CN101329343A - New generation early diagnosis prostate gland cancer reagent kit and preparation method and detection process thereof - Google Patents
New generation early diagnosis prostate gland cancer reagent kit and preparation method and detection process thereof Download PDFInfo
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Abstract
The invention provides a kit, a preparation method and a detection method used for the new-generation early diagnosis of prostate cancer; the method of the invention is a new method used for quantitatively detecting the compound of prostate specific antigen and Alpha 1-antichymotrypsin (PSA-ACT) in the blood serum of prostate patients, comprising the preparation method and the detection method. The invention uses an immune enzyme-linked dual-antibody sandwich method to measure a knub marker: the compound of the prostate specific antigen and the Alpha 1-antichymotrypsin, which is closely relevant to the prostate cancer. The dual-antibody comprises a single-clone antibody which captures total prostate specific antigen (total PSA) (free prostate specific antigen (Free PSA)) and complexed prostate specific antigen (complexed PSA), and a detection single-clone antibody which is specifically bonded to the compound protein of the prostate specific antigen and the Alpha 1- antichymotrypsin. The kit of the invention is used for detecting the compound protein of the prostate specific antigen and the Alpha 1-antichymotrypsin (PSA-ACT) in the blood serum of the prostate patients and has high sensitiveness, high specificity and high accuracy. The method of the invention can be widely applied to the screening, the clinic diagnosis and the curative effect tracking observation of the prostate patients.
Description
Kit of prostate specific antigen and α 1-ACT compound protein label and preparation method thereof and detection method in a kind of new quantitative test prostatosis human serum.
Technical field
The present invention is a kind of new quantitative detecting method, detects prostate specific antigen and α 1-ACT compound label method (comprising preparation method and detection method) among the prostate cancer patients serum.
Technical background
Prostate cancer (prostate cancer) incidence of disease in developed country is very high, and mortality ratio is at second.With the U.S. is example, and 180,000 above new cases are arranged every year.And China is developing country, shows according to statistics in 2005, has every year 1500 10000 the male sex more than 50 years old to carry out the coherence check of prostate cancer.The current generaI investigation of generally using in the world and the method for diagnosing prostate cancer are the total prostate specific antigen detection methods.Total prostate specific antigen comprises free prostate gland specific antigen (Free PSA or fPSA and compound prostate specific antigen (complexed PSA or cPSA).It is believed that normal adult male prostate organ produces free prostate gland specific-antigen PSA (Wang et al.Invest Urol 1979; 17:159-63; Watt et al., Proc Natl Acad Sci USA 1986; 83:3166-70), this free prostate gland specific antigen is proteinase (Akiyama et al., FEBS Letters 1987; 225:168-72; Christenssonet al., Eur J Biochem 1990; 194:755-763; Lilja et al.J Clin Invest 1985; 76:1899-903), when the free prostate gland specific antigen soaks in the patients'blood from patient's prostate, cause inflammation.At this moment, suppress free prostate gland specific antigen protein in the blood and (comprise α 1-ACT, alpha1-antitrypsin and alpha2-macroglobulin) be attached on the free prostate gland specific antigen, form compound, for example prostate specific antigen and α 1-ACT compound (PSA-ACT), prostate specific antigen and alpha1-antitrypsin compound (PSA-AT), prostate specific antigen and alpha2-macroglobulin compound (PSA-A2M), to suppress the active reaction of free prostate gland specific antigen (Stenman et al.Cancer Res 1991,51:222-226; Zhou et al.Clin Chem1993,39:2483-91; Leinonen et al.Clin Chem 1993,39:2098-2103; Christensson etal.J Urol 1993,150:100-105; Lilja H.Urol Clin North Am 1993,20 (4): 681-686; Lilja et al.Clin Chem 1991,37:1618-1625; Bjork et al.Urology 1994,43:427-434; Allard et al.Clin Chem 1998,44:1216-23).Make free prostate gland specific antigen proteinase lose the effect of Profilin enzymatic activity.The prostate specific antigen complex concentration of this formation becomes positive correlation with the prostate state of an illness.Though several Profilin multienzyme complexs that combine with the free prostate gland specific antigen are arranged, comprising 1) prostate specific antigen and α 1-ACT compound (PSA-ACT), 2) prostate specific antigen and alpha1-antitrypsin compound (PSA-AT) and 3) prostate specific antigen and the compound compound of alpha2-macroglobulin (PSA-A2M).Wherein prostate specific antigen and alpha1-antitrypsin compound content seldom account for 1-3%; And prostate specific antigen and alpha2-macroglobulin compound can not be discerned by antibody, have only prostate specific antigen and α 1-ACT compound to be discerned by antibody.And occupy very high concentration (Stenman et al.Cancer Res 1991,51:222-226; Zhou et al.ClinChem 1993:39:2483-91; Allard et al.Clin Chem 1998,44:1216-23).
