CN102346184A - 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 among the north america male sex, and the patient that the U.S. in 2008 newly is diagnosed as prostate cancer accounts for this year and newly is 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 present focus of research.Desirable tumour serum mark should have following characteristics: 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 tumor treatment recruitment evaluation, can be used in the transfer and the recurrence of prediction tumour.
PSA uses prostate cancer blood serum designated object the most widely at present.Although the PSA examination has brought many benefits, also endure dispute and query in recent years to the fullest extent.In the last few years, find that the specificity to prostate cancer of PSA detection is relatively 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.Also have some marks, perhaps clinical practice is comparatively limited to, and perhaps effect is not very desirable.Searching can substituting PS A or the serodiagnosis mark that remedies the PSA defective be very necessary.
(Mindin DIL-1) belongs to the extracellular matrix proteins class, because of differential expression in the alveolar cell of canceration and non-canceration is gained the name to vertebra albumen 2 for SPON2, Spondin-2.The amino acid sequence of vertebra albumen 2 is SEQ ID NO:1.
The amino acid sequence of prostate specific antigen (PSA) is shown in SEQ ID NO:2.
Summary of the invention
An object of the present invention is to provide a kind of anti-vertebra albumen 2 antibody, detect the new purposes of the kit of vertebra albumen 2.
The new purposes of the antibody of anti-vertebra albumen 2 is the antibody of anti-vertebra albumen 2 is used for the kit of auxiliary diagnosis prostate cancer in preparation application.
The antibody of said anti-vertebra albumen 2 is the monoclonal antibody of anti-vertebra albumen 2 and/or the polyclonal antibody of anti-vertebra albumen 2;
The monoclonal antibody of said anti-vertebra albumen 2 is the monoclonal antibody of the anti-vertebra albumen 2 in mouse source; The polyclonal antibody of said anti-vertebra albumen 2 is the polyclonal antibody of the anti-vertebra albumen 2 in sheep source.
Above-mentioned antibody all is the antibody that obtains from commercial sources, or cultivates the antibody that obtains through cells in vitro; The polyclonal antibody of the anti-vertebra albumen 2 in sheep source specifically can be available from R&D company, and catalog number is AF-2609; The monoclonal antibody of the anti-vertebra albumen 2 in mouse source specifically can be available from Abnova company, and catalog number is H00010417-M01J.
The new purposes that detects the kit of vertebra albumen 2 is the kit of detection vertebra albumen 2 is used for the kit of auxiliary diagnosis prostate cancer in preparation application.
The kit that detects vertebra albumen 2 is enzyme linked immunological kit that detects vertebra albumen 2 or the SABC kit that detects vertebra albumen 2.
Vertebra albumen 2 also belongs to protection scope of the present invention in the application of kit that design and/or preparation are used for the auxiliary diagnosis prostate cancer.
The enzyme linked immunological kit of above-mentioned arbitrary said detection vertebra albumen 2 is used in the application of kit of auxiliary diagnosis prostate cancer in preparation, and said prostate cancer is following 1) or 2) shown in:
1) prostate cancer that prostate cancer that Gleason scoring is 4～6 minutes prostate cancer, prostate cancer that the Gleason scoring is 7 minutes, the Gleason scoring is 8 minutes or Gleason scoring are 9～10 minutes;
2) the negative prostate cancer of prostate specific antigen;
The SABC kit that the kit that detects vertebra albumen 2 detects vertebra albumen 2 is used in the application of kit of auxiliary diagnosis prostate cancer in preparation, and said prostate cancer is shown in following I or II or the III:
I, Gleason scoring are 7～8 minutes prostate cancer or≤6 minutes prostate cancer of Gleason scoring;
II, WHO scoring are 2 grades prostate cancer;
III, the prostate cancer of metastasis is arranged.
The enzyme linked immunological kit of above-mentioned arbitrary said detection vertebra albumen 2 is used in the application of kit of auxiliary diagnosis prostate cancer in preparation, said prostate specific antigen feminine gender be in the serum prostate specific antigen concentration smaller or equal to 10ng/ml.
The enzyme linked immunological kit of above-mentioned arbitrary said detection vertebra albumen 2 is used in the application of kit of auxiliary diagnosis prostate cancer in preparation, the enzyme linked immunological kit of said detection vertebra albumen 2 by polyclonal antibody, the standard solution of the anti-vertebra albumen 2 in the monoclonal antibody of the anti-vertebra albumen 2 in mouse source, sheep source, encapsulate damping fluid, confining liquid, lavation buffer solution, stop buffer, substrate solution and enzyme labelled antibody and form;
The said damping fluid that encapsulates is made up of sodium carbonate, soda mint and water; Said sodium carbonate is 1.59g/L in the said concentration that encapsulates in the damping fluid, and said soda mint is 2.93g/L in the said concentration that encapsulates in the damping fluid;
Said lavation buffer solution is made up of sodium chloride, potassium chloride, sodium hydrogen phosphate, potassium dihydrogen phosphate, Tween 20 and water; The concentration of sodium chloride in said lavation buffer solution is 8g/L; The concentration of potassium chloride in said lavation buffer solution is 0.2g/L; The concentration of sodium hydrogen phosphate in said lavation buffer solution is 1.44g g/L; The concentration of potassium dihydrogen phosphate in said lavation buffer solution is 0.24g/L, and the concentration of Tween 20 in said lavation buffer solution is 0.1% (percent by volume);
Said confining liquid is made up of said lavation buffer solution and bovine serum albumin(BSA), and the concentration of bovine serum albumin(BSA) in said confining liquid is 10g/L;
Said stop buffer is the aqueous sulfuric acid of 2M;
Said enzyme labelled antibody is the anti-mouse antibodies of HRP labelled goat;
Said standard solution is that said confining liquid and said vertebra albumen 2 are formed the different following solution of vertebra albumen 2 concentration: 100,50,25,12.5,6.3,3.1 and 1.6ng/ml.
