CN102346184B - Novel application of Spondin-2 (SPON2) - Google Patents
Novel application of Spondin-2 (SPON2) Download PDFInfo
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Abstract
The invention discloses novel application of Spondin-2 (SPON2). The novel application is application of an antibody for the SPON2 in preparation of a kit for aided diagnosis of prostatic cancer. An experiment shows that: an enzyme coupled immune kit for detecting the SPON2 or an immunohistochemistry kit for detecting the SPON2 are used for diagnosing the prostatic cancer, so a diagnosis result is more precise and accurate; and the novel application is superior to the conventional prostatic specific antigen (PSA) diagnosis method.
Description
Technical field
The present invention relates to the new purposes of SPON2.
Background technology
Prostate cancer is one of modal malignant tumour in the north america male sex, and the patient that the U.S. in 2008 is newly diagnosed as prostate cancer accounts for this year and is newly diagnosed as 25% of malignant tumor patient.In China, along with progressively aging of population structure, the incidence of disease of prostate cancer is also increasing year by year.
Tumour serum mark be the current focus of research.Desirable tumour serum mark should have following feature: can distinguish high-risk and healthy population, can be used in the diagnosis of tumour, method is simple, with low cost, can be used in the clinical stages assessment of tumour, can be used in the result for the treatment of assessment of tumour, can be used in transfer and the recurrence of predicting tumors.
PSA is the prostate cancer blood serum designated object being most widely used at present.Although PSA examination has brought many benefits, also endure in recent years dispute to the fullest extent and query.In the last few years, find that the specificity to prostate cancer of PSA detection is poor, the blood-serum P SA of benign prostatic hyperplasis and prostatitis patient also can raise to some extent.In addition, Serum PSA level belongs to gray area between 4～10ng/ml, is difficult to distinguish prostatic benign lesion and malignant change.Some marks, or clinical practice in addition comparatively limit to, or effect is not very desirable.Searching can substituting PS A or the serodiagnosis mark that makes up PSA defect be very necessary.
SPON2 (SPON2, Spondin-2, Mindin, DIL-1) belongs to extracellular matrix proteins class, because differential expression in the alveolar cell in canceration and non-canceration is gained the name.The amino acid sequence of SPON2 is SEQ ID NO:1.
The amino acid sequence of prostate specific antigen (PSA) is as shown in SEQ ID NO:2.
Summary of the invention
An object of the present invention is to provide a kind of antibody of anti-SPON2, the new purposes of the kit of detection SPON2.
The application of the antibody that the new purposes of the antibody of anti-SPON2 is anti-SPON2 in the kit for the preparation of auxiliary diagnosis prostate cancer.
The antibody of described anti-SPON2 is the monoclonal antibody of anti-SPON2 and/or the polyclonal antibody of anti-SPON2;
The monoclonal antibody of described anti-SPON2 is the monoclonal antibody of the anti-SPON2 in mouse source; The polyclonal antibody of described anti-SPON2 is the polyclonal antibody of the anti-SPON2 in sheep source.
Above-mentioned antibody is all the antibody obtaining from commercial channels, or cultivates through cells in vitro the antibody obtaining; The polyclonal antibody of the anti-SPON2 in sheep source specifically can be purchased from R & D company, and catalog number is AF-2609; The monoclonal antibody of the anti-SPON2 in mouse source specifically can be purchased from Abnova company, and catalog number is H00010417-M01J.
The new purposes that detects the kit of SPON2 is the application of kit in the kit for the preparation of auxiliary diagnosis prostate cancer that detects SPON2.
Detect the kit of SPON2 for detecting the enzyme linked immunological kit of SPON2 or the SABC kit of detection SPON2.
SPON2 is in design and/or also belong to protection scope of the present invention for the preparation of the application in the kit of auxiliary diagnosis prostate cancer.
In the application of the enzyme linked immunological kit of above-mentioned arbitrary described detection SPON2 in the kit for the preparation of auxiliary diagnosis prostate cancer, described prostate cancer is following 1) or 2) shown in:
1) prostate cancer that the prostate cancer that Gleason scoring is the prostate cancer of 4～6 minutes, prostate cancer that Gleason scoring is 7 minutes, Gleason scoring is 8 minutes or Gleason scoring are 9～10 minutes;
2) prostate cancer of prostate specific antigen feminine gender;
In the application of the SABC kit of the kit detection SPON2 of detection SPON2 in the kit for the preparation of auxiliary diagnosis prostate cancer, described prostate cancer is shown in following I or II or III:
I, Gleason scoring is prostate cancer or the Gleason scoring prostate cancer of≤6 minutes of 7～8 minutes;
II, WHO scoring is the prostate cancer of 2 grades;
III, there is the prostate cancer of metastasis.
In the application of the enzyme linked immunological kit of above-mentioned arbitrary described detection SPON2 in the kit for the preparation of auxiliary diagnosis prostate cancer, described prostate specific antigen is negative is that in serum, prostate specific antigen concentration is less than or equal to 10ng/ml.