The present total prostate specific antigen detection method of widespread usage clinically, when its susceptibility 90% the time, its specificity has only about 25% (10-31%), especially when the total prostate specific antigen concentration level is between the 4-10 nanograms/milliliter, this detection method is difficult to distinguish prostate cancer and hyperplasia of prostate (Zhou et al.Clin Chem 1993,39:2483-91; Allard et al.Clin Chem 1998,44:1216-23).So low method for detecting specificity is difficult to practical application in diagnosis and generaI investigation prostatic disorders.In recent years, also adopted a kind of analytical approach in the clinical diagnosis in free prostate gland specific antigen and total prostate specific antigen ratio, improve detection specificity (the Mitrunen et al.Clin Chem 1995,41 (8): 1115-1120 of total prostate specific antigen; Catalona et al.JAMA 1995,274 (15): 1214-1220; Prestigiacomo and Stamey.Scan J Clin Lab Invest 1995,55Supple 221:32-34).Be to contain greater than 25% free prostate gland specific antigen in all prostatosis human serums to be judged as normally, and content is judged as the prostate cancer patient less than 25% free prostate gland specific antigen.But the method need detect total prostate specific antigen and free prostate gland specific antigen simultaneously, add free prostate gland specific antigen content seldom with technical error, (susceptibility was at 90% o'clock, and its specificity is at (between the 10-45%) about 28% to cause the method that detection prostatosis human specific is not had remarkable improvement.At present, Bayer AG has invented the compound prostate specific antigen total prostate specific antigen of a kind of indirect detection and has comprised free prostate gland specific antigen and compound prostate specific antigen (complexed PSA orcPSA) method (Allard et al.Clin.Chem 1998; 44:1216-23; Allard et al.US PatentNo.:5,840,501), be used for clinical in approval in 2000 by U.S. FDA.But the method is the content of indirect detection composite PSA in serum, and promptly the total prostate specific antigen content of Jian Ceing deducts free prostate gland specific antigen content and obtains compound prostate specific antigen content.The method can not guarantee that all free prostate gland specific antigens are closed, so the susceptibility of method is at 85% o'clock, specificity only is about 32%, does not significantly improve the specificity that the prostate cancer patient detects.In addition, other several PSA-ACT detection methods because of the excessive concentration of its antibody deficiency specificity and detection, do not significantly improve specificity (Wang et al.Poster presentation at 1994 ISOBMmeeting of clinical prostate medical diagnosis on disease; Chan et al.Clin Chem 1996,42 (6): S255; Wang et al.Clin Chem 1997,43 (6): S225).
The inventive method adopts immuno-enzymatic connection double antibody sandwich method, and its capture antibody is anti-T-PSA monoclonal antibody.Select anti-prostate-specific-antigen and α 1-ACT compound protein monoclonal antibody for use, (Bjorket al.Urol 1994,43 (4): 427-434 to detect in the prostatosis human serum prostate specific antigen main and that be closely related with prostate cancer and α 1-ACT compound protein label; Allard et al.Clin.Chem 1998; 44:1216-23; Allardet al.US Patent No.:5,840,501), be high specific detection method of new generation.Preliminary clinical trial shows, compares with the clinical detection method of current widespread usage, and this method has significantly improved the specificity that detects prostate specific antigen and α 1-ACT compound in the prostatosis human serum, brings up to about 68% from about 25%.The kit that prostate specific antigen and α 1-ACT compound label detect in a new generation has hypersensitivity, high specific and high accuracy, can be used as prostate patient screening survey with and curative effect follow the trail of and diagnose usefulness.
Summary of the invention
Ultimate principle of the present invention
Prostate specific antigen and α 1-ACT compound protein method ultimate principle (see figure 1) detect in the present invention a new generation.Capture antibody can be discerned free prostate gland specific antigen and prostate specific antigen and the α 1-ACT compound protein in patients serum's sample specifically.When adding when being detected antibody with α 1-ACT compound protein, have only in the blood serum sample prostate specific antigen and α 1-ACT compound protein molecule to be joined double antibody sandwich method and detect by immuno-enzymatic of new generation of the present invention by the anti-prostate-specific-antigen on the enzyme labeling.And the high-load of prostate specific antigen and α 1-ACT compound protein is considered to closely related with the prostate cancer patient.This is the nucleus of detection method of new generation.