The enzyme linked immunological kit and the SABC kit of above-mentioned detection vertebra albumen 2 also can obtain from commercial sources; The SABC kit of above-mentioned detection vertebra albumen 2 specifically can be goat two step method SABC kit, specifically can be available from biotech firm of middle China fir Golden Bridge, and catalog number is PV-9003.
The enzyme linked immunological kit of above-mentioned arbitrary said detection vertebra albumen 2 is used in the application of kit of auxiliary diagnosis prostate cancer in preparation; In the enzyme linked immunological kit of said detection vertebra albumen 2, it is the solution of 630ng/ml that the said polyclonal antibody that encapsulates the anti-vertebra albumen 2 in damping fluid and said sheep source is formed antibody concentration; It is the solution of 5ug/ml that the monoclonal antibody of the anti-vertebra albumen 2 in said confining liquid and mouse source is formed antibody concentration; Said confining liquid and enzyme labelled antibody constitutive enzyme labeling antibody concentration are the solution of 80ng/ml;
The amino acid sequence of said vertebra albumen 2 is shown in SEQ ID NO:1; The amino acid sequence of said prostate specific antigen is shown in SEQ ID NO:2.
Another object of the present invention provides a kind of enzyme linked immunological kit that is used for the auxiliary diagnosis prostate cancer.
The enzyme linked immunological kit that is used for the auxiliary diagnosis prostate cancer provided by the present invention is made up of following material: the antibody of above-mentioned arbitrary said anti-vertebra albumen 2, standard solution, encapsulate damping fluid, confining liquid, lavation buffer solution, stop buffer and enzyme labelled antibody.
In the above-mentioned enzyme linked immunological kit, said antibody is the polyclonal antibody of monoclonal antibody and the sheep of the anti-vertebra albumen 2 in the mouse source anti-vertebra albumen 2 of originating;
The said damping fluid that encapsulates is made up of sodium carbonate, soda mint and water; Said sodium carbonate is 1.59g/L in the said concentration that encapsulates in the damping fluid, and said soda mint is 2.93g/L in the said concentration that encapsulates in the damping fluid;
Said lavation buffer solution is made up of sodium chloride, potassium chloride, sodium hydrogen phosphate, potassium dihydrogen phosphate, Tween 20 and water; The concentration of sodium chloride in said lavation buffer solution is 8g/L; The concentration of potassium chloride in said lavation buffer solution is 0.2g/L; The concentration of sodium hydrogen phosphate in said lavation buffer solution is 1.44g g/L; The concentration of potassium dihydrogen phosphate in said lavation buffer solution is 0.24g/L, and the concentration of Tween 20 in said lavation buffer solution is 0.1% (percent by volume);
Said confining liquid is made up of said lavation buffer solution and bovine serum albumin(BSA), and the concentration of bovine serum albumin(BSA) in said confining liquid is 10g/L;
Said stop buffer is the aqueous sulfuric acid of 2M;
Said enzyme labelled antibody is the anti-mouse antibodies of HRP labelled goat;
Said standard solution is that said confining liquid and said vertebra albumen 2 are formed the different following solution of vertebra albumen 2 concentration: 100,50,25,12.5,6.3,3.1 and 1.6ng/ml.
In above-mentioned arbitrary said enzyme linked immunological kit, it is the solution of 630ng/ml that the said polyclonal antibody that encapsulates the anti-vertebra albumen 2 in damping fluid and said sheep source is formed antibody concentration; It is the solution of 5ug/ml that the monoclonal antibody of the anti-vertebra albumen 2 in said confining liquid and mouse source is formed antibody concentration; Said confining liquid and enzyme labelled antibody constitutive enzyme labeling antibody concentration are the solution of 80ng/ml.
The experiment proof; Use the enzyme linked immunological kit that detects SPON2 to 13 routine normal persons; 4 routine benign prostatic hyperplasiss; SPON2 in the 72 routine patients with prostate cancer serum detects; The content of SPON2 is significantly higher than normal person or benign prostatic hyperplasis person in the patients with prostate cancer as a result, shows that the content through detecting SPON2 in the serum can diagnosing prostate cancer; Especially outstanding is; Content and the normal person difference of SPON2 in 4～6 minutes patients with prostate cancer serum of Gleason scoring is extremely remarkable; SPON2 is extremely remarkable less than the content in the 10ng/ml patients with prostate cancer serum and normal person's difference in the PSA level, and it is higher more accurate to prove with SPON2 diagnosing prostate cancer degree of accuracy.
