CN104897900A - Prostateexosomal protein antigen, antibody and application thereof - Google Patents
Prostateexosomal protein antigen, antibody and application thereof Download PDFInfo
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Abstract
The invention relates to a prostateexosomal protein (PESP) antigen, a specific corresponding antibody and application thereof, and on the basis further acquires a kit based on prostasome leakage protein quantitative indirect enzyme-linked immunoadsorption so as to effectively detect and diagnose chronic prostatitis. By means of reaction system precision detection, stability test and destructive test on the urine detection kit based on PESP antibody-antigen reaction, the reagent quality shows high batch production probability. The application is truly noninvasive, the operation is simple, and the kit has high sensitivity and specificity. The kit can alleviate the patient pain caused by chronic pelvic pain syndrome/chronic prostatitis, and also early diagnosis can further strengthen prevention and treatment of prostate cancer, therefore the kit has wide application prospect.
Description
Technical field
The present invention relates to a kind of Prostasomes to leak proteantigen, be specifically related to this Prostasomes a kind of and leak proteantigen and preparation method thereof and application.
Background technology
Chronic prostatitis (ChronicProstatitis, CP) is one of modal disease of Urology Surgery Lipid on Young-middle Male, it is reported that it accounts for 30% of Number of Outpatients.According to epidemiology survey, in China adult human male crowd, more than 10% has prostatitis sample symptom, and 50% male sex's different times all suffered from prostatitis.Potential chronic prostatitis or chronic pelvic pain (CPPS) patients pool are described.Meanwhile, important factor in the inflammatory process caused by chemicals, physical factor or biopreparate or the pathogenesis of human cancer.In prostate cancer, after the existence of inflammation and cell epithelia tissue damage, regenerate the key factor (DeMarzoetal., 2007) being considered to vicious transformation.Prostata tissue is subject to the infiltration of inflammatory cell, thus discharges various active substance, and the release of these materials is also increased with bacterium and virus infections, uric acid and to eat prostate carcinogenic substance relevant.In the past in 10 years, the contact between inflammation and cancer, and prostatitis has been studied fully as the hypothesis of prostate cancer high risk factor and has been confirmed on epidemiology, clinical, morphology, aetology, cell and molecular level.In fact, point out in document, about more than 12% prostatitis, as treatment can be converted into prostate cancer not in time.So prostatitis has a strong impact on men's health (the refined etal. of Chen Zhi, 2009; That man of virtue and ability group etal., 2011).
Prostatitis clinical manifestation lacks specificity because of patient individual difference, and therefore result for the treatment of is unsatisfied with (Kriegeretal., 1999 always clinically; Engeleretal., 2012; That man of virtue and ability group etal., 2011).According to 1997 NIH (NIH) to prostatitic classification, i.e. NIH classification (Kriegeretal., 1999; The refined etal. of Chen Zhi, 2009), comprise I type: acute bacterial prostatitis (ABP); II type: chronic bacterial prostatitis (CBP); Type III: be divided into IIIA again, chronic non-bacterial prostatitis (CAP) and IIIB, chronic pelvic pain syndrome (CPPS); IV type: asymptomatic struvite prostatitis (AIP).In clinical practice, usually common with III type chronic prostatitis, account for about 90%, so chronic prostatitis mainly refers to type III chronic prostatitis.In clinical practice work, first 3 of the inspection method (multiselect) that CAP and the CPPS patient that uropoiesis andrology doctor examines head selects is routine urinalysis (80.3%), massage of prostate fluid inspection (71.0%), health check-up (comprising digital rectal examination) (63.2%).Massage of prostate fluid inspection and digital rectal examination have certain invasive to patient, and great majority diagnosis still depends on Clinical signs and doctors experience, is therefore starved of easy without invasive, while molecular diagnosis method accurately and reliably.
China application CN102998455A discloses the kit of a kind of detection or diagnosing prostate cancer, and it comprises δ-catenin adsorb antibodies, bag is buffered liquid, lavation buffer solution, Block buffer, δ-catenin detect antibody, enzyme labelled antibody.Or comprise δ-catenin adsorb antibodies, bag be buffered liquid, lavation buffer solution, Block buffer, δ-catenin detect antibody, nano-quantum point mark.Described kit can detect by ELISA method the δ-catenin albumen becoming high correlation with prostate cancer from patient urine, and ELISA method is ripe, Sensitivity and Specificity is higher, is noninvasive to patient, can repeat to take, patient takes like a shot.Nano-quantum point (QD) labelling method sampling amount is little, is combined with QD by antibody used in ELISA method, can detect the change of δ-catenin albumen in urine more delicately, thus for diagnosing prostate cancer.But this kit is only applicable to prostate cancer and detects, and can not distinguish chronic prostatitis syndrome patient and normal person's sample very well, therefore be not suitable for prostatitis detection.