Technical scheme of the present invention
1. reagent constituents of the present invention
It is anti-that immuno-enzymatic connection double antibody sandwich method kit of the present invention is used for detection by quantitative prostatosis human serum prostate-specific
Former and α 1-ACT compound protein label, its component comprises as follows:
A. capture antibody.Capture antibody is immunoglobulin G monoclonal antibody (IgG1), can discern free prostate gland specific antigen and prostate specific antigen and α 1-ACT compound protein in the blood serum sample specifically, this capture antibody bag is finished the preparation of capture antibody bag quilt by to elisa plate.
B. certification mark antibody.Its antibody also is immunoglobulin G monoclonal antibody (IgG1), can discern prostate specific antigen and α 1-ACT compound protein in the blood serum sample specifically, can not discern prostate specific antigen or α 1-ACT separately.This monoclonal antibody available bases acid phosphatase (Alkaline Phosphatase or AP) or horseradish peroxidase (Horseradish Peroxidase or HRP) lotus root connection form enzyme receipts or other documents in duplicate resistive connection compound.With prostate specific antigen in its recognition sample and α 1-ACT compound, release signal.Its release signal can be converted into detected molecular conecentration, is used for quantitative test.
C. prostate specific antigen and α 1-ACT compound protein (Chen et al.Clin Chem 1995,41 (9): 1273-82).Prostate specific antigen and α 1-ACT compound protein are used as the typical curve for preparing quantitative test.Its prostate specific antigen and α 1-ACT complex antigen concentration gradient are 0,3.125,6.25,12.50,31.25,78.125,156.25 and 312.50 nanograms/milliliter (the prostate specific antigen concentration gradient is 0,2,4,10,25,50 and 100 nanograms/milliliter), prostate specific antigen and first kind chymotrypsin compound are kept in 10 mM sodium acetates/150 mM sodium chloride damping fluids, and pH 5.6.Available PBS phosphate buffer (100 mM phosphoric acid/150 mM sodium chloride, pH 7.2, Pierce, Rockford, IL) and normal women's serum dilution prostate specific antigen and α 1-ACT compound (PSA-ACT), normal women's serum does not contain the former and α 1-ACT compound of prostate-specific.Calculate prostate specific antigen and α 1-ACT complex concentration in the sample with its typical curve.
D. the phosphate buffer cleansing solution that contains 0.05% tween.
E. the blocking agent that contains 2% bSA stops the association reaction of non-specific disturbing molecule and prostate specific antigen and α 1-ACT compound.
F. use PBS phosphate buffer diluted sample.
G. negative control for normal women's serum, does not contain any prostate specific antigen and α 1-ACT compound and homologue thereof.
H. positive control is for the carcinoma of prostate patients serum is contained a certain amount of prostate specific antigen and α 1-ACT compound protein.
2. the preparation method of kit of the present invention
The present invention is used for detecting the prostate specific antigen and the α 1-ACT compound protein content of prostatosis human serum, and the preparation method of its kit specifically describes as follows:
A. catch the elisa plate preparation of total prostate specific antigen monoclonal antibody bag quilt: capture antibody is added in the phosphate buffer evenly, add in the enzyme linked plate holes, every hole 100 microlitres (down with).Hatch under the room temperature after two hours and discard liquid in the hole,, add the 2% bSA solution (250 microlitres/hole) that is dissolved among the TRIS after elisa plate pats dry again, to get rid of nonspecific reaction with the phosphate buffer washing that contains 0.05% tween three times.After at room temperature hatching one hour, discard the liquid in the plate hole, wash three times, be affixed on the elisa plate, put into 4 degree refrigerators and preserve stand-by with the adhesive sticker sheet.
B. the preparation of prostate specific antigen and α 1-ACT compound protein antigen typical curve: free prostate gland specific antigen lotus root is linked on the first kind chymotrypsin molecule, form prostate specific antigen and α 1-ACT compound protein (Chen et al.Clin Chem 1995,41 (9): 1273-82).The prostate specific antigen and the α 1-ACT compound protein that are purified the back acquisition are kept in 10 mM sodium acetates/150 mM sodium chloride damping fluids (pH 5.6) standby.
C. the preparation of antibody conjugates is surveyed in the enzyme joint inspection:
Alkaline phosphatase or horseradish peroxidase are attached on anti-prostate-specific-antigen and the α 1-ACT compound antibody, the enzyme joint inspection survey antibody conjugates that is purified the back acquisition is kept at the slow salt (RABiosources in liquid of PBS phosphoric acid, Belmont, CA).