Experiment also proves, with the SABC kit that detects SPON2 normal and prostate cancer beside organism, 19 routine benign prostatic hyperplasiss and 44 routine prostate cancer tissues detections to 10 examples.SPON2 obviously raises than positive rate in normal structure and the benign prostatic hyperplasis tissue in prostate cancer tissue as a result, expresses to strengthen; 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 takes place to shift strengthens more remarkable; The staining power of SPON2 and PSA are proportionate in the prostate cancer; But it is expressed and the benign lesion obvious difference, is far superior to PSA.It is higher more accurate with SPON2 diagnosing prostate cancer degree of accuracy to prove.
Description of drawings
Fig. 1 is the typical curve that double fastener heart ELISA method detects SPON2 concentration.
Fig. 2 obviously raises than normal person and Benign Prostatic Hypertrophy in patients with prostate cancer serum for SPON2.Horizontal line is represented meta numerical value, and difference is carried out statistical study with Willcoxon Two-Sample Test.
Fig. 3 helps the diagnosis of Serum PSA level less than the 10ng/ml patients with prostate cancer for SPON2.
Fig. 4 only expresses in the positive prostate cancer cell line of AR for sxemiquantitative RT-PCR confirms SPON2.
Fig. 5 only expresses in the positive prostate cancer cell line of AR for extracellular protein Western Blot confirms SPON2.
Fig. 6 is that the staining power of PSA in the tissue specimen of patients with prostate cancer and the staining power of SPON2 are proportionate.The P value is calculated with Spearman is relevant.(r=Spearman related coefficient).
Fig. 7 be SPON2 and PSA at normal prostate tissue, the benign prostatic hyperplasis tissue is marked with different Gleason, the expression in different WHO classification and the TNM prostate cancer tissue by stages.As reference, photo is depicted as the same position of same interlacing point on the organization chip of serial section with HE dyeing.
Embodiment
Employed experimental technique is conventional method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
The nutrient culture media that uses among the following embodiment, antibody, chemical reagent are following:
Title place of production article No.
The homemade analysis of sodium chloride is pure
The homemade analysis of potassium chloride is pure
The homemade analysis of sodium hydrogen phosphate is pure
The homemade analysis of potassium dihydrogen phosphate is pure
The ZB-2305 of China fir Golden Bridge in the HRP mark goat anti-mouse
IgG(H+L)
The SPON2 sheep polyclonal antibody R&D AF-2609 of company
(Anti-human?Mindin
Antibody)
The biological PV-9003 of China fir Golden Bridge in the goat two step method SABC kit
The biological ZLI-9061 of China fir Golden Bridge in the special-purpose phosphate buffer of SABC
(0.01M?PH＝7.2～7.4)
Citrate antigen retrieval liquid (the biological ZLI9064 of China fir Golden Bridge in 0.01
M?PH＝6.0)
The biological ZLI-9031 of China fir Golden Bridge in the DAB chromogenic reagent box
The ultra English biotechnology in prostate cancer tissue chip Shaanxi (numbering PR807)
The homemade analysis of xylene is pure
The homemade analysis of neutral gum is pure
The biological ZLI-9610 of China fir Golden Bridge in the haematoxylin
The homemade analysis of ammoniacal liquor is pure
The homemade analysis of soda mint is pure
The RF101 of bovine serum albumin (BSA) U.S. Proliant company
Solubility single component tmb substrate dissolves a day root science and technology PA107-01
Liquid
The homemade 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 that uses among the following embodiment is following:
The biological ZLI-9305 of China fir Golden Bridge in the SABC pen
96 hole ELISA Plate Corning Costar 9018
IX70 fluorescence inverted microscope Olympus
Enzyme joins detector Bio-Rad
In the present embodiment, the content value of SPON2 or PSA is all used M (Q in the detection sample
R) expression, M representes the median of SPON2 content in the sample; Q
RThe interquartile range of SPON2 content, i.e. Q in the expression sample
RThe absolute value of the difference of=upper quartile value and lower quartile numerical value.
One, kit is formed
1, coated antibody: the polyclonal antibody (SPON2 sheep polyclonal antibody) of the anti-vertebra albumen 2 in sheep source.
2, binding antibody: the monoclonal antibody (SPON2 mouse monoclonal antibody) of the anti-vertebra albumen 2 in mouse source.
3, standard items: vertebra albumen 2.Confining liquid and vertebra albumen 2 is formed the different following solution of vertebra albumen 2 concentration: 100,50,25,12.5,6.3,3.1 and 1.6ng/ml.
