliliter ultracentrifugation pipe, 100,000g is centrifuged 120 minutes under 4 °C of low temperature；Supernatant is removed, takes precipitation to add 2.5 milliliters PH7.8 TBS buffer solutions and protease inhibitors, mix；100,000g is centrifuged 120 minutes under 4 °C of low temperature, removes supernatant Liquid, precipitation is taken to be dissolved in 2.5 milliliter of 0.5 % NP-40,80mM the mM Tris of NaCl, 0.2% Deoxycholate, 50 respectively, PH 8.0 and protease inhibitors；100,000g is centrifuged 20 minutes under 4 °C of low temperature；Take supernatant, loading TSKgel G3000PW, 280 nanometers of readings, take in 40kDa plots peak albumen, -80 °C of freezen protectives, produce.
- The preparation method of proteantigen 2. a kind of Prostasomes leak, it is characterised in that500 milliliters of chronic prostatitis syndrome patient's urina sanguinis midstream urines are taken, add equivalent pH7.3 HEPES buffer solution and protease Inhibitor, use filtered through gauze；Filtered fluid is taken to be respectively placed in 10 50 milliliters of centrifuge tubes, 400g centrifuges 10 points under 4 °C of low temperature Clock, go to precipitate, take supernatant 10,000g under 4 °C of low temperature to centrifuge 20 minutes, go to precipitate, take supernatant to be respectively placed in 200 3 Milliliter ultracentrifugation pipe, 100,000g is centrifuged 120 minutes under 4 °C of low temperature；Supernatant is removed, takes precipitation to add 2.5 milliliters PH7.8 TBS buffer solutions and protease inhibitors, mix；100,000g is centrifuged 120 minutes under 4 °C of low temperature, removes supernatant Liquid, precipitation is taken to be dissolved in 2.5 milliliter of 0.5 % NP-40,80mM the mM Tris of NaCl, 0.2% Deoxycholate, 50 respectively, PH 8.0 and protease inhibitors；100,000g is centrifuged 20 minutes under 4 °C of low temperature；Take supernatant, loading TSKgel G3000PW, 280 nanometers of readings, take in 40kDa plots peak albumen, -80 °C of freezen protectives, produce.
- A kind of 3. antibody corresponding with antigen-specific described in claim 1.
- 4. antibody described in antigen described in claim 1 and claim 3 is preparing detection or the prostatitic reagent of diagnosing chronic Application in box.
- 5. application according to claim 4, it is characterised in that：Described kit uses ELISA method or collaurum legal system It is standby to form.
- 6. application according to claim 5, it is characterised in that：Described kit includes to leak egg for Prostasomes Bai Kangyuan monoclonal detection antibody, coating buffer solution, lavation buffer solution, Block buffer, enzyme labelled antibody.
- 7. application according to claim 6, it is characterised in that：Characterized in that, Prostasomes leak, proteantigen is examined It is that monoclonal mouse anti-human's Prostasomes leak the antibody of proteantigen to survey antibody.
- 8. application according to claim 6, it is characterised in that：Characterized in that, also include standard items and negative control；Institute It is that the Protein Detection antibody that leaked to Prostasomes produces the human prostate corpusculum of immune response and leaked proteantigen to state standard items.
- 9. application according to claim 6, it is characterised in that：The coating buffer solution is carbonate or dPBS；It is described to wash It is the dPBS containing detergent to wash buffer solution.
- 10. kit according to claim 6, it is characterised in that the detection antibody identification Prostasomes leak egg Bai Kangyuan.
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| CN110456044B (en) * | 2019-08-20 | 2021-07-23 | 郑州安图生物工程股份有限公司 | Kit for prostatitis detection |
| CN112574302B (en) * | 2019-09-29 | 2022-05-06 | 昂科生物医学技术(苏州)有限公司 | Hybridoma cell LCZ8A3, monoclonal antibody secreted by hybridoma cell LCZ8A3 and application of monoclonal antibody |
| CN112574955B (en) * | 2019-09-29 | 2022-10-25 | 昂科生物医学技术(苏州)有限公司 | Hybridoma cell LCZ9H4, monoclonal antibody secreted by same and application of monoclonal antibody |
| CN115184616A (en) * | 2022-06-23 | 2022-10-14 | 昂科生物医学技术(苏州)有限公司 | Application of prostasome exosmosis protein antigen and antibody thereof in preparation of benign prostatic hyperplasia diagnostic kit |
| CN115128280B (en) * | 2022-06-28 | 2023-12-05 | 昂科生物医学技术(苏州)有限公司 | Fluorescent chromatography test strip and kit for detecting prostatic small body leakage protein, and preparation method and application thereof |
| CN115420893A (en) * | 2022-07-11 | 2022-12-02 | 昂科生物医学技术(苏州)有限公司 | PSEP colloidal gold test paper, kit, preparation method and application thereof |
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