D. the preparation of other component of kit:
A. cleansing solution is the phosphate cleansing solution that contains 0.05% tween,
The zymolyte of the usefulness that b. develops the color (blue phosphorus, Blue Phos) is the substrate of alkaline phosphatase; ABTS is the horseradish peroxidase substrate,
C. the color reaction stop buffer is the sulfuric acid solution of 2 equivalents,
D. negative control is normal women's serum, does not contain prostate specific antigen and α 1-ACT compound serum sample,
E. positive control is the prostatosis human serum sample that contains prostate specific antigen and α 1-ACT compound.
3. the determination step of the inventive method
The present invention is used for detecting the concentration of the prostate specific antigen and the α 1-ACT compound protein label of prostatosis human serum, and its determination step is as follows:
A. point sample: every hole adds 100 microlitre blood serum samples, prostate specific antigen and α 1-ACT compound protein antigen samples and standard control sample (comprising the positive, negative control and blank) in the enzyme linked plate holes that contains capture antibody bag quilt.With PBS phosphate buffer dilute serum sample and typical curve sample, each sample double repeats.After at room temperature hatching two hours, discard liquid in the hole, wash three times, pat dry.
B. add the enzyme joint inspection and survey antibody conjugates.Except the blank hole, every hole adds the anti-prostate-specific-antigen and the α 1-ACT compound protein bond of 100 microlitres, 2 millimicro grams per milliliter alkaline phosphatases or horseradish peroxidase lotus root connection, after hatching two hours under the room temperature, discard plate hole liquid, wash three times, pat dry, be affixed on elisa plate with the adhesive sticker sheet.
C. chromogenic reaction.Every hole adds 100 microlitre zymolytes (blue phosphorus or ABTS substrate) solution, places and observes the chromogenic reaction degree under the room temperature.
D. color development stopping reaction.Every hole adds the 2 equivalent sulfuric acid stop buffers (dilute with water sulfuric acid liquid) of 100 microlitre equivalent, and liquid is 1: 1 ratio (finally being the sulfuric acid liquid of 1 equivalent) with developing the color.
E. light absorption OD pH-value determination pH.With the OD value after the microplate reader working sample colour developing cessation reaction,, measure it with the 595-650nm wavelength and stop the OD value if used blue phosphorus substrate and the reaction of alkaline phosphatase antibody conjugates; If, measure it with the 405nm wavelength and stop the OD value with the termination product of ABTS substrate and the reaction of horseradish peroxidase antibody conjugates.
F. data analysis.The concentration that mainly comprises prostate specific antigen and first kind chymotrypsin compound in the general biostatistics assay determination sample, deviation, related coefficient etc.
Clinical susceptibility of colony and specificity in the G.ROC analysis and judgement patients serum sample.
Experimental result
The present invention adopts prostate specific antigen and the α 1-ACT compound protein label in the immuno-enzymatic connection double antibody sandwich method quantitative measurement prostatosis human serum.One, catching monoclonal antibody and catching the total prostate specific antigen molecule specifically of employing comprises free prostate gland specific antigen and compound prostate specific antigen molecule; Its two employing can be discerned the detection monoclonal antibody of prostate specific antigen and α 1-ACT compound protein specifically, this monoclonal antibody can not single identification prostate specific antigen molecule, α 1-ACT, alpha1-antitrypsin and alpha2-macroglobulin etc. or nonspecific proteins molecule bond.Its prostate specific antigen and α 1-ACT compound are the compositions that mainly is detected in the total prostate specific antigen.
The clinical trial result of the inventive method:
Compare with the prostate specific antigen method that detects commonly used, the inventive method in detecting the prostate cancer patients serum prostate specific antigen and during first-1 ACT compound susceptibility higher, generally between 70-95%.And, significantly improved the specificity (when susceptibility reached 88%, the specificity of this method reached 68%) between difference prostate cancer and the non-prostate cancer in the total prostate specific antigen grey area of 2.5-10 nanograms/milliliter.The inventive method prostate specific antigen and α 1-ACT compound in measuring prostatosis human serum sample reach＞and during 10 nanograms/milliliter, its susceptibility is near 100%.