Summary of the invention
Namely the first object of the present invention is to provide a kind of and can be used for Prostasomes that prostatitis detects and to leak proteantigen.
Prostasomes of the present invention leaks proteantigen, adopts and is prepared from the following method:
Get 500 milliliters of chronic prostatitis syndrome patient urina sanguinis midstream urines, add HEPES damping fluid and the protease inhibitors of equivalent pH7.3, by filtered through gauze; Get filtered fluid and be placed in 10 50 milliliters of centrifuge tubes respectively, centrifugal 10 minutes of 400g under 4 DEG C of low temperature, go precipitation, get supernatant under 4 DEG C of low temperature 10,000g centrifugal 20 minutes, go precipitation, get supernatant and be placed in 200 3 milliliters of ultracentrifugation pipes respectively, under 4 DEG C of low temperature 100,000g centrifugal 120 minutes; Remove supernatant, get TBS damping fluid and protease inhibitors that precipitation adds 2.5 milliliters of pH7.8, mixing; Under 4 DEG C of low temperature 100,000g centrifugal 120 minutes, remove supernatant, get precipitation and be dissolved in 2.5 milliliters of 0.5%NP-40,80mMNaCl, 0.2%Deoxycholate, 50mMTris, pH8.0 and protease inhibitors respectively; Under 4 DEG C of low temperature 100,000g centrifugal 20 minutes; Get supernatant, loading TSKgelG3000PW, 280 nanometer readings, be taken at 40kDa plots peak albumen ,-80 DEG C of freezen protective, to obtain final product.
Meanwhile, the present invention protects above-mentioned Prostasomes further and to leak the preparation method of proteantigen:
Get 500 milliliters of chronic prostatitis syndrome patient urina sanguinis midstream urines, add HEPES damping fluid and the protease inhibitors of equivalent pH7.3, by filtered through gauze; Get filtered fluid and be placed in 10 50 milliliters of centrifuge tubes respectively, centrifugal 10 minutes of 400g under 4 DEG C of low temperature, go precipitation, get supernatant under 4 DEG C of low temperature 10,000g centrifugal 20 minutes, go precipitation, get supernatant and be placed in 200 3 milliliters of ultracentrifugation pipes respectively, under 4 DEG C of low temperature 100,000g centrifugal 120 minutes; Remove supernatant, get TBS damping fluid and protease inhibitors that precipitation adds 2.5 milliliters of pH7.8, mixing; Under 4 DEG C of low temperature 100,000g centrifugal 120 minutes, remove supernatant, get precipitation and be dissolved in 2.5 milliliters of 0.5%NP-40,80mMNaCl, 0.2%Deoxycholate, 50mMTris, pH8.0 and protease inhibitors respectively; Under 4 DEG C of low temperature 100,000g centrifugal 20 minutes; Get supernatant, loading TSKgelG3000PW, 280 nanometer readings, be taken at 40kDa plots peak albumen ,-80 DEG C of freezen protective, to obtain final product.
Above-mentioned preparation method can to leak proteantigen by the Prostasomes albumen described in rapid batch preparation, and described method is stable can be reproduced, easy to utilize.
Another object of the present invention is to protect the antibody corresponding with above-mentioned antigen-specific.
Another object of the present invention is to protect above-mentioned antigen and the application of antibody in the kit preparing detection or diagnosing prostate cancer.
Mentioned reagent box can adopt multiple preparation method disclosed in prior art to be prepared from, those skilled in the art can know, when obtain Prostasomes of the present invention leak proteantigen and antibody thereof, based on this special antigen and antibody, Prostasomes is leaked albumen detection effect accurately, no matter adopt which kind of preparation method, the kit that can be used for detection or diagnosing prostate cancer can be obtained.
The kit employing ELISA method that the present invention is preferably described or colloidal gold method are prepared from.
Kit of the present invention comprise for Prostasomes leak proteantigen monoclonal detect antibody, bag be buffered liquid, lavation buffer solution, Block buffer, enzyme labelled antibody.Specifically consist of those skilled in the art understood, the present invention is not particularly limited this.
Wherein, Prostasomes of the present invention leaks, and to detect antibody be that monoclonal mouse anti-human's Prostasomes leaks the antibody of proteantigen to proteantigen.
Further, this kit also comprises standard items and negative control; Described standard items to leak proteantigen for the Protein Detection antibody that leaks to Prostasomes produces immunoreactive human prostate corpusculum.
Wherein, described kit bag is buffered liquid is carbonate or dPBS; Described lavation buffer solution is the dPBS containing washing agent.
Described detection antibody recognition Prostasomes leaks proteantigen.