4, encapsulate damping fluid: form by sodium carbonate, soda mint and water; Said sodium carbonate is 1.59g/L in the said concentration that encapsulates in the damping fluid, and said soda mint is 2.93g/L in the said concentration that encapsulates in the damping fluid;
5, lavation buffer solution: form by sodium chloride, potassium chloride, sodium hydrogen phosphate, potassium dihydrogen phosphate, Tween 20 and water; The concentration of sodium chloride in said lavation buffer solution is 8g/L; The concentration of potassium chloride in said lavation buffer solution is 0.2g/L; The concentration of sodium hydrogen phosphate in said lavation buffer solution is 1.44g/L; The concentration of potassium dihydrogen phosphate in said lavation buffer solution is 0.24g/L, and the concentration of Tween 20 in said lavation buffer solution is 0.1% (percent by volume);
6, confining liquid: be made up of said lavation buffer solution and bovine serum albumin(BSA), the concentration of bovine serum albumin(BSA) in said confining liquid is 10g/L.
7, the aqueous sulfuric acid of stop buffer: 2M;
8, the composition of substrate solution: tetramethyl benzidine (3,3 ', 5,5 '-Tetramethylbenzidine, aqueous solution TMB).
9, enzyme labelled antibody: the anti-mouse antibodies of HRP labelled goat.
10, following solution:
1) solution of coated antibody: by encapsulating damping fluid and coated antibody is formed, the concentration of coated antibody in solution is 630ng/ml;
2) solution of binding antibody: be made up of confining liquid and binding antibody, the concentration of binding antibody in solution is 5ug/ml;
3) solution of enzyme labelled antibody: be made up of confining liquid and enzyme labelled antibody, the concentration of enzyme labelled antibody in solution is 80ng/ml.
Two, kit preparation
Carbonate encapsulates damping fluid and prepares according to following method: in the 900ml deionized water, add 1.59g sodium carbonate and 2.93g soda mint, deionized water is settled to 1L.
ELISA lavation buffer solution (PBST) is prepared according to following method: in 900ml distilled water, add 8g sodium chloride; 0.2g potassium chloride; 1.44g sodium hydrogen phosphate; 0.24g potassium dihydrogen phosphate; Transfer pH value to 7.4; Be settled to 1L with distilled water, be cooled to room temperature behind the autoclaving, use behind adding 0.1% (percent by volume) Tween 20 reagent mixings.
ELISA confining liquid: add 10g bovine serum albumin(BSA) (BSA) to above-mentioned ELISA lavation buffer solution, be settled to 1L with the ELISA lavation buffer solution.
ELISA reaction terminating liquid: 2M aqueous sulfuric acid.
Three, the application of kit
(1) foundation of SPON2 double fastener heart ELISA typical curve
1, encapsulates damping fluid dilution SPON2 sheep polyclonal antibody (Anti-human Mindin Antibody) 630ng/ml with carbonate, place the every hole 100 μ l of elisa plate to encapsulate for 4 ℃ and spend the night;
2, wash elisa plate 3 times with the ELISA lavation buffer solution;
3, add the every hole of ELISA confining liquid 100 μ l, 37 ℃ of 2h;
4, be respectively 100,50,25 with ELISA confining liquid gradient dilution SPON2 standard items (SPON2 Recombinant Protein (P01)) concentration; 12.5,6.3,3.1 and 1.6ng/ml; Get do not add standard items the ELISA confining liquid as blank, each concentration is done two repetitions.Every hole 100 μ l place elisa plate, 37 ℃ of 2h;
5, wash elisa plate 3 times with the ELISA lavation buffer solution;
6, dilute SPON2 mouse monoclonal antibody (SPON2monoclonal antibody (M01J), clone 3C6) to 5 μ g/ml with the ELISA confining liquid, every hole 100 μ l place elisa plate, 37 ℃ of 2h;
7, wash elisa plate 3 times with the ELISA lavation buffer solution;
8, dilute HRP mark goat anti-mouse IgG (H+L) to 80ng/ml with the ELISA confining liquid, every hole 100 μ l place elisa plate, 37 ℃ of 2h;
9, wash elisa plate 3 times with the ELISA lavation buffer solution;
10, every hole adds 100 μ l soluble T MB single component substrate solutions, lucifuge 15-20min;
11, add the every hole of solution stopping 100 μ l stopped reactions among the ELISA;
12, read every hole 450nm light absorption reading, deduct 570nm light absorption reading, be this hole reading, get the reading of the mean value in two holes as this concentration;
13, after the reading of each concentration deducts the blank reading, take the logarithm, with the logarithm fit line sexual intercourse of standard items concentration.
The typical curve that obtains as shown in Figure 1.Y＝1.5763x+2.5237，R
2＝0.9768.
(2) detect patient's serum with the double fastener heart ELISA method of setting up
Detect 89 parts of human serum samples, comprising: the normal the elderly (Normal) of 13 examples, 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 the elderly of serum specimen.