In the application of the enzyme linked immunological kit of above-mentioned arbitrary described detection SPON2 in the kit for the preparation of auxiliary diagnosis prostate cancer, the polyclonal antibody of the anti-SPON2 in the monoclonal antibody of the anti-SPON2 that the enzyme linked immunological kit of described detection SPON2 is originated by mouse, sheep source, standard solution, coated damping fluid, confining liquid, lavation buffer solution, stop buffer, substrate solution and enzyme labelled antibody form;
Described coated damping fluid is comprised of sodium carbonate, sodium bicarbonate and water; The concentration of described sodium carbonate in described coated damping fluid is 1.59g/L, and the concentration of described sodium bicarbonate in described coated damping fluid is 2.93g/L;
Described lavation buffer solution is comprised of sodium chloride, potassium chloride, sodium hydrogen phosphate, potassium dihydrogen phosphate, Tween 20 and water; The concentration of sodium chloride in described lavation buffer solution is 8g/L, the concentration of potassium chloride in described lavation buffer solution is 0.2g/L, the concentration of sodium hydrogen phosphate in described lavation buffer solution is 1.44g g/L, the concentration of potassium dihydrogen phosphate in described lavation buffer solution is 0.24g/L, and the concentration of Tween 20 in described lavation buffer solution is 0.1% (percent by volume);
Described confining liquid is comprised of described lavation buffer solution and bovine serum albumin(BSA), and the concentration of bovine serum albumin(BSA) in described confining liquid is 10g/L;
Described stop buffer is the aqueous sulfuric acid of 2M;
Described enzyme labelled antibody is the goat anti-mouse antibody of HRP mark;
To be described confining liquid form from described SPON2 the following solution that SPON2 concentration is different to described standard solution: 100,50,25,12.5,6.3,3.1 and 1.6ng/ml.
The enzyme linked immunological kit of above-mentioned detection SPON2 and SABC kit also can obtain from commercial channels; The SABC kit of above-mentioned detection SPON2 specifically can be goat two step method SABC kit, specifically can be purchased from biotech firm of Zhong Shan Golden Bridge, and catalog number is PV-9003.
In the application of the enzyme linked immunological kit of above-mentioned arbitrary described detection SPON2 in the kit for the preparation of auxiliary diagnosis prostate cancer, in the enzyme linked immunological kit of described detection SPON2, the polyclonal antibody of the anti-SPON2 in described coated damping fluid and described sheep source forms the solution that antibody concentration is 630ng/ml; The monoclonal antibody of the anti-SPON2 in described confining liquid and mouse source forms the solution that antibody concentration is 5ug/ml; The solution that described confining liquid and enzyme labelled antibody constitutive enzyme labeling antibody concentration are 80ng/ml;
The amino acid sequence of described SPON2 is as shown in SEQ ID NO:1; The amino acid sequence of described prostate specific antigen is as shown in SEQ ID NO:2.
Another object of the present invention is to provide a kind of enzyme linked immunological kit for auxiliary diagnosis prostate cancer.
Enzyme linked immunological kit for auxiliary diagnosis prostate cancer provided by the present invention is comprised of following material: the antibody of above-mentioned arbitrary described anti-SPON2, standard solution, coated damping fluid, confining liquid, lavation buffer solution, stop buffer and enzyme labelled antibody.
In above-mentioned enzyme linked immunological kit, described antibody is the monoclonal antibody of anti-SPON2 in mouse source and the polyclonal antibody of the anti-SPON2 in sheep source;
Described coated damping fluid is comprised of sodium carbonate, sodium bicarbonate and water; The concentration of described sodium carbonate in described coated damping fluid is 1.59g/L, and the concentration of described sodium bicarbonate in described coated damping fluid is 2.93g/L;
Described lavation buffer solution is comprised of sodium chloride, potassium chloride, sodium hydrogen phosphate, potassium dihydrogen phosphate, Tween 20 and water; The concentration of sodium chloride in described lavation buffer solution is 8g/L, the concentration of potassium chloride in described lavation buffer solution is 0.2g/L, the concentration of sodium hydrogen phosphate in described lavation buffer solution is 1.44g g/L, the concentration of potassium dihydrogen phosphate in described lavation buffer solution is 0.24g/L, and the concentration of Tween 20 in described lavation buffer solution is 0.1% (percent by volume);
Described confining liquid is comprised of described lavation buffer solution and bovine serum albumin(BSA), and the concentration of bovine serum albumin(BSA) in described confining liquid is 10g/L;
Described stop buffer is the aqueous sulfuric acid of 2M;
Described enzyme labelled antibody is the goat anti-mouse antibody of HRP mark;
To be described confining liquid form from described SPON2 the following solution that SPON2 concentration is different to described standard solution: 100,50,25,12.5,6.3,3.1 and 1.6ng/ml.
In above-mentioned arbitrary described enzyme linked immunological kit, the polyclonal antibody of the anti-SPON2 in described coated damping fluid and described sheep source forms the solution that antibody concentration is 630ng/ml; The monoclonal antibody of the anti-SPON2 in described confining liquid and mouse source forms the solution that antibody concentration is 5ug/ml; The solution that described confining liquid and enzyme labelled antibody constitutive enzyme labeling antibody concentration are 80ng/ml.