Kit of the present invention, during use, concrete flow process is as follows:
1, prepare:
1. from 2-8 DEG C of cold storage environment, kit is taken out, BSA pulvis is poured into premade closure liquid in PBS damping fluid bottle (sucks PBS damping fluid in BSA pulvis pipe with pipettor more if desired after turned letter pulvis, pour in PBS damping fluid bottle after shaking up again, in order to avoid pulvis sticks on tube wall), fully mix.
2. by 20X concentrated cleaning solutions 50ml(two bottles) shake up rear pour into wash in the bottle for handling liquid toilet or cosmetic substance of trigger completely (dissolve under 37 DEG C of conditions can being placed in if any precipitating crystalline), then pour purified water (resistivity is at more than 15M Ω) into, be settled to 1000ml, fully mix.
2, number: from aluminium foil bag, take out microwell plate.A1 is blank well, and A2-A6 hole bag is by 5 working concentration: 0.1ng of positive criteria product; 1ng; 2ng; 5ng; 10ng.All the other each hole serial numbers as required.
3, application of sample: except positive criteria hole and blank well, all the other add urine sample (patting microwell plate edge to have prevented bubble) by number respectively in corresponding hole.The every Kong Jiayi of 90 person-portions/box do not carry out adding urine sample by inspection people urine sample 100 μ l(blank control wells, close, add primary antibodie, add two operations such as anti-, and respectively step operation is identical for all the other).With after shrouding film shrouding, microwell plate being put into 37 DEG C of constant temperature ovens (or water bath) one hour.
4, clean: wash 5 times (optionally each every hole cleaning fluid 300 μ about l of setting with cleaning fluid washing on trigger, soak time 5 second), washed plate is taken out, pat on paper handkerchief if desired, to remove raffinate in hole (comprising all clappers below all can not prop up at the bottom of plate with finger, to prevent from affecting last mensuration effect).
5, close: except blank well, in each hole (containing positive gauge orifice, not containing blank control wells), add the prefabricated confining liquid of 100 μ l, with after shrouding film shrouding, microwell plate being put into 37 DEG C of constant temperature ovens (or water bath) 40 minutes.
6, clean: wash 5 times (optionally each every hole cleaning fluid 300 μ about the l of setting, soak time 5 second) with cleaning fluid washing on trigger, washed plate is taken out, pats on paper handkerchief if desired, to remove raffinate in hole.
7, PSEP antibody is added: pat microwell plate edge to have prevented pore to adding PSEP antibody 50 μ l(in each hole (containing positive gauge orifice, not containing blank control wells)).With after shrouding film shrouding, microwell plate being put into 37 DEG C of constant temperature ovens (or water bath) 40 minutes.
8, clean: wash 5 times (optionally each every hole cleaning fluid 300 μ about the l of setting, soak time 5 second) with cleaning fluid washing on trigger, washed plate is taken out, pats on paper handkerchief if desired, to remove raffinate in hole.
9, enzyme labelled antibody is added: pat microwell plate edge to have prevented pore to adding enzyme labelled antibody 100 μ l(in each hole (containing positive gauge orifice, not containing blank control wells)).With after shrouding film shrouding, microwell plate being put into 37 DEG C of constant temperature ovens (or water bath) 20 minutes.
10, clean: wash 5 times (optionally each every hole cleaning fluid 300 μ about the l of setting, soak time 5 second) with cleaning fluid washing on trigger, washed plate is taken out, pats on paper handkerchief if desired, to remove raffinate in hole.
11, develop the color: inject 50 μ l nitrite ion A and 50 μ l nitrite ion B(to every hole (containing positive gauge orifice and blank well) and pat microwell plate edge and make it mix and prevented pore), its normal temperature lucifuge is placed 20 minutes.
12, stop: inject 50 μ l stop buffers (patting microwell plate edge make it mix and prevented pore) to every hole (containing positive gauge orifice and blank well).
13, measure: microwell plate is put into enzyme non-analysis meter, use dual wavelength 450nm/630nm measure each hole OD value and record result.
14, result judges: according to PSEP antibody test feature, PSEP positive findings: assess tested sample with typical curve, concentration value > 1.20ng/ml;
PSEP negative findings: assess tested sample with typical curve, concentration value≤1.20ng/ml; It is 0.10 invalid that OD value is less than.
The invention provides a kind of Prostasomes leak proteantigen and with its special corresponding antibody, and the kit of Immunofluorescent antibody of the protein quantification that can obtain further leaking based on Prostasomes, to be effective to diagnosis and detection chronic prostatitis.Prostasomes (prostasomes) is the ultrastructure of diameter about 30 ~ 150 nanometer be present in prostatic fluid, (exocytosis) is told or leak (excretion) is formed by prostatic epithelium or interstitial cell etc. are outer, corpusculum (exosome) structure is told with outer in its hetero-organization, size, composition is similar but not identical.