Detection method is following:
1, encapsulates damping fluid dilution SPON2 sheep polyclonal antibody (Anti-human Mindin Antibody) 630ng/ml with carbonate, place the every hole 100 μ l of elisa plate to encapsulate for 4 ℃ and spend the night;
2, wash elisa plate 3 times with the ELISA lavation buffer solution;
3, add the every hole of ELISA confining liquid 100 μ l, 37 ℃ of 2h;
4, press test serum: ELISA confining liquid=1: 2 dilution test serum, get do not add standard items the ELISA confining liquid as blank.Every hole 100 μ l place elisa plate, 37 ℃ of 2h;
5, wash elisa plate 3 times with the ELISA lavation buffer solution;
6, dilute SPON2 mouse monoclonal antibody (SPON2monoclonal antibody (M01J), clone 3C6) to 5 μ g/ml with the ELISA confining liquid, every hole 100 μ l place elisa plate, 37 ℃ of 2h;
7, wash elisa plate 3 times with the ELISA lavation buffer solution;
8, dilute HRP mark goat anti-mouse IgG (H+L) to 80ng/ml with the ELISA confining liquid, every hole 100 μ l place elisa plate, 37 ℃ of 2h;
9, wash elisa plate 3 times with the ELISA lavation buffer solution;
10, every hole adds 100 μ l soluble T MB single component substrate solutions, lucifuge 15-20min;
11, add the every hole of solution stopping 100 μ l stopped reactions among the ELISA;
12, read every hole 450nm light absorption reading, deduct 570nm light absorption reading, be this hole reading;
13, after the reading in each hole deducts the blank reading, take the logarithm, the substitution typical curve is got 3 * lg
(1)(substitution curve income value) is serum SPON2 concentration.
Whether the content difference with SPON2 in the content of SPON2 in the Willcoxon Two-Sample Test statistical study normal population and the patients with prostate cancer colony is remarkable.
Result such as table 1 are with shown in Figure 2.The result: SPON2 content has tangible rise (* * *, P is less than 0.001) than normal person or all benign lesion persons in the patients with prostate cancer serum.Use Willcoxon Two-Sample Test and confirm that its difference has statistical significance.Show, can use this kit diagnosing prostate cancer.
Table 1. is used double fastener heart ELISA and is detected the differential expression (unit ng/ml) of SPON2 in normal person, hyperplasia of prostate person and patients with prostate cancer serum
(3) the mark serum SPON2 concentration of patients with prostate cancer of the SPON2 concentration in the normal human serum and different Gleason relatively
The Gleason 70 routine patients with prostate cancer of marking are divided into grade described in the table 2.Whether the content difference with SPON2 in the patients with prostate cancer colony of the content of SPON2 in SNK method (q check) the statistical study normal population and different Gleason scorings is remarkable.
The serum SPON2 concentration of SPON2 concentration in the normal human serum with different Gleason scoring patients with prostate cancer is compared.The prostate cancer serum SPON2 expression that the result shows each Gleason scoring (4～6 minutes, 7 minutes, 8 minutes, 9～10 minutes) like table 2:SNK method (q check) is apparently higher than normal person (*), and has statistical significance; And Gleason scoring to be 4～6 minutes patients with prostate cancer (#) serum SPON2 express is higher than other Gleason scoring groups (7 minutes, 8 minutes, 9～10 minutes) and has statistical significance.
Table 2, application double fastener heart ELISA detect the differential expression (unit ng/ml) of SPON2 in the patients with prostate cancer serum of normal person and different Gleason scoring
(4) the serum SPON2 concentration ratio of the SPON2 concentration in the normal human serum and blood-serum P SA≤10ng/ml patients with prostate cancer
The detection method of prostate specific antigen PSA concentration in the serum: Applied Electrochemistry luminescence method; Use RocheElecsys 2010 fully automatic electric chemical illumination immunity analysis instruments and supporting PSA reagent thereof (the German Luo Shi diagnosis company limited by forming a complete production network with instrument provides), all use in the phase in effect.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 the normal human serum and blood-serum P SA≤10ng/ml patients with prostate cancer is compared, and the blood-serum P SA concentration to them also compares simultaneously.The SPON2 level is all apparently higher than normal the elderly in the patients with prostate cancer serum of the patients with prostate cancer of result: PSA≤10ng/ml (*) and PSA＞10ng/ml (*).Use Willcoxon Two-Sample Test and confirm that it has statistical significance (p equals 0.0013 and 0.0001 respectively).And in the patient of PSA≤10ng/ml, PSA level and normal the elderly and no significant difference (p=0.2852).Illustrate: SPON2 helps the diagnosis of Serum PSA level less than the prostate patient of 10ng/ml, and the Serum PSA level of these patients with prostate cancer and normal person and 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 the 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 that the prostate cancer bone shifts, and androgen receptor is negative; C4-2B is developed by LNCaP, and the clone for bone shifts equally also has the character of the non-dependence of androgen, and AR is positive; DU145 is the clone that the prostate cancer brain shifts, and androgen receptor is negative.
Result such as Fig. 4 and shown in Figure 5.The result shows that SPON2 has the prostate cancer cell line specificity, only in the positive prostate cancer cell line of AR, expresses.