Experimental results show that, with the enzyme linked immunological kit that detects SPON2 to 13 routine normal persons, 4 routine benign prostatic hyperplasiss, SPON2 in 72 routine serum of patients with prostate cancers detects, in result patients with prostate cancer, the content of SPON2 is significantly higher than normal person or benign prostatic hyperplasis person, and showing can diagnosing prostate cancer by detecting the content of SPON2 in serum; Especially outstanding is, content and the normal person difference of SPON2 in the Gleason scoring serum of patients with prostate cancer of 4～6 minutes is extremely remarkable, content and normal person's difference that SPON2 is less than in 10ng/ml serum of patients with prostate cancer in PSA level are extremely remarkable, and it is higher more accurate to prove by SPON2 diagnosing prostate cancer degree of accuracy.
Experiment also proves, with SABC kit normal and prostate cancer beside organism, 19 routine benign prostatic hyperplasiss and the 44 routine prostate cancer tissues detections to 10 examples that detect SPON2.Result SPON2 obviously raises than positive rate in normal structure and Benign Prostatic Hyperplasia Tissues: in prostate cancer tissue, expresses and strengthens; More outstanding is, SPON2 dyeing in the prostate cancer of 2 grades of diagnosis Gleason scorings (7～8), WHO classification, the prostate cancer tissue that occurs to shift strengthens more remarkable, in prostate cancer, the staining power of SPON2 and PSA are proportionate, but it is obvious that it is expressed with benign lesion difference, is far superior to PSA.Proof is higher more accurate by SPON2 diagnosing prostate cancer degree of accuracy.
Accompanying drawing explanation
Fig. 1 is the typical curve that two sandwich ELISA methods detect SPON2 concentration.
Fig. 2 is that SPON2 calibration ordinary person and Benign Prostatic Hypertrophy in serum of patients with prostate cancer obviously raise.Horizontal line represents meta numerical value, and difference is carried out statistical study with Willcoxon Two-Sample Test.
Fig. 3 is that SPON2 contributes to Serum PSA level to be less than the diagnosis of 10ng/ml patients with prostate cancer.
Fig. 4 is that sxemiquantitative RT-PCR confirms that SPON2 only expresses in the prostate cancer cell line of the AR positive.
Fig. 5 is that extracellular protein Western Blot confirms that SPON2 only expresses in the prostate cancer cell line of the AR positive.
Fig. 6 is that in the tissue specimen of patients with prostate cancer, the staining power of PSA and the staining power of SPON2 are proportionate.P value is calculated with Spearman is relevant.(r=Spearman related coefficient).
Fig. 7 be SPON2 and PSA at normal prostate tissue, the expression in Benign Prostatic Hyperplasia Tissues: and different Gleason scoring, different WHO classification and TNM prostate cancer tissue by stages.With HE dyeing, as reference, photo is depicted as the same position of same interlacing point on the organization chip of serial section.
Embodiment
The experimental technique using in following embodiment if no special instructions, is conventional method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The nutrient culture media using in following embodiment, antibody, chemical reagent are as follows:
Title place of production article No.
The domestic analysis of sodium chloride is pure
The domestic analysis of potassium chloride is pure
The domestic analysis of sodium hydrogen phosphate is pure
The domestic analysis of potassium dihydrogen phosphate is pure
The domestic analysis of Tween 20 is pure
The HRP mark goat anti-mouse Zhong Shan ZB-2305 of Golden Bridge
IgG(H+L)
The SPON2 sheep polyclonal antibody R & D AF-2609 of company
(Anti-human?Mindin
Antibody)
The biological PV-9003 of goat two step method SABC kit Zhong Shan Golden Bridge
The biological ZLI-9061 of the special-purpose phosphate buffer Zhong Shan of SABC Golden Bridge
(0.01M?PH＝7.2～7.4)
Citrate antigen retrieval liquid (the biological ZLI9064 of 0.01 Zhong Shan Golden Bridge
M?PH＝6.0)
The biological ZLI-9031 of DAB chromogenic reagent He Zhong China fir Golden Bridge
The super English biotechnology in prostate cancer tissue chip Shaanxi (numbering PR807)
The domestic analysis of dimethylbenzene is pure
The domestic analysis of neutral gum is pure
The biological ZLI-9610 of haematoxylin Zhong Shan Golden Bridge
The domestic analysis of ammoniacal liquor is pure
The domestic analysis of sodium bicarbonate is pure
The RF101 of bovine serum albumin (BSA) U.S. Proliant company
Molten day root science and technology PA107-01 of solubility single component tmb substrate
Liquid
The domestic analysis of the concentrated sulphuric acid is pure
SPON2 mouse monoclonal antibody Abnova H00010417-M01J
(SPON2?monoclonal
antibody(M01J)，clone?3C6)
SPON2 standard items (SPON2 Abnova H00010417-P01
Recombinant?Protein(P01))
The PSA sheep polyclonal antibody R & D AF1344 of company
(Anti-human?Kallikrein
3/PSA?Antibody)
The key instrument equipment using in following embodiment is as follows:
The biological ZLI-9305 of SABC Bi Zhong China fir Golden Bridge
96 hole ELISA Plate Corning Costar 9018
IX70 fluorescence inverted microscope Olympus
Enzyme connection detector Bio-Rad
In the present embodiment, in detection sample, the content value of SPON2 or PSA is all used M (Q
r) represent, M represents the median of SPON2 content in sample; Q
rthe interquartile range that represents SPON2 content in sample, i.e. Q
rthe absolute value of the difference of=upper quartile value and lower quartile numerical value.