Although tell approach outer at present, signal transduction mechanism is understood very few, and Recent study finds, there is nucleic acid, albumen, carbohydrate in Prostasomes, fat, small molecule metabolites etc.The Prostasomes albumen (its called after prostateexosomal protein, or be called for short PSEP) that leaks is very many, identifies (Lietal2008 not yet completely at present; Luetal., 2009; ).The application is antigen from the Prostasomes that Patients with Chronic Prostatitis is separated, screen, obtaining special antibody, is to leak the humanized murine antibodies of albumen (PSEP) for anti-Prostasomes, for studying its sensitivity detected for chronic prostatitis and specificity further.
Current diagnosis prostatitis EPS method used, use massage of prostate liquid, very large infringement and discomfort is had to patient, and it is reliably easy to the present invention is based on the special Prostasomes detection method that proteantigen provides that leaks, sensitivity reaches 85%, a kind ofly can be used for the Noninvasive of a kind of novelty of Diagnosis of Chronic Prostatitis, painless molecule measuring means.
Accompanying drawing explanation
Fig. 1 is the evaluation to PSEP kit Test of accuracy (ROC tracing analysis), and wherein horizontal ordinate is specificity, and ordinate is sensitivity.
Embodiment
To give an actual example description to the principle of the invention and feature below, example, only for explaining the present invention, is not intended to limit scope of the present invention.
Embodiment 1
Prostasomes leaks the preparation of proteantigen (PSEP): get 500 milliliters of chronic prostatitis syndrome patient urina sanguinis midstream urines, add equivalent HEPES damping fluid (pH7.3) and protease inhibitors (Sigma Co., USA),, use filtered through gauze.Get filtered fluid, be placed in 10 50 milliliters of centrifuge tubes respectively, centrifugal 10 minutes of 400g under 4 DEG C of low temperature.Go precipitation, get supernatant.Under 4 DEG C of low temperature 10,000g centrifugal 20 minutes.Go precipitation, get supernatant.Be placed in 200 3 milliliters of ultracentrifugation pipes respectively, under 4 DEG C of low temperature 100,000g centrifugal 120 minutes.Remove supernatant, get precipitation.Add 2.5 milliliters of TBS damping fluids (pH7.8) and protease inhibitors (Sigma Co., USA), mixing.Under 4 DEG C of low temperature 100,000g centrifugal 120 minutes.Remove supernatant, get precipitation.Be dissolved in 2.5 milliliters of 0.5%NP-40,80mMNaCl, 0.2%Deoxycholate, 50mMTris, pH8.0 and protease inhibitors (Sigma Co., USA) respectively.Under 4 DEG C of low temperature 100,000g centrifugal 20 minutes.Get supernatant, loading TSKgelG3000PW (Sigma Co., USA), 280 nanometer readings, be taken at 40kDa plots peak albumen.-80 DEG C of freezen protective.
Embodiment 2
Described in the present embodiment, kit is composed as follows: the mud-stream urine of prostatitis patient; Confining liquid; Prostasomes leaks proteantigen (PSEP); PSEP detects antibody; Cleaning fluid; Developer; Stop buffer.
Utilize the concrete grammar of mentioned reagent box to prostatitic detection or diagnosis as follows:
1. Specimen origin: according to prostatitic diagnostic criteria (that man of virtue and ability group etal. in " Chinese urological diseases diagnoses and treatment companion ", 2011) collect the mud-stream urine 213 parts of prostatitis patient, select chronic prostatitis 176 example meeting Comprehensive Clinical diagnosis completely.Age 16-72 year, 34 years old mean age.Normal person's health check-up urine 100 parts, age 20-68 year.34 years old mean age.Routine urinalysis is all normal.Urine detects after collecting immediately, or puts-20 DEG C of Refrigerator stores.
2. Prostasomes leaks the preparation of proteantigen (PSEP): get 500 milliliters of chronic prostatitis syndrome patient urina sanguinis midstream urines, add equivalent HEPES damping fluid (pH7.3) and protease inhibitors (Sigma Co., USA),, use filtered through gauze.Get filtered fluid, be placed in 10 50 milliliters of centrifuge tubes respectively, centrifugal 10 minutes of 400g under 4 DEG C of low temperature.Go precipitation, get supernatant.Under 4 DEG C of low temperature 10,000g centrifugal 20 minutes.Go precipitation, get supernatant.Be placed in 200 3 milliliters of ultracentrifugation pipes respectively, under 4 DEG C of low temperature 100,000g centrifugal 120 minutes.Remove supernatant, get precipitation.Add 2.5 milliliters of TBS damping fluids (pH7.8) and protease inhibitors (Sigma Co., USA), mixing.Under 4 DEG C of low temperature 100,000g centrifugal 120 minutes.Remove supernatant, get precipitation.Be dissolved in 2.5 milliliters of 0.5%NP-40,80mMNaCl, 0.2%Deoxycholate, 50mMTris, pH8.0 and protease inhibitors (Sigma Co., USA) respectively.Under 4 DEG C of low temperature 100,000g centrifugal 20 minutes.Get supernatant, loading TSKgelG3000PW (Sigma Co., USA), 280 nanometer readings, be taken at 40kDa plots peak albumen.-80 DEG C of freezen protective.