1. measure the mark curve (see figure 2) that prostate specific antigen and α 1-ACT compound protein content are used
Preserve in the damping fluid (pH 5.6) of 10 mM sodium acetates/150 mM sodium chloride with highly purified (＞98% purity) prostate specific antigen and α 1-ACT compound protein, become the sample of variable concentrations with PBS phosphate buffer or normal women's serum dilution prostate specific antigen with α 1-ACT compound protein.Its concentration is 0,3.125,6.25,12.5,31.25,78.125,156,312.50 nanograms/milliliter, and corresponding to 0,1,2,4,10,25,50,100 nanograms/milliliter free prostate gland specific antigen concentration.Free prostate gland specific antigen molecular weight is 32 kilodaltons, α 1-ACT composite molecular weight is 68 kilodaltons, prostate specific antigen and α 1-ACT composite molecular weight are 100 kilodaltons, and the ratio of free prostate gland specific antigen and prostate specific antigen and α 1-ACT compound is 3.125 (32/100=3.125).The capture antibody bag is 2 mcg/ml by the concentration of elisa plate, and the detection antibody concentration of alkaline phosphatase lotus root connection is 2.5 nanograms/milliliter.During as substrate reactions, the detection wavelength is 595-650nm with blue phosphorus; As product, measure its OD value with the 405nm wavelength with ABTS substrate and the reaction of horseradish peroxidase antibody conjugates.
2.ROC analyze the susceptibility and the specificity (see figure 3) of the inventive method
60 parts of prostatosis human serum samples comprise 35 prostate cancer patients, 25 hyperplasia of prostate patients.Solid diamond symbolic representation prostate specific antigen and α 1-ACT compound sample distribution curve, square hollow symbolic representation total prostate specific antigen sample distribution curve.All clinical prostate patients confirm through biopsy that all sample obtains from New York medical college of Rochester University, the double blind random sampling.Figure three shows that ordinate is represented the susceptibility percent; Horizontal ordinate is represented specificity percent (1-specificity decimal multiply by 100 again).For example, (1-0.3) multiply by the 100%=70% specificity.Show that as figure three when detected sample reached 88.2% susceptibility, its specificity reached 68.2%; And when its susceptibility reached 86%, its specificity reached about 80%.
3. prostate cancer patient and non-prostate cancer patient's testing result distribution (see figure 4)
Detect 100 carcinoma of prostate patients and non-carcinoma of prostate patients serum sample altogether, comprised 30 carcinoma of prostate patients, 30 hyperplasia of prostate patients and 40 normal males (more than 50 years old) control serum sample.All carcinoma of prostate patients and non-carcinoma of prostate patients serum sample standard deviation confirm that through biopsy sample obtains from New York medical college of Rochester University, the double blind random sampling.Carcinoma of prostate patient's sample total prostate specific antigen content is all in (prostate specific antigen and α 1-ACT compound content are more than 30 nanograms/milliliter) more than 10 nanograms/milliliter.Solid triangle is expressed as the carcinoma of prostate patient's sample, and empty solid squares is represented the hyperplasia of prostate patient's sample, and the open squares symbol is the normal male sample.Figure four gradient ordinates are all carcinoma of prostate patients and non-carcinoma of prostate patients serum sample total prostate specific antigen content, are unit with the nanograms/milliliter, with the index mark number.Figure four shows, prostate specific antigen and α 1-ACT compound content (refer to prostate specific antigen content when 10 nanograms/milliliter, it distinguishes carcinoma of prostate patient and non-carcinoma of prostate patient's critical standard), its susceptibility of the inventive method is near 100%, and specificity reaches about 95%.
Description of drawings
Fig. 1: prostate specific antigen and α 1-ACT compound protein (PSA-ACT) immuno-enzymatic connection double antibody sandwich method ultimate principle detect in a new generation
The enzyme connection double antibody sandwich method principle schematic of detection by quantitative PSA-ACT compound protein
PSA is a prostate specific antigen; ACT is a α 1-ACT; HRP or AP is that horseradish peroxidase or alkaline phosphatase lotus root are linked on anti-prostate-specific-antigen and the α 1-ACT compound protein antibody molecule, forms anti-kind of chymotrypsin compound protein bond of prostate specific antigen and α 1-, does to detect and uses.