One, kit forms
1, coated antibody: the polyclonal antibody (SPON2 sheep polyclonal antibody) of the anti-SPON2 in sheep source.
2, binding antibody: the monoclonal antibody (SPON2 mouse monoclonal antibody) of the anti-SPON2 in mouse source.
3, standard items: SPON2.Confining liquid and SPON2 form the following solution that SPON2 concentration is different: 100,50,25,12.5,6.3,3.1 and 1.6ng/ml.
4, coated damping fluid: formed by sodium carbonate, sodium bicarbonate and water; The concentration of described sodium carbonate in described coated damping fluid is 1.59g/L, and the concentration of described sodium bicarbonate in described coated damping fluid is 2.93g/L;
5, lavation buffer solution: formed by sodium chloride, potassium chloride, sodium hydrogen phosphate, potassium dihydrogen phosphate, Tween 20 and water; The concentration of sodium chloride in described lavation buffer solution is 8g/L, the concentration of potassium chloride in described lavation buffer solution is 0.2g/L, the concentration of sodium hydrogen phosphate in described lavation buffer solution is 1.44g/L, the concentration of potassium dihydrogen phosphate in described lavation buffer solution is 0.24g/L, and the concentration of Tween 20 in described lavation buffer solution is 0.1% (percent by volume);
6, confining liquid: be comprised of described lavation buffer solution and bovine serum albumin(BSA), the concentration of bovine serum albumin(BSA) in described confining liquid is 10g/L.
7, the aqueous sulfuric acid of stop buffer: 2M;
8, the composition of substrate solution: the aqueous solution of tetramethyl benzidine (3,3 ', 5,5 '-Tetramethylbenzidine, TMB).
9, enzyme labelled antibody: the goat anti-mouse antibody of HRP mark.
10, following solution:
1) solution of coated antibody: be comprised of coated damping fluid and coated antibody, the concentration of coated antibody in solution is 630ng/ml;
2) solution of binding antibody: be comprised of confining liquid and binding antibody, the concentration of binding antibody in solution is 5ug/ml;
3) solution of enzyme labelled antibody: be comprised of confining liquid and enzyme labelled antibody, the concentration of enzyme labelled antibody in solution is 80ng/ml.
Two, kit preparation
The coated damping fluid of carbonate is prepared as follows: in 900ml deionized water, add 1.59g sodium carbonate and 2.93g sodium bicarbonate, deionized water is settled to 1L.
ELISA lavation buffer solution (PBST) is prepared as follows: in 900ml distilled water, add 8g sodium chloride, 0.2g potassium chloride, 1.44g sodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, adjust pH value to 7.4, with distilled water, be settled to 1L, after autoclaving, be cooled to room temperature, add 0.1% (percent by volume) Tween, 20 reagent to mix rear use.
ELISA confining liquid: add 10g bovine serum albumin(BSA) (BSA) to above-mentioned ELISA lavation buffer solution, be settled to 1L with ELISA lavation buffer solution.
ELISA reaction terminating liquid: 2M aqueous sulfuric acid.
Three, the application of kit
(1) foundation of the two sandwich ELISA typical curves of SPON2
1, with the coated damping fluid dilution of carbonate SPON2 sheep polyclonal antibody (Anti-human Mindin Antibody) 630ng/ml, be placed in 4 ℃ of coated spending the night of the every hole 100 μ l of elisa plate;
2, with ELISA lavation buffer solution, wash elisa plate 3 times;
3, add the ELISA confining liquid 100 every holes of μ l, 37 ℃ of 2h;
4, by ELISA confining liquid gradient dilution SPON2 standard items (SPON2 Recombinant Protein (P01)) concentration, be respectively 100,50,25,12.5,6.3,3.1 and 1.6ng/ml, get do not add standard items ELISA confining liquid as blank, each concentration is done two repetitions.Every hole 100 μ l are placed in elisa plate, 37 ℃ of 2h;
5, with ELISA lavation buffer solution, wash elisa plate 3 times;
6, with ELISA confining liquid, dilute SPON2 mouse monoclonal antibody (SPON2monoclonal antibody (M01J), clone 3C6) to 5 μ g/ml, every hole 100 μ l are placed in elisa plate, 37 ℃ of 2h;
7, with ELISA lavation buffer solution, wash elisa plate 3 times;
8, with ELISA confining liquid, dilute HRP mark goat anti-mouse IgG (H+L) to 80ng/ml, every hole 100 μ l are placed in elisa plate, 37 ℃ of 2h;
9, with ELISA lavation buffer solution, wash elisa plate 3 times;
10, every hole adds 100 μ l soluble T MB single component substrate solutions, lucifuge 15-20min;
11, add ELISA to end the every hole of liquid 100 μ l stopped reactions;
12, read every hole 450nm light absorption reading, deduct 570nm light absorption reading, be this hole reading, get the mean value in two holes as the reading of this concentration;
13, the reading of each concentration deducts after blank reading, takes the logarithm, with the logarithm fit line sexual intercourse of standard items concentration.
The typical curve obtaining as shown in Figure 1.Y＝1.5763x+2.5237，R
2＝0.9768.
