CN103091499B - Preparation and application of tumor marker calreticulin detection kit - Google Patents
Preparation and application of tumor marker calreticulin detection kit Download PDFInfo
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- CN103091499B CN103091499B CN201310025418.3A CN201310025418A CN103091499B CN 103091499 B CN103091499 B CN 103091499B CN 201310025418 A CN201310025418 A CN 201310025418A CN 103091499 B CN103091499 B CN 103091499B
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Abstract
The invention discloses preparation and application of a tumor marker calreticulin detection kit, and belongs to the fields of genetic engineering and clinical diagnosis. A monoclonal antibody and a polyclonal antibody of the CRT (calreticulin) are prepared by recombinant CRT genetic immune BALB/c mice and new Zealand white rabbits; and an ELISA (enzyme-linked immunosorbent assay) kit with high specificity and sensitivity of which the lowest CRT content is 0.3125ng/ml and the detection limit is 40-0.3125ng/ml is built. The collected clinical sample is detected through the prepared ELISA kit; the result shows that CRT in blood serum of a patient with lung cancer is obviously higher than that in normal blood serum; significant statistic difference exists (P is smaller than 0.01); and an experimental method is provided for diagnosis of the clinical patients with lung cancer.
Description
Technical field
The invention belongs to genetic engineering and clinical diagnosis field, relate to a kind of preparations and applicatio of tumor markers calprotectin detection kit.
Background technology
Calprotectin (Calreticulin, CRT) be the ER molecular chaperones with various biological function of 46KD size, except being distributed in endoplasmic reticulum, in nucleus, nuclear membrane, surface of cell membrane and extracellular matrix, all there is expression, multiple biochemical physiological effect can be played.When tumour occurs, the CRT of secretion property is on the rise in the serum of hepatocarcinoma patient and the urine of bladder cancer patients, in addition, increases to some extent the expression that gynecological tumor compares normal healthy controls CRT as the research of breast cancer, cervical carcinoma also shows; With the patient finding the CRT positive during immunohistochemical staining research cancer of the stomach often with transfer, tumor-microvessel is assembled and five-year survival rate the is low symptom of distant place lymph node.But because the mechanism of tumor development is not yet illustrated so far, therefore the early detection of tumour also becomes more and more important.
In the research and clinical practice of tumour, early detection, early diagnosis, early treatment are crucial.Tumor markers (Tumor Marker, TM) is at cancer screening, diagnosis, judging prognosis and lapse to, evaluate treatment curative effect and people at highest risk's follow-up observation etc. in all there is larger practical value.Along with the continuous maturation of monoclonal antibody technique, there is the monoclonal antibody of a large amount of antitumor marks, and combine with the immunology detection technology (RIA, IRMA, ELISA, CLIA, IFA, TRFIA etc.) of special sensitivity, develop numerous tumor markers test items and be constantly applied to clinical, having become an important Index for examination of tumor patient.Based on above result of study, selection CRT is carried out methodological foundation and clinical testing as tumor markers by the present invention.The present invention applies a pair specific monoclonal antibody and the how anti-ELISA kit setting up hypersensitivity, detects for clinical tumor patient and normal