Fig. 2: the mark curve of measuring prostate specific antigen and α 1-ACT compound content
Fig. 3: ROC analyzes the susceptibility and the specificity of the inventive method
Fig. 4: the testing result of prostate cancer patient and non-cancer patient serum sample distributes
Embodiment
1. the present invention is immuno-enzymatic connection double antibody sandwich method, prostate specific antigen and α 1-ACT compound content in the detection by quantitative prostatosis human serum, its reagent constituents embodiment
A. capture antibody.Capture antibody is immunoglobulin G monoclonal antibody (IgG1), can discern free prostate specific antigen and prostate specific antigen and α 1-ACT compound protein in the blood serum sample specifically.With PBS phosphate buffer diluted sample, the capture antibody bag is arrived (NUNC EIA 96-hole elisa plate in the immune enzyme linked plate holes, 96FMaxisorp, Nalge NUNC, Rochester, NY), after wet box is hatched two hours, discard the liquid in the enzyme linked plate holes, wash three times, add 2% bSA (2%BSA).Wet box discards liquid in the plate hole after hatching 1 hour, wash three times, with adhesive sticker sheet capping elisa plate, finishes the preparation of capture antibody bag quilt.
B. certification mark antibody.Its antibody also is immunoglobulin G monoclonal antibody (IgG1), can only discern prostate specific antigen and α 1-ACT compound protein in the blood serum sample specifically, can not discern prostate specific antigen or α 1-ACT separately.This monoclonal antibody available bases acid phosphatase (AP) or horseradish peroxidase (HRP) lotus root connection form enzyme receipts or other documents in duplicate resistive connection compound.With prostate specific antigen and the α 1-ACT compound in its lotus root connection formation enzyme receipts or other documents in duplicate resistive connection compound recognition sample.Its release signal can be converted into detected molecular conecentration, is used for quantitative test.
C. prostate specific antigen and α 1-ACT compound protein (Chen et al.Clin Chem.41 (9): 1273-82).Prostate specific antigen and α 1-ACT compound protein are used as the typical curve sample for preparing quantitative test.Its prostate specific antigen and α 1-ACT compound protein concentration are 0,3.125,6.25,12.50,31.25,78.125,156.25 and 312.50 nanograms/milliliter, being converted into prostate specific antigen concentration is 0,2,4,10,25,50 and 100 nanograms/milliliter.Prostate specific antigen and α 1-ACT compound are kept in 10 mM sodium acetates/150 mM sodium chloride damping fluids (pH 5.6).Prostate specific antigen and α 1-ACT compound can dilute with PBS phosphate buffer or normal women's serum.Prostate specific antigen and α 1-ACT compound are made typical curve and are used after dilution.Calculate the concentration that detects prostate specific antigen and α 1-ACT compound in the sample with its typical curve.
D. the phosphate buffer cleansing solution that contains 0.05% tween.
E. block the association reaction of non-specific disturbing molecule and prostate specific antigen and α 1-ACT compound with the blocking agent of 2% bSA, with dissolving of PBS phosphate buffer and dilution.
The F.PBS phosphate buffer is used for diluted sample (PBS:0.0078M Na
2HPO
47H
2O, 0.0022M KH
2PO
4, 0.137MNaCl, 0.002M KCl, 0.05%Tween-20/1%BSA, 0.02%azide).
G. negative control for normal women's serum, does not contain any prostate specific antigen and α 1-ACT compound and homologue thereof.
H. positive control contains a certain amount of prostate specific antigen and α 1-ACT compound protein for the carcinoma of prostate patients serum.
2. the concrete grammar of kit preparation of the present invention
Kit of the present invention is used for detecting prostatosis human serum prostate specific antigen and α 1-ACT compound protein content, and the preparation method is as follows for its kit:
A. catch the elisa plate preparation of T-PSA monoclonal antibody bag quilt
Capture antibody is added in the phosphate buffer evenly, add to every hole 100 microlitres, 2 millimicro grams per milliliters in the enzyme linked plate holes.After hatching two hours under the room temperature.Discard liquid in the hole,, add the 2% bSA solution that is dissolved among the TRIS after elisa plate pats dry again, be used to stop nonspecific reaction with containing 0.05% Tween-20 phosphate buffer washing three times.After at room temperature hatching one hour, discard the liquid in the plate hole, wash three times, be affixed on the elisa plate, put into 2-8 degree refrigerator and preserve stand-by with the adhesive sticker sheet.
B. the preparation of prostate specific antigen and α 1-ACT compound protein antigen typical curve
Free prostate gland specific antigen lotus root is linked on the α 1-ACT molecule, forms prostate specific antigen and α 1-ACT compound protein.The prostate specific antigen and the α 1-ACT compound protein that are purified the back acquisition are kept in 10 mM sodium acetates/150 mM sodium chloride damping fluids (pH 5.6) standby.