3.PSEP detects the preparation of antibody: thaw PSEP albumen, by BCA (U.S. ThomasFisherScientific) company kit measurement concentration.Get 6 BALB/c mouse, every only first time injection 50 micrograms.After 2 weeks, again inject.After 10 days, get mouse blood, make ELISA titer determination with 96 orifice plates of PSEP albumen 50 nanogram bed board.Then third time injecting immune is made.After 2 weeks, titre reaches 1:10, and more than 000, do the 4th booster shots.After 10 days, spleen and the SP2/0 murine myeloma cell of getting 2 the highest mouse of titre merge.Get cultured cell supernatant to screen PSEP protein ELISA, obtain PSEP specific antibody clone strain.Expand at RPMI1640+ calf serum and cultivate rear chronic prostatitis syndrome urine checking positive antibody and IgG is obtained to immunoglobulin (Ig) somatotype.Nutrient solution expands 3000 milliliters to, gets supernatant, is loaded onto albumin A/G chromatographic column carries out purifying to obtain PSEP antibody purification.
2. reagent and instrument: 96 hole polystyrene inspection plates are provided by U.S. Becton-Dickson and U.S. ThomasFisher Scientific; The sheep anti-mouse antibody IgG available from Sigma of HRP mark; Cleaning fluid, developer, stop buffer adopt this room preparation, and compare with Shanghai Kehua Bio-technology Co., Ltd product.TMB, carbamide peroxide, polysorbas20, citric acid, sodium acetate, EDTA, thiosulfuric acid etc. are all purchased from traditional Chinese medicines group chemical reagent Suzhou company limited.
DEM-III automatic enzyme label washing plate, adopts Beijing Tuo Pu Analytical Instrument Co., Ltd.Microplate reader (680 type) is purchased from BIO-RAD company.WHS type intelligence constant humidity constant temperature oven, purchased from the south of the River, Ningbo instrument plant.Micropipettor, adopts Eppendorf, Gilson brand.
Two, method
1.ELISA detects: (indirect ELISA method)
Dilution is coated on (A2-A6 hole) in 96 orifice plates for a certain proportion of PSEP protein standard substance, and all the other holes add urine specimen, and 37 DEG C of constant temperature ovens hatch 1 hour, wash after trigger washes plate 5 times, add 1%BSA and close, every hole 100 microlitre, 37 DEG C of constant temperature ovens hatch 40 minutes, wash plate 5 times.Add specificity PSEP and detect antibody, 37 DEG C of constant temperature ovens hatch 40 minutes, after washing plate, and add the sheep anti-mouse igg of HRP mark, 37 DEG C of constant temperature ovens hatch 20 minutes, and after washing plate, lucifuge develops the color 20 minutes, adds stop buffer, cessation reaction.The OD value of each reacting hole at double wave 450nm/630nm place is measured by microplate reader.Standard items OD value according to variable concentrations draws typical curve, is compared by the numerical value of patient with positive criteria hole count value, thus draws the actual PSEP concentration of testee's urine sample.
2. autogamy cleaning fluid, developer, stop buffer: cleaning fluid is the PBS solution containing 0.05% Tween-20; Developer A is containing citric acid, sodium acetate, urea peroxide; Developer B is containing citric acid, EDTA, TMB hydrochloride, 1% hypo solution; Stop buffer is 2mol/LH2SO4.
3. pattern detection: detect prostatitis and normal person's urine sample with indirect ELISA method.Detect true positives a, false positive b, false negative c, true negative d according to clinical sample, obtain detection sensitivity, specificity, positive desired value, negative desired value calculate Cutoff value.
4. methodology qualification:
4.1 analytical precisions: variation within batch: by sample (inflammation and normal person) each duplicate detection 10 times of two concentration levels, calculate the mean value M and the standard deviation SD that measure concentration results for 10 times, draw coefficient of variation CV.
Difference between batch: detect same two parts of samples respectively with the kit of 3 lot numbers, each repetition 10 times, calculates mean value M and the standard deviation SD of 30 results, obtains coefficient of variation CV.
The stability experiment of 4.2 each ingredients: detect elisa plate, specific antibody, enzyme labelled antibody, developer, positive criteria product etc. 37 DEG C of OD values that preservation is measured after 3 days, 5 days, 7 days respectively.And each ingredient is put into 4 DEG C of refrigerators, monthly detect once, observe valid period.