(2) by two sandwich ELISA methods of setting up, detect patients serum
Detect 89 parts of human serum samples, comprising: 13 routine normal aging peoples (Normal), 4 routine Benign Prostatic Hypertrophy (BPH), the serum of 72 routine patients with prostate cancer (PCa).The agreement of taking all to pass through patient and normal aging people of serum specimen.
Detection method is as follows:
1, with the coated damping fluid dilution of carbonate SPON2 sheep polyclonal antibody (Anti-human Mindin Antibody) 630ng/ml, be placed in 4 ℃ of coated spending the night of the every hole 100 μ l of elisa plate;
2, with ELISA lavation buffer solution, wash elisa plate 3 times;
3, add the ELISA confining liquid 100 every holes of μ l, 37 ℃ of 2h;
4, press test serum: ELISA confining liquid=1: 2 dilution test serums, get do not add standard items ELISA confining liquid as blank.Every hole 100 μ l are placed in elisa plate, 37 ℃ of 2h;
5, with ELISA lavation buffer solution, wash elisa plate 3 times;
6, with ELISA confining liquid, dilute SPON2 mouse monoclonal antibody (SPON2monoclonal antibody (M01J), clone 3C6) to 5 μ g/ml, every hole 100 μ l are placed in elisa plate, 37 ℃ of 2h;
7, with ELISA lavation buffer solution, wash elisa plate 3 times;
8, with ELISA confining liquid, dilute HRP mark goat anti-mouse IgG (H+L) to 80ng/ml, every hole 100 μ l are placed in elisa plate, 37 ℃ of 2h;
9, with ELISA lavation buffer solution, wash elisa plate 3 times;
10, every hole adds 100 μ l soluble T MB single component substrate solutions, lucifuge 15-20min;
11, add ELISA to end the every hole of liquid 100 μ l stopped reactions;
12, read every hole 450nm light absorption reading, deduct 570nm light absorption reading, be this hole reading;
13, the reading in each hole deducts after blank reading, takes the logarithm, and substitution typical curve, gets 3 * lg
(1)(substitution curve income value) is serum SPON2 concentration.
Whether remarkable with the content difference of SPON2 in the content of SPON2 in Willcoxon Two-Sample Test statistical study normal population and patients with prostate cancer colony.
Result is as shown in table 1 and Fig. 2.Result: in serum of patients with prostate cancer, SPON2 content calibration ordinary person or all benign lesion persons have obvious rise (* * *, P is less than 0.001).Application Willcoxon Two-Sample Test confirms that its difference has statistical significance.Show, can be with this kit diagnosing prostate cancer.
The two sandwich ELISAs of table 1. application detect the differential expression (unit ng/ml) of SPON2 in normal person, hyperplasia of prostate person and serum of patients with prostate cancer
(3) the mark serum SPON2 concentration of patients with prostate cancer of the SPON2 concentration in normal human serum and different Gleason relatively
The Gleason 70 routine patients with prostate cancer of marking, are divided into grade described in table 2.Whether remarkable with the content difference of SPON2 in the content of SPON2 in SNK method (q check) statistical study normal population and the patients with prostate cancer colony of different Gleason scorings.
SPON2 concentration in normal human serum is compared from the serum SPON2 concentration of different Gleason scoring patients with prostate cancer.The prostate cancer serum SPON2 expression that result shows each Gleason scoring (4～6 minutes, 7 minutes, 8 minutes, 9～10 minutes) as table 2:SNK method (q check) is apparently higher than normal person (*), and has statistical significance; And patients with prostate cancer (#) the serum SPON2 that Gleason scoring is 4～6 minutes expresses higher than other Gleason scoring groups (7 minutes, 8 minutes, 9～10 minutes) and has statistical significance.
Table 2, the two sandwich ELISAs of application detect the differential expression (unit ng/ml) of SPON2 in the serum of patients with prostate cancer of normal person and different Gleason scoring
(4) the serum SPON2 concentration ratio of the SPON2 concentration in normal human serum and blood-serum P SA≤10ng/ml patients with prostate cancer
The detection method of prostate specific antigen PSA concentration in serum: Applied Electrochemistry luminescence method, RocheElecsys 2010 fully automatic electric chemical illumination immunity analysis instruments and supporting PSA reagent thereof (by the German Roche diagnosis company limited forming a complete production network with instrument, being provided) are provided, all in effect, in the phase, use.The preparation of typical curve and serum detect and carry out in strict accordance with operating process.
The serum SPON2 concentration of the SPON2 concentration in normal human serum and blood-serum P SA≤10ng/ml patients with prostate cancer is compared, their blood-serum P SA concentration is also compared simultaneously.In the serum of patients with prostate cancer of the patients with prostate cancer of result: PSA≤10ng/ml (*) and PSA > 10ng/ml (*), SPON2 level is all apparently higher than normal aging people.Application Willcoxon Two-Sample Test determines that it has statistical significance (p equals respectively 0.0013 and 0.0001).And in the patient of PSA≤10ng/ml, PSA level and normal aging people no significant difference (p=0.2852).Illustrate: SPON2 contributes to Serum PSA level to be less than the diagnosis of the prostate patient of 10ng/ml, and the Serum PSA level of these patients with prostate cancer and normal person no significant difference (Fig. 3 and table 3).