Embodiment 3, SABC method diagnosing prostate cancer
One, immunohistochemical staining analysis (organization chip) method of SPON2 in prostata tissue is following:
1, roasting sheet dewaxes to water: xylene I20min, xylene II 20min, 100% ethanol 5min, 100% ethanol 5min, 95% ethanol 5min, 90% ethanol 5min, 85% ethanol 5min, 75% ethanol 5min, deionized water 5min then;
2, repair liquid with citrate and carry out antigen retrieval;
Citrate is repaired the liquid storage liquid:
A.0.1M citric acid soln: take by weighing 21.01g citric acid (C
6H
8O
7H
2O) be dissolved in the 1000ml distilled water.
B.0.1M liquor sodii citratis: take by weighing 29.41g sodium citrate (C
6H
5Na
3O
72H
2O) be dissolved in the 1000ml distilled water.
Citrate is repaired the liquid working fluid: get 9mlA liquid and 41ml B liquid and add in the 450ml distilled water, the pH value of solution value should be 6.0).
3, behind the groupization pen delineation conversion zone, drip oxydol 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 mixing and gets final product) in the groupization box, wash each 5min 3 times;
5, China fir Golden Bridge antibody diluent dilutes the R&D SPON2 of company sheep polyclonal antibody (Anti-human Mindin Antibody) to working concentration 4ug/ml in the usefulness;
Middle China fir 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 transfers 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% (promptly 1: 10-20 doubly dilutes) promptly obtains antibody diluent.The serum of the animal that is chosen as two anti-sources of general serum kind.
6, one anti-(being the SPON2 of R&D company sheep polyclonal antibody working fluid) dripped and made to the sample center (30-50 μ l), and after wet box shook up shaking table low speed 5min, 4 ℃ were spent the night;
7, the PBS washing lotion is washed in the groupization box 3 times, each 5min;
8, drip in the goat two step method kit one of reagent 1 (polymerization assistant), dial with the rifle head even, shaking table 5min, 37 ℃ of 20min;
9, the PBS washing lotion is washed in the groupization box 3 times, each 5min;
10, add one of reagent 2 (the anti-goat IgG polymer of horseradish enzyme labeling), dial with the rifle head even, shaking table 5min, 37 ℃ of 20min;
11, the PBS washing lotion is washed in the groupization box 3 times, each 5min;
12, by specification requires to carry out the DAB colour developing; The DAB liquid making method that develops the color: with a small amount of TBS dissolving 50mg3,3 '-diaminobenzidine, four hydrochlorides (DAB) are supplied 100ml with TBS then earlier, abundant mixing, and the final concentration that makes DAB is 0.05%, adds 30%H before filtering the back colour developing
2O
235 μ L filter out sediment and get final product.
13, change in the haematoxylin box dyeing 1-2min over to;
14, with tap water flush away haematoxylin loose colour, about 2-3 time, deionization bubbly water 5min;
15, (add 15 milliliters of 1mmol/L aqueous hydrochloric acid solutions among the 70% ethanol water 985mL) in the differentiation liquid and soaked 10 seconds, reduce background;
16, strengthen haematoxylin dyeing intensity in the alkaline solution; (70% ethanol water and ammoniacal liquor are with 100 milliliters: the 5min mixed liquor of 0.5 ml volumes ratio), deionized water rinsing 3 times to add SABC nuclear staining enhancing 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, xylene II 20min, xylene I20min;
18,2 neutral resinss are dripped with 1 milliliter of rifle head in the sample center, build cover glass, note not producing bubble;
19, air-dry more than 2 hours, mirror is observed down.
PSA detects: method is with above-mentioned consistent, different is one anti-be the PSA of R&D company sheep polyclonal antibody, with in China fir Golden Bridge antibody diluent be diluted to working concentration 5ug/ml.
Two, test material and statistical study as a result
1, the organization chip analysis of SPON2
Among 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.Flake wherein takes place in the 6 routine prostate cancer samples or do not see prostate cancer tissue, flake appears 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 is as contrast.
(1) at first under low power lens, whether each interlacing point SPON2 dyeed and judge; With its branch negative (), the weak positive (±) and positive (+); Its statistics such as table 4; Statistics employing Fisher rigorous examination method (Fisher ' s Exact Test) to analyze, the positive rate of finding SPON2 in patients with prostate cancer is apparently higher than normal person and benign prostate cancer hyperplasia patient.
(2) under 40 * object lens,, take pictures after the white balance to 5 points of each sample picked at random on the chip.Utilization Image-Pro Plus 6.0 computed in software are chosen the integral optical density of 5 points, and (Integral optical density, IOD) sum are averaged as the IOD SUM of each sample.Utilization SAS 6.12 softwares are to normal structure (comprising cancer beside organism), and the IOD SUM of benign prostatic hyperplasis tissue and prostate cancer tissue analyzes (table 5), and staining power is M (Q with median (interquartile range)
R) expression, the difference between each group of P (CHi Square)＜0.05 interval scale has conspicuousness.Normal tissue of result's SPON2 dyeing in the prostate cancer tissue sample and hyperplastic prostate tissue 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 SPON2 dyeing obviously is better than normal and hyperplasia of prostate prostata tissue in the prostate cancer, and has statistical significance.