4.3 orthogonal experiments: specific binding antibody is diluted according to 1:50,1:100; Enzyme labelled antibody dilutes according to 1:2000,1:2500,1:3000,1:5000, then first add 37 DEG C, sample and hatch one hour, wash plate close after, after adding the specific antibody of variable concentrations, hatch 40 minutes for 37 DEG C, add the enzyme labelled antibody of variable concentrations after washing plate 5 times, 37 DEG C hatch 20 minutes after, wash plate colour developing.Best specific antibody and the working concentration of enzyme labelled antibody is selected according to OD Value Data.
The checking of 4.4 sample incubation times: the incubation time of sample will directly affect test effect, therefore measure the incubation time of sample under the prerequisite determining antibody used, reagent.The present embodiment compares different incubation time (2 hours, 1 hour, the 40 minutes) impacts on testing result, thus finds out the best incubation time to clinical detection.
The comparison of 4.5 different developers: the developer of the present embodiment Shanghai China of section in contrast, detects the color developing of the developer prepared.
The comparison (ELISA double-antibody method and indirect method) of 4.6 different detection methods: the determination of detection method has vital effect for Detection results, determining antibody and reagent and must screen detection method after the test duration, the present embodiment compares double-antibody method and indirect method to the impact of testing result.
5. statistical procedures: adopt SPSS17.0 statistical software to carry out statistical procedures.Inflammatory disease patients compares employing rank test with the differences between samples of normal person.P < 0.05 has statistical significance for difference.
Test findings
One, clinical sample testing result: apply the indirect ELISA detection method that this test is set up, respectively the urine sample of 176 parts of patients with chronic prostatitis and the urine sample of 100 routine normal persons are detected, result is as follows: patients with chronic prostatitis is via clinical symptoms, prostatitis massage liquid lecithin reduces, and is reference without other relevant diseases.Normal population reference is that routine urinalysis is normal without clinical symptoms.
PSEP protein content contrast in table 1 patients with chronic prostatitis and contrast crowd
* show note: PSEP content is ng/ml, according to the OD value detecting absorbance, converted by typical curve and obtain.Meanwhile, each Data duplication is measured for three times, calculating of averaging.
PESP comparision contents (ng/ml) in table 2 patient and normal population
| Group | N | Mean | SD | Median |
| Case | 176 | 3.013 | 2.199 | 2.34 |
| Control | 100 | 0.734 | 0.574 | 0.605 |
* note is shown: Z=10.74P<0.05
PSEP content data analysis display in patient and contrast crowd, with regard to current data, PSEP content in prostatitis patient and in normal person in skewed distribution.Adopt rank test to be compared two groups of data, in display patient and control group, the distribution of PSEP content exists and shows sex differernce, and the concentration of the PSEP detected in clinical samples is higher than the concentration of PSEP in control group.
According to Fig. 1, draw from this research, if critical point is set to 1.17ng/ml, the sensitivity that PSEP kit detects chronic prostatitis syndrome can reach 82.39%, and specificity can reach 87%.Under ROC region, area can reach 0.89. and represents that PSEP kit has good diagnostic value to chronic prostatitis.
The sensitivity of the different truncation points of table 3ROC curve and specificity
| AreaundertheROCcurve(AUC) | 0.890 |
| StandardError a | 0.0192 |
| 95%ConfidenceInterval b | 0.847to0.924 |
| zstatistic | 20.276 |
| SignificancelevelP(Area=0.5) | <0.0001 |
The intermediate value of the some correspondence selecting Youden index maximum, as the diagnostic criteria of chronic prostatitis, show that the diagnostic points of PSEP is 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 |
Two, PSEP detection side science of law qualification
1. analytical precision: by the sample of two concentration levels (chronic prostatitis is positive and negative) each duplicate detection 10 times, calculates mean value M and the standard deviation SD of 10 measurements concentration results (OD), show that coefficient of variation CV is less than 8%.Detect same two parts of samples respectively with the kit of 3 lot numbers, (positive and negative) is each repeats 10 times, and mean value M and standard deviation SD, its coefficient of variation CV of calculating 30 results are 10.1%.The results are shown in Table 5.Can find out that this kit reaction system has good precision from following data.
The precision of table 5 reaction system detects
2. kit is in the evaluation of 37 DEG C of condition stability inferiors: the prostatitis PSEP protein detection kit assembled is placed in 37 DEG C and carries out the test that accelerates the failure, the results are shown in Table 6.Find in 37 DEG C of constant temperature ovens when 7 days, its positive sample OD value is 81%, P/N>4 of reagent preparation detection on the same day.As being scaled ng value according to each typical curve, the positive sample numerical value after 7 days is still 96% of the positive sample numerical value of 0 day.Can find out that 37 DEG C of preservations are after 7 days from experimental result, experimental result kept stable.The antigen standard be coated on elisa plate puts into 37 DEG C of constant temperature ovens after 3 days, 5 days, 7 days respectively, the typical curve R of positive criteria product
2value is all greater than 0.99.Have document point out 37 DEG C often preserve within one day, be equivalent to 4 DEG C preserve 45 days, so can calculate kit principal ingredient can keep Cord blood many months stablize.