Sxemiquantitative RT-PCR detects the expression of SPON2 in various prostate cancer cell lines.Extracellular protein Western Blot detects the expression of SPON2 in various prostate cancer cell lines.
BPH is benign prostatic hyperplasis model.LNCaP and C4-2 cell are the prostate cancer research cell models of admitting in the world.PC3 is the cell model of prostate cancer with osseous metastasis, and androgen receptor is negative; C4-2B is developed by LNCaP, is the clone that bone shifts, and equally also has the character of the non-dependence of androgen, and AR is positive; DU145 is the clone that prostate cancer brain shifts, and androgen receptor is negative.
Result as shown in Figure 4 and Figure 5.Result shows, SPON2 has prostate cancer cell line specificity, only in the prostate cancer cell line of the AR positive, expresses.
One, immunohistochemical staining analysis (organization chip) method of SPON2 in prostata tissue is as follows:
1, roasting sheet, then dewaxes to water: dimethylbenzene I20min, dimethylbenzene II 20min, 100% ethanol 5min, 100% ethanol 5min, 95% ethanol 5min, 90% ethanol 5min, 85% ethanol 5min, 75% ethanol 5min, deionized water 5min;
2, with citrate, repair liquid and carry out antigen retrieval;
Citrate is repaired liquid storage liquid:
A.0.1M citric acid soln: take 21.01g citric acid (C
6h
8o
7h
2o) be dissolved in 1000ml distilled water.
B.0.1M liquor sodii citratis: take 29.41g sodium citrate (C
6h
5na
3o
72H
2o) be dissolved in 1000ml distilled water.
Citrate is repaired liquid working fluid: get 9mlA liquid and 41ml B liquid adds in 450ml distilled water, pH should be 6.0).
3, after groupization pen delineation conversion zone, drip hydrogen peroxide normal-temperature reaction 10min;
4, PBS washing lotion (sodium chloride (NaCl) 8g, potassium chloride (KCl) 0.2g, 12 hypophosphite monohydrate disodium hydrogen (Na
2hPO
412H
2o) 3.58g, potassium dihydrogen phosphate (KH
2pO
4) 0.24g, deionized water is settled to 1L, adds 1ml Tween20 to mix) in groupization box, wash each 5min 3 times;
5, the Yong Zhong China fir Golden Bridge antibody diluent dilution R & D SPON2 of company sheep polyclonal antibody (Anti-human Mindin Antibody) is to working concentration 4ug/ml;
Zhong Shan Golden Bridge antibody diluent: with TBS (Tris damping fluid (TBS) (25mol/L Tris damping fluid), deionized water 800ml, sodium chloride 8g, Tris alkali 3g, Kcl 0.2g adjusts PH to 7.4 with Hcl, add water and be settled to 1L, autoclaving, room temperature preservation) or PBS the NIS of intact animal is made into concentration is that the working fluid of 5-10% (1: 10-20 doubly dilutes) obtains antibody diluent.The serum of the animal that is chosen as two anti-sources of general serum kind.
6, dripping is to sample center (30-50 μ l) for primary antibodie (being the R & D SPON2 of company sheep polyclonal antibody working fluid), and wet box shakes up after shaking table low speed 5min, and 4 ℃ are spent the night;
7, PBS washing lotion is washed 3 times in groupization box, each 5min;
8, drip in goat two step method kit one of reagent 1 (polymerization assistant), with rifle head, dial even, shaking table 5min, 37 ℃ of 20min;
9, PBS washing lotion is washed 3 times in groupization box, each 5min;
10, reagent adding 2 (the anti-goat IgG polymer of horseradish enzyme labeling) is one, with rifle head, dial even, shaking table 5min, 37 ℃ of 20min;
11, PBS washing lotion is washed 3 times in groupization box, each 5min;
12, by specification requires to carry out DAB colour developing; DAB nitrite ion compound method: first dissolve 50mg3 with a small amount of TBS, 3 '-diaminobenzidine, four hydrochlorides (DAB), then supply 100ml with TBS, fully mix, and the final concentration that makes DAB is 0.05%, adds 30%H before colour developing after filtering
2o
235 μ L, filter out sediment.
13, proceed in haematoxylin box dyeing 1-2min;
14, with tap water, wash away haematoxylin loose colour, about 2-3 time, deionization bubbly water 5min;
15, in differentiation liquid, (in 70% ethanol water 985mL, adding 15 milliliters of 1mmol/L aqueous hydrochloric acid solutions) soaks 10 seconds, reduces background;
16, in alkaline solution, strengthen haematoxylin dyeing intensity; (70% ethanol water and ammoniacal liquor are with 100 milliliters: the mixed liquor of 0.5 ml volumes ratio) 5min, deionized water rinsing 3 times to add SABC nuclear staining to strengthen liquid (returning indigo plant);
17, mounting dehydration: 75% ethanol 5min, 85% ethanol 5min, 90% ethanol 5min, 95% ethanol 5min, 100% ethanol 5min, 100% ethanol 5min, dimethylbenzene II 20min, dimethylbenzene I20min;
18,2 neutral resinss are dripped with 1 milliliter of rifle head in sample center, build cover glass, note not producing bubble;
19, air-dry more than 2 hours, Microscopic observation.