(3) utilization SAS 6.12 softwares are to normal structure (comprising cancer beside organism), and the benign prostatic hyperplasis tissue is analyzed (table 6) with the IOD SUM of the prostate cancer tissue of different WHO classifications (WHO Grade), and staining power is with M (Q
R) expression, the difference between each group of P (CHi Square)＜0.05 interval scale has conspicuousness.As a result, SPON2 is classified as in 2 grades the tissue 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 that SPON2 dyeing obviously is better than normal and hyperplasia of prostate prostata tissue in 2 grades the prostate cancer, and has statistical significance.
(4) utilization SAS 6.12 softwares are to normal structure (comprising cancer beside organism), and benign prostatic hyperplasis tissue uses SAS software to analyze (table 7) with the IOD SUM of the prostate cancer tissue of different Gleason scorings (Gleason Score), and staining power is with M (Q
R) expression, the difference between each group of P (CHi Square)＜0.05 interval scale has conspicuousness.As a result, SPON2 is that dyeing strengthens the most obviously in 7～8 minutes the tissue 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 SPON2 dyeing obviously is better than normal prostate tissue in 7～8 minutes the prostate cancer, and has statistical significance.
#:SNK method (q check) shows that SPON2 dyes in all Gleason scoring≤6 minutes and 7～8 minutes the prostate cancer and all obviously is better than the hyperplasia of prostate prostata tissue, and has statistical significance.
(5) utilization SAS 6.12 softwares are to normal structure (comprising cancer beside organism), benign prostatic hyperplasis tissue and metastasis is arranged and the IOD SUM of the prostate cancer tissue of no metastasis uses SAS software to analyze (table 8), and staining power is with M (Q
R) expression, the difference between each group of P (CHi Square)＜0.05 interval scale has conspicuousness.As a result, SPON2 dyes in the prostate cancer tissue of metastasis is arranged and strengthens more obviously, and the differential expression of PSA between each group do not have conspicuousness.
*: SNK method (q check) shows that SPON2 dyeing obviously is better than the hyperplasia of prostate prostata tissue in the prostate cancer that transfer is arranged, and has statistical significance.
(6) corresponding PSA of each sample in the 44 routine prostate cancer tissues and the IOD SUM of SPON2 are analyzed the expression of the two as a result be proportionate (Fig. 6).
(7) chosen normal structure; Benign prostatic hyperplasis tissue and different WHO classification; Different Gleason mark, and have or not representational totally 6 examples of prostate cancer tissue of transfer, the map under 40 * object lens of PSA dyeing, SPON2 dyeing and HE dyeing same position in serial section.Result such as Fig. 7.
Claims (10)
1. the antibody of anti-vertebra albumen 2 is used for the application of the kit of auxiliary diagnosis prostate cancer in preparation.
2. application according to claim 1 is characterized in that: the antibody of said anti-vertebra albumen 2 is the monoclonal antibody of anti-vertebra albumen 2 and/or the polyclonal antibody of anti-vertebra albumen 2;
The monoclonal antibody of said anti-vertebra albumen 2 is the monoclonal antibody of the anti-vertebra albumen 2 in mouse source; The polyclonal antibody of said anti-vertebra albumen 2 is the polyclonal antibody of the anti-vertebra albumen 2 in sheep source.
3. the kit that detects vertebra albumen 2 is used for the application of the kit of auxiliary diagnosis prostate cancer in preparation;
Or vertebra albumen 2 is used for the application of the kit of auxiliary diagnosis prostate cancer in design and/or preparation.
4. according to arbitrary described application among the claim 1-3, it is characterized in that: the kit of said detection vertebra albumen 2 is enzyme linked immunological kit that detects vertebra albumen 2 or the SABC kit that detects vertebra albumen 2.
5. according to arbitrary described application among the claim 1-4, it is characterized in that: the kit of said detection vertebra albumen 2 is for detecting the enzyme linked immunological kit of vertebra albumen 2, and said prostate cancer is following 1) or 2) shown in:
1) prostate cancer that prostate cancer that Gleason scoring is 4～6 minutes prostate cancer, prostate cancer that the Gleason scoring is 7 minutes, the Gleason scoring is 8 minutes or Gleason scoring are 9～10 minutes;
2) the negative prostate cancer of prostate specific antigen;
The kit of said detection vertebra albumen 2 is for detecting the SABC kit of vertebra albumen 2, and said prostate cancer is shown in following I or II or the III:
I, Gleason scoring are 7～8 minutes prostate cancer or≤6 minutes prostate cancer of Gleason scoring;
II, WHO scoring are 2 grades prostate cancer;
III, the prostate cancer of metastasis is arranged.