Table 637 DEG C destructive test result
3. the kit stability experiment evaluation of preserving under 4 DEG C of conditions:
Prostatitis PSEPELISA kit is put kept dry under 4 DEG C of conditions by the present embodiment further, and monthly regularly sampling observation is until 12 months, detects prostatitis positive sample and normal person's sample.The results are shown in Table 7.The data visible 1-12 month from table, positive sample ng value is monthly without obviously reducing, and negative sample numerical stability, all at below 1ng.P/N>4.7。The standard items curve R of each moon
2value all more than 0.99, closely 1.In preservation after 12 months, positive sample ng value still reaches 90% of normal condition.Illustrate that each composition of kit can keep stable for 12 months at Cord blood.
Table 7 kit 4 DEG C preserves experiment
The selection of 4.PSEP specific antibodies and enzyme labelled antibody optimum dilution degree:
Select the PSEP specific antibodies of variable concentrations and enzyme labelled antibody to the impact of testing result in table 8.
The reaction result of table 8 variable concentrations PSEP specific antibodies and enzyme labelled antibody
As can be seen from table in we, when enzyme labelled antibody concentration height, negative sample OD value is higher, easily causes false positive to occur; And when enzyme labelled antibody concentration is in 1:5000 dilution proportion, positive sample numerical value is on the low side.Consider, select PSEP specific antibodies 1:100 dilution, enzyme labelled antibody 1:2500 dilutes as well.
5. different incubation time is on the impact of testing result:
The present embodiment compares three different incubation times to the impact of testing result.Find positive sample.Hatch 2h and 1h for 37 DEG C and there is no notable difference, p > 0.05.Hatch 2h compared with 40 minutes, have and show difference, p < 0.05 for 37 DEG C.Concerning negative sample, hatch between 2h and 1h and 40 minute for 37 DEG C and compare, equal indifference, p > 0.05.Therefore the incubative time of detection kit is chosen as 37 DEG C of 1h.
The different incubation time of table 9 is on the impact of 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 comparing of autogamy developer and Shanghai Xiamen Kehua developer: the OD value of the patient that we measure with autogamy developer and normal person's sample is all higher than the OD value that the Shanghai Xiamen Kehua nitrite ion of outsourcing is measured, but no difference of science of statistics between the two, P>0.05, illustrates that the nitrite ion of autogamy has good color developing effect.
Table 10 autogamy developer compares (OD) with Shanghai Xiamen Kehua developer
In prostatitis patient and control group normal person urine, the distribution of PSEP content exists and shows sex differernce as can be seen from the above results, and the concentration of the PSEP detected in clinical samples is apparently higher than the concentration of PSEP in control group.The detection of secreting albumen outside PSEP can provide a reliable basis for the diagnosis of clinical prostate inflammation.This kit preserves experiment under damaging experiment and 4 DEG C of conditions through strict 37 DEG C for a long time simultaneously, and testing result is stablized, and meets state food drug surveilance office external diagnosis reagent appraisal standards.
Chronic pelvic pain syndrome/chronic prostatitis (CPPS/CP) is that the urological department male sex goes to a doctor one of principal disease, affects the common disease of Lipid on Young-middle Male health especially.They are syndromes of a kind of inhomogeneity, are defined as " having 3 months pelvis area discomforts or pain in the past in 6 months at least, subsidiary uropoiesis/reproductive system functional disturbance " and without any identifiable cancer, infect and the existence of anatomic abnormalities pathology.They also usually cause passive cognitive, behavior, property, or Hypoemotivity.In addition, cancer gene group view research prove convincingly promotion sensitivity gene sudden change from one's youth namely class set.Chronic pelvic pain syndrome/chronic prostatitis is common in 20 ~ 50 years old male sex, just can embody carcinoma of prostate symptom for many years.According in prostate cancer, after the existence of inflammation and cell epithelia tissue damage, regenerate the key factor being considered to vicious transformation.Obviously, early stage diagnosis and treatment chronic pelvic pain syndrome/chronic prostatitis is significant to reducing or preventing and treating prostate cancer.
At present, chronic pelvic pain syndrome/Diagnosis of Chronic Prostatitis is mainly rejected based on clinical experience and symptom.Be used to guide or auxiliary diagnosis without any simple and feasible molecule or immunization method in clinical detection, cause clinical diagnosis subjective, inaccurate and mistaken diagnosis.This not only causes treatment difficulty further, also unfavorable searching treatment new drug development.So, the urine detection kit that the present invention is based on PSEP is chronic pelvic pain syndrome/chronic prostatitis molecular immune diagnostic kit that the whole world is initiated, precision through reaction system detects, stability test, and its reagent quality of destructive test all shows and highly can be mass.Its application is real Noninvasive, is easy to operation, easy, and has high sensitivity and specificity.It is by painful for the patient not only alleviated due to chronic pelvic pain syndrome/chronic prostatitis, and early stage diagnosis and treatment will be strengthened further to the control of prostate cancer, are with a wide range of applications.