The C. preparation of oxidation horseradish peroxidase (HRP)-detection antibody conjugates (RA Biosources, Inc., Belmont, CA)
D. the preparation of other component of kit:
A. cleansing solution is the phosphate cleansing solution (10mM Trizma/0.139M NaCl/2.0mMKCl/0.05%Tween-20, pH 7.2-7.4with 0.02%azide) that contains 0.1% tween;
B. colour developing is that blue phosphorus (Blue Phos) is alkaline phosphatase substrate with zymolyte: Blue Phos MicrowellPhosphatase Substrate System contains 2 components (A, B). component A is solubility 1g/L BCIP (5-bromo-4chloro-3-indolyl-phosphate), B component is tetrazolium, KPL, Gaithersburg, MD); At the bottom of ABTS is horseradish peroxidase: ABST Peroxidase SubstrateSystem contain 2 components (A, B). component A is 0.3g/L 2,2 '-azino-di-3ethylbenzthiazoline-6-sulfonate)
Glycerine/citrate buffer solution, B component are 0.02%H
2O
2, KPL, Gaithersburg, MD);
C. the color reaction stop buffer is the sulfuric acid solution of 2 equivalents;
D. negative control is normal women's serum, does not contain prostate specific antigen and α 1-ACT compound serum sample;
E. positive control is the prostatosis human serum sample that contains prostate specific antigen and α 1-ACT compound.
3. prostate specific antigen and α 1-ACT complex concentration concrete grammar and step in the inventive method detection by quantitative prostatosis human serum
A. point sample: every hole adds 100 microlitre blood serum samples and prostate specific antigen and α 1-ACT compound protein antigen samples and standard control sample (comprising the positive, negative control and blank) in the enzyme linked plate holes that contains capture antibody bag quilt.Its prostate specific antigen and α 1-ACT complex antigen concentration are 0,3.125,6.25,12.50,31.25,78.125,156.25 and 312.50 nanograms/milliliter, being converted into prostate specific antigen concentration is 0,2,4,10,25,50 and 100 nanograms/milliliter.Prostate specific antigen and α 1-ACT compound are kept in 10 mM sodium acetates/150 mM sodium chloride damping fluids (pH 5.6).With PBS phosphate buffer dilute serum sample and typical curve sample, each sample double repeats.After at room temperature hatching two hours, discard liquid in the hole, wash three times, pat dry.
B. add the enzyme joint inspection and survey antibody conjugates.Except the blank hole, every hole adds the anti-prostate-specific-antigen and the α 1-ACT compound protein bond of 100 microlitres, 2.5 millimicro grams per milliliter peroxidase lotus roots connection, hatch two hours under the room temperature after, discard plate hole liquid, wash three times, pat dry, be affixed on elisa plate with the adhesive sticker sheet.
C. chromogenic reaction.Every hole adds 100 microlitre zymolytes (blue phosphorus or ABTS) solution, places and observes the chromogenic reaction degree under the room temperature.
D. color development stopping reaction.Every hole adds the 2 equivalent sulfuric acid stop buffers (dilute with water sulfuric acid liquid) of 100 microlitre equivalent, and liquid is 1: 1 ratio (finally being the sulfuric acid liquid of 1 equivalent) with developing the color.
E. light absorption OD pH-value determination pH.With the OD value after the microplate reader working sample colour developing cessation reaction, measure the termination product OD value of its ABTS substrate and the reaction of horseradish peroxidase antibody conjugates with the 405nm wavelength.Microplate reader is ELISAMicroplate Reader, Model:VERS max, and Molecular Devices, Sunnyvale, CA)
F. data analysis.The biostatistics analytical approach mainly comprises the concentration of prostate specific antigen and α 1-ACT compound in the working sample, deviation, related coefficient, analytical approach etc.
G. clinical susceptibility of colony and specificity in clinical susceptibility and specificity analyses model (ROC) the analysis and judgement patients serum sample.
Claims (3)
1. an immuno-enzymatic that is used for detection by quantitative prostatosis human serum prostate specific antigen and α 1-ACT compound protein label joins the double antibody sandwich method kit, and its component comprises:
1) monoclonal antibody of anti-total prostate specific antigen (antigen comprises free prostate gland specific antigen and compound prostate specific antigen) is as immuno-enzymatic connection double antibody sandwich method kit capture antibody;
2) be dissolved in bSA (2%) in the phosphate buffer, to get rid of nonspecific reaction;
3) prostate specific antigen and α 1-ACT compound (PSA-ACT) are done typical curve, are used for quantitative test;
4) anti-prostate specific antigen and α 1-ACT compound antibody and alkaline phosphatase or horseradish mistake thing enzyme lotus root connect compound, are used for marker detection prostate specific antigen and α 1-ACT compound protein;
5) contain the phosphoric acid washing liquid of tween (0.05%TWEEN-20);
6) phosphate buffer of dilute sample;
7) negative control is normal women's blood serum sample (not containing prostate specific antigen and α 1-ACT compound exists);
8) positive control contains the prostatosis human serum sample of prostate specific antigen and α 1-ACT compound.