6. according to arbitrary described application among the claim 1-5, it is characterized in that:
The kit of said detection vertebra albumen 2 is for detecting the enzyme linked immunological kit of vertebra albumen 2, said prostate specific antigen feminine gender be in the serum prostate specific antigen concentration smaller or equal to 10ng/ml;
The enzyme linked immunological kit of said detection vertebra albumen 2 by polyclonal antibody, the standard solution of the anti-vertebra albumen 2 in the monoclonal antibody of the anti-vertebra albumen 2 in mouse source, sheep source, encapsulate damping fluid, confining liquid, lavation buffer solution, stop buffer, substrate solution and enzyme labelled antibody and form;
The said damping fluid that encapsulates is made up of sodium carbonate, soda mint and water; Said sodium carbonate is 1.59g/L in the said concentration that encapsulates in the damping fluid, and said soda mint is 2.93g/L in the said concentration that encapsulates in the damping fluid;
Said lavation buffer solution is made up of sodium chloride, potassium chloride, sodium hydrogen phosphate, potassium dihydrogen phosphate, Tween 20 and water; The concentration of sodium chloride in said lavation buffer solution is 8g/L; The concentration of potassium chloride in said lavation buffer solution is 0.2g/L; The concentration of sodium hydrogen phosphate in said lavation buffer solution is 1.44g g/L; The concentration of potassium dihydrogen phosphate in said lavation buffer solution is 0.24g/L, and the concentration of Tween 20 in said lavation buffer solution is 0.1% (percent by volume);
Said confining liquid is made up of said lavation buffer solution and bovine serum albumin(BSA), and the concentration of bovine serum albumin(BSA) in said confining liquid is 10g/L;
Said stop buffer is the aqueous sulfuric acid of 2M;
Said enzyme labelled antibody is the anti-mouse antibodies of HRP labelled goat;
Said standard solution is that said confining liquid and said vertebra albumen 2 are formed the different following solution of vertebra albumen 2 concentration: 100,50,25,12.5,6.3,3.1 and 1.6ng/ml.
7. according to arbitrary described application among the claim 1-6, it is characterized in that:
In the enzyme linked immunological kit of said detection vertebra albumen 2, it is the solution of 630ng/ml that the said polyclonal antibody that encapsulates the anti-vertebra albumen 2 in damping fluid and said sheep source is formed antibody concentration; It is the solution of 5ug/ml that the monoclonal antibody of the anti-vertebra albumen 2 in said confining liquid and mouse source is formed antibody concentration; Said confining liquid and enzyme labelled antibody constitutive enzyme labeling antibody concentration are the solution of 80ng/ml;
The amino acid sequence of said vertebra albumen 2 is shown in SEQ ID NO:1; The amino acid sequence of said prostate specific antigen is shown in SEQ ID NO:2.
8. enzyme linked immunological kit that is used for the auxiliary diagnosis prostate cancer is made up of following material: the antibody of anti-vertebra albumen 2, standard solution, encapsulate damping fluid, confining liquid, lavation buffer solution, stop buffer and enzyme labelled antibody.
9. enzyme linked immunological kit according to claim 8 is characterized in that: said antibody is the polyclonal antibody of monoclonal antibody and the sheep of the anti-vertebra albumen 2 in the mouse source anti-vertebra albumen 2 of originating;
The said damping fluid that encapsulates is made up of sodium carbonate, soda mint and water; Said sodium carbonate is 1.59g/L in the said concentration that encapsulates in the damping fluid, and said soda mint is 2.93g/L in the said concentration that encapsulates in the damping fluid;
Said lavation buffer solution is made up of sodium chloride, potassium chloride, sodium hydrogen phosphate, potassium dihydrogen phosphate, Tween 20 and water; The concentration of sodium chloride in said lavation buffer solution is 8g/L; The concentration of potassium chloride in said lavation buffer solution is 0.2g/L; The concentration of sodium hydrogen phosphate in said lavation buffer solution is 1.44g g/L; The concentration of potassium dihydrogen phosphate in said lavation buffer solution is 0.24g/L, and the concentration of Tween 20 in said lavation buffer solution is 0.1% (percent by volume);
Said confining liquid is made up of said lavation buffer solution and bovine serum albumin(BSA), and the concentration of bovine serum albumin(BSA) in said confining liquid is 10g/L;
Said stop buffer is the aqueous sulfuric acid of 2M; Said enzyme labelled antibody is the anti-mouse antibodies of HRP labelled goat;
Said standard solution is that said confining liquid and said vertebra albumen 2 are formed the different following solution of vertebra albumen 2 concentration: 100,50,25,12.5,6.3,3.1 and 1.6ng/ml.
10. it is characterized in that according to Claim 8 or 9 described enzyme linked immunological kits: it is the solution of 630ng/ml that the said polyclonal antibody that encapsulates the anti-vertebra albumen 2 in damping fluid and said sheep source is formed antibody concentration; It is the solution of 5ug/ml that the monoclonal antibody of the anti-vertebra albumen 2 in said confining liquid and mouse source is formed antibody concentration; Said confining liquid and enzyme labelled antibody constitutive enzyme labeling antibody concentration are the solution of 80ng/ml.
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