Although describe the present invention in detail with most preferred embodiment above, those skilled in the art should know, under the prerequisite not departing from inventive concept and spirit, any improvement make the present invention and modification, still belong within the scope of protection of present invention.
Claims (10)
1. Prostasomes leaks a proteantigen, it is characterized in that, adopts and be prepared from the following method:
Get 500 milliliters of chronic prostatitis syndrome patient urina sanguinis midstream urines, add HEPES damping fluid and the protease inhibitors of equivalent pH7.3, by filtered through gauze; Get filtered fluid and be placed in 10 50 milliliters of centrifuge tubes respectively, centrifugal 10 minutes of 400g under 4 ° of C low temperature, go precipitation, get supernatant under 4 ° of C low temperature 10, centrifugal 20 minutes of 000g, goes precipitation, gets supernatant and be placed in 200 3 milliliters of ultracentrifugation pipes respectively, under 4 ° of C low temperature 100,000g centrifugal 120 minutes; Remove supernatant, get TBS damping fluid and protease inhibitors that precipitation adds 2.5 milliliters of pH7.8, mixing; Under 4 ° of C low temperature 100,000g centrifugal 120 minutes, remove supernatant, get precipitation and be dissolved in 2.5 milliliter of 0.5 % NP-40 respectively, 80mM NaCl, 0.2% Deoxycholate, 50 mM Tris, pH 8.0 and protease inhibitors; Under 4 ° of C low temperature 100,000g centrifugal 20 minutes; Get supernatant, loading TSKgel G3000PW, 280 nanometer readings, be taken at 40kDa plots peak albumen ,-80 ° of C freezen protective, to obtain final product.
2. Prostasomes leaks a preparation method for proteantigen, it is characterized in that,
Get 500 milliliters of chronic prostatitis syndrome patient urina sanguinis midstream urines, add HEPES damping fluid and the protease inhibitors of equivalent pH7.3, by filtered through gauze; Get filtered fluid and be placed in 10 50 milliliters of centrifuge tubes respectively, centrifugal 10 minutes of 400g under 4 ° of C low temperature, go precipitation, get supernatant under 4 ° of C low temperature 10, centrifugal 20 minutes of 000g, goes precipitation, gets supernatant and be placed in 200 3 milliliters of ultracentrifugation pipes respectively, under 4 ° of C low temperature 100,000g centrifugal 120 minutes; Remove supernatant, get TBS damping fluid and protease inhibitors that precipitation adds 2.5 milliliters of pH7.8, mixing; Under 4 ° of C low temperature 100,000g centrifugal 120 minutes, remove supernatant, get precipitation and be dissolved in 2.5 milliliter of 0.5 % NP-40 respectively, 80mM NaCl, 0.2% Deoxycholate, 50 mM Tris, pH 8.0 and protease inhibitors; Under 4 ° of C low temperature 100,000g centrifugal 20 minutes; Get supernatant, loading TSKgel G3000PW, 280 nanometer readings, be taken at 40kDa plots peak albumen ,-80 ° of C freezen protective, to obtain final product.
3. an antibody corresponding with antigen-specific described in claim 1.
4. antibody described in antigen described in claim 1 and claim 3 preparation detect or diagnosing prostate cancer kit in application.
5. application according to claim 5, is characterized in that: described kit adopts ELISA method or colloidal gold method to be prepared from.
6. application according to claim 5, is characterized in that: described kit comprise for Prostasomes leak proteantigen monoclonal detect antibody, bag be buffered liquid, lavation buffer solution, Block buffer, enzyme labelled antibody.
7. application according to claim 6, is characterized in that: it is characterized in that, Prostasomes leaks, and to detect antibody be that monoclonal mouse anti-human's Prostasomes leaks the antibody of proteantigen to proteantigen.
8. application according to claim 6, is characterized in that: it is characterized in that, also comprises standard items and negative control; Described standard items to leak proteantigen for the Protein Detection antibody that leaks to Prostasomes produces immunoreactive human prostate corpusculum.
9. application according to claim 6, is characterized in that: it is carbonate or dPBS that described bag is buffered liquid; Described lavation buffer solution is the dPBS containing washing agent.
10. kit according to claim 6, is characterized in that, described detection antibody recognition Prostasomes leaks proteantigen.
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| CN110456044A (en) * | 2019-08-20 | 2019-11-15 | 郑州安图生物工程股份有限公司 | A kind of kit for prostatitis detection |
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