2. the preparation method of prostate specific antigen and α 1-ACT compound kit in the new detection by quantitative prostatosis human serum, its feature is as follows:
1) catches the elisa plate preparation of total PSA monoclonal antibody bag quilt
Capture antibody is added in the PBS phosphate buffer evenly, adds in the enzyme linked plate holes (100 microlitres/hole), hatch two hours under the room temperature after, discard liquid in the hole.With the phosphate buffer washing that contains 0.05% tween three times, elisa plate is patted dry, add the 2% bSA solution (250 microlitres/hole) that is dissolved among the TRIS again.After at room temperature hatching one hour, discard the liquid in the plate hole, wash three times, be affixed on the elisa plate, put into 2-8 degree refrigerator and preserve stand-by with the adhesive sticker sheet.
2) preparation prostate specific antigen and α 1-ACT compound protein, with its conduct in typical curve
Free prostate gland specific antigen lotus root is linked on the α 1-ACT molecule, forms prostate specific antigen and α 1-ACT compound protein (PSA-ACT).The prostate specific antigen and α 1-ACT compound (PSA-ACT) label of purified acquisition are kept at 10 mM sodium acetates/150 mM sodium chloride damping fluids/0.05%sodium azide (w/v), standby among the pH 5.6.
3) preparation of antibody conjugates is surveyed in the enzyme joint inspection
Alkaline phosphatase or horseradish peroxidase are attached on anti-prostate-specific-antigen and the α 1-ACT compound antibody, and it is stand-by to obtain highly purified enzyme joint inspection survey antibody conjugates.
4) preparation of other reagent constituents
A. cleansing solution is the phosphate cleansing solution that contains 0.05% tween;
The zymolyte of the usefulness that B. develops the color (blue phosphorus, Blue Phos) is the substrate of alkaline phosphatase; ABTS is the horseradish peroxidase substrate;
C. the color reaction stop buffer is the sulfuric acid solution of 2 equivalents;
D. negative control is normal women's serum, does not contain prostate specific antigen and α 1-ACT compound serum sample;
E. positive control is the prostatosis human serum sample that contains prostate specific antigen and α 1-ACT compound.
3. new detection method that is used for detection by quantitative blood serum sample prostate specific antigen and α 1-ACT compound, its step is as follows:
A. point sample: blood serum sample, prostate specific antigen and α 1-ACT compound protein sample and reference standard sample (comprising the positive, negative control and blank) join in the elisa plate of capture antibody bag quilt.With phosphate buffer dilute serum sample and typical curve sample, each sample double repeats.After at room temperature hatching two hours, discard liquid in the hole, wash three times, pat dry.
B. add the enzyme joint inspection and survey antibody conjugates (100 microlitres/hole).Except that blank, every hole adds the anti-prostate-specific-antigen and the α 1-ACT compound protein antibody conjugates of alkaline phosphatase or horseradish peroxidase lotus root connection, at room temperature hatch two hours after, discard enzyme linked plate holes liquid, wash three times, pat dry, be affixed on elisa plate with the adhesive sticker sheet.
C. chromogenic reaction.Every hole adds 100 microlitre enzyme substrate solutions, places under the room temperature and observes chromogenic reaction.
D. color development stopping reaction.Every hole adds the stop buffer (the sulfuric acid liquid of 2 equivalents) of equivalent
E. light absorption OD pH-value determination pH.With the termination OD value after the microplate reader working sample colour developing cessation reaction, if with blue phosphorus as alkaline phosphatase substrate, value is measured the value with the reacted termination of alkaline phosphatase antibody conjugates OD with the 595-650nm wavelength; If as the horseradish peroxidase substrate, measure and horseradish peroxidase antibody conjugates reaction back OD value with the 405nm wavelength with ABTS.
F. data analysis.The concentration that mainly comprises general biostatistics assay determination sample prostate specific antigen and α 1-ACT compound, deviation, related coefficient etc.
G. colony's susceptibility and specificity among clinical susceptibility and specificity analyses model (ROC, Receiver Operator Characteristic) the analysis and judgement prostatosis human serum sample.
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