PSA detects: method is with above-mentioned consistent, and different is that primary antibodie is the R & D PSA of company sheep polyclonal antibody, and Yong Zhong China fir Golden Bridge antibody diluent is diluted to working concentration 5ug/ml.
Two, test material and result statistical study
1, the tissue microarray assay of SPON2
In the prostata tissue chip PR807 that Shaanxi Chaoying Biotechnology Co., Ltd. provides, contain normal prostate tissue 3 examples, prostate cancer cancer beside organism 7 examples, benign prostatic hyperplasis (BPH) sample 20 examples, prostate cancer (PCa) sample 50 examples.Wherein in 6 routine prostate cancer samples, flake occurring or do not see prostate cancer tissue, there is flake in 1 routine benign prostatic hyperplasis sample.This experiment has been carried out immunohistochemical staining with SPON2 and PSA antibody respectively to the chip of two serial section, and the HE dyeing of the identical organization chip that company provides in contrast.
(1) first under low power lens, whether each interlacing point SPON2 is dyeed and judged, by its minute negative (one), the weak positive (±) and positive (+), its statistics is as table 4, statistics employing Fisher rigorous examination method (Fisher ' s Exact Test) analyze, find that the positive rate of SPON2 in patients with prostate cancer is apparently higher than normal person and benign prostate cancer hyperplasia patient.
(2) under 40 * object lens, each sample on chip is chosen to 5 points at random, after white balance, take pictures.Use Image-Pro Plus 6.0 softwares to calculate integral optical density (Integral optical density, the IOD) sum of choosing 5 points, average as the IOD SUM of each sample.Use SAS 6.12 software normal tissues (comprising cancer beside organism), Benign Prostatic Hyperplasia Tissues:, and the IOD SUM of prostate cancer tissue analyzes (table 5), median for staining power (interquartile range) is M (Q
r) represent, the difference between each group of P (CHi Square) < 0.05 interval scale has conspicuousness.Result SPON2 dyeing compared with normal tissue and hyperplastic prostate tissue in prostate cancer tissue sample all have obvious enhancing, and the differential expression of PSA does not have conspicuousness.Showing that this SABC detects can be used for diagnosing prostate cancer.
*: SNK method (q check) shows that in prostate cancer, SPON2 dyeing is obviously better than normal and hyperplasia of prostate prostata tissue, and has statistical significance.
(3) use SAS 6.12 software normal tissues (comprising cancer beside organism), Benign Prostatic Hyperplasia Tissues:, and the IOD SUM of the prostate cancer tissue of different WHO classification (WHO Grade) analyzes (table 6), (Q of M for staining power
r) represent, the difference between each group of P (CHi Square) < 0.05 interval scale has conspicuousness.As a result, SPON2 is classified as in the tissue of 2 grades dyeing at WHO and strengthens the most obviously, and the differential expression of PSA between each group do not have conspicuousness.
*: SNK method (q check) shows that WHO is classified as SPON2 dyeing in the prostate cancer of 2 grades and is obviously better than normal and hyperplasia of prostate prostata tissue, and has statistical significance.
(4) use SAS 6.12 software normal tissues (comprising cancer beside organism), Benign Prostatic Hyperplasia Tissues:, use SAS software to analyze (table 7), (Q of M for staining power with the IOD SUM of the prostate cancer tissue of different Gleason scorings (Gleason Score)
r) represent, the difference between each group of P (CHi Square) < 0.05 interval scale has conspicuousness.As a result, SPON2 is that in the tissue of 7～8 minutes, dyeing strengthens the most obviously in Gleason scoring, and the differential expression of PSA between each group do not have conspicuousness.
*: SNK method (q check) shows that Gleason scoring is that in the prostate cancer of 7～8 minutes, SPON2 dyeing is obviously better than normal prostate tissue, and has statistical significance.
#:SNK method (q check) shows in the prostate cancer of all Gleason scoring≤6 minutes and 7～8 minutes that SPON2 dyes and is all obviously better than hyperplasia of prostate prostata tissue, and has statistical significance.
(5) use SAS 6.12 software normal tissues (comprising cancer beside organism), Benign Prostatic Hyperplasia Tissues:, with have metastasis and use SAS software to analyze (table 8), (Q of M for staining power without the IOD SUM of the prostate cancer tissue of metastasis
r) represent, the difference between each group of P (CHi Square) < 0.05 interval scale has conspicuousness.As a result, SPON2 dyes and strengthens more obviously in having the prostate cancer tissue of metastasis, and the differential expression of PSA between each group do not have conspicuousness.
*: SNK method (q check) shows to have SPON2 dyeing in the prostate cancer of transfer to be obviously better than hyperplasia of prostate prostata tissue, and has statistical significance.
(6) PSA that in 44 routine prostate cancer tissues, each sample is corresponding and the IOD SUM of SPON2 are analyzed to the two expression of result be proportionate (Fig. 6).
(7) chosen normal structure, Benign Prostatic Hyperplasia Tissues: and different WHO classification, different Gleason mark, and have or not representational totally 6 examples of prostate cancer tissue of transfer, the contrast figure under 40 * object lens of PSA dyeing, SPON2 dyeing and HE dyeing same position in serial section.Result is as Fig. 7.
Claims (2)
1. the application of the kit of detection SPON2 in the kit for the preparation of auxiliary diagnosis prostate cancer; The kit of described detection SPON2 is for detecting the enzyme linked immunological kit of SPON2; The amino acid sequence of described SPON2 is as shown in SEQ ID NO:1;
When the kit of described detection SPON2 is for detecting the enzyme linked immunological kit of SPON2, described prostate cancer is following 1) or 2) shown in:
1) prostate cancer that the prostate cancer that Gleason scoring is the prostate cancer of 4～6 minutes, prostate cancer that Gleason scoring is 7 minutes, Gleason scoring is 8 minutes or Gleason scoring are 9～10 minutes;
2) prostate cancer of prostate specific antigen feminine gender; Described prostate specific antigen is negative is that in serum, prostate specific antigen concentration is less than or equal to 10ng/ml; The amino acid sequence of described prostate specific antigen is as shown in SEQ ID NO:2;
The polyclonal antibody of the anti-SPON2 in the monoclonal antibody of the anti-SPON2 that the enzyme linked immunological kit of described detection SPON2 is originated by mouse, sheep source, standard solution, coated damping fluid, confining liquid, lavation buffer solution, stop buffer, substrate solution and enzyme labelled antibody form;
Described coated damping fluid is comprised of sodium carbonate, sodium bicarbonate and water; The concentration of described sodium carbonate in described coated damping fluid is 1.59g/L, and the concentration of described sodium bicarbonate in described coated damping fluid is 2.93g/L;
Described lavation buffer solution is comprised of sodium chloride, potassium chloride, sodium hydrogen phosphate, potassium dihydrogen phosphate, Tween20 and water; The concentration of sodium chloride in described lavation buffer solution is 8g/L, the concentration of potassium chloride in described lavation buffer solution is 0.2g/L, the concentration of sodium hydrogen phosphate in described lavation buffer solution is 1.44g g/L, the concentration of potassium dihydrogen phosphate in described lavation buffer solution is 0.24g/L, and the concentration of Tween20 in described lavation buffer solution is 0.1%(percent by volume);
Described confining liquid is comprised of described lavation buffer solution and bovine serum albumin(BSA), and the concentration of bovine serum albumin(BSA) in described confining liquid is 10g/L;
Described stop buffer is the aqueous sulfuric acid of 2M;
Described enzyme labelled antibody is the goat anti-mouse antibody of HRP mark;
To be described confining liquid form from described SPON2 the following solution that SPON2 concentration is different to described standard solution: 100,50,25,12.5,6.3,3.1 and 1.6ng/ml;
In the enzyme linked immunological kit of described detection SPON2, the polyclonal antibody of the anti-SPON2 in described coated damping fluid and described sheep source forms the solution that antibody concentration is 630ng/ml; The monoclonal antibody of the anti-SPON2 in described confining liquid and mouse source forms the solution that antibody concentration is 5ug/ml; The solution that described confining liquid and enzyme labelled antibody constitutive enzyme labeling antibody concentration are 80ng/ml.
2. for an enzyme linked immunological kit for auxiliary diagnosis prostate cancer, by following material, formed: the antibody of anti-SPON2, standard solution, coated damping fluid, confining liquid, lavation buffer solution, stop buffer and enzyme labelled antibody; Described antibody is the monoclonal antibody of anti-SPON2 in mouse source and the polyclonal antibody of the anti-SPON2 in sheep source;
Described coated damping fluid is comprised of sodium carbonate, sodium bicarbonate and water; The concentration of described sodium carbonate in described coated damping fluid is 1.59g/L, and the concentration of described sodium bicarbonate in described coated damping fluid is 2.93g/L;
Described lavation buffer solution is comprised of sodium chloride, potassium chloride, sodium hydrogen phosphate, potassium dihydrogen phosphate, Tween20 and water; The concentration of sodium chloride in described lavation buffer solution is 8g/L, the concentration of potassium chloride in described lavation buffer solution is 0.2g/L, the concentration of sodium hydrogen phosphate in described lavation buffer solution is 1.44g g/L, the concentration of potassium dihydrogen phosphate in described lavation buffer solution is 0.24g/L, and the concentration expressed in percentage by volume of Tween20 in described lavation buffer solution is 0.1%;
Described confining liquid is comprised of described lavation buffer solution and bovine serum albumin(BSA), and the concentration of bovine serum albumin(BSA) in described confining liquid is 10g/L;
Described stop buffer is the aqueous sulfuric acid of 2M; Described enzyme labelled antibody is the goat anti-mouse antibody of HRP mark;
To be described confining liquid form from described SPON2 the following solution that SPON2 concentration is different to described standard solution: 100,50,25,12.5,6.3,3.1 and 1.6ng/ml; The polyclonal antibody of the anti-SPON2 in described coated damping fluid and described sheep source forms the solution that antibody concentration is 630ng/ml; The monoclonal antibody of the anti-SPON2 in described confining liquid and mouse source forms the solution that antibody concentration is 5ug/ml; The solution that described confining liquid and enzyme labelled antibody constitutive enzyme labeling antibody concentration are 80ng/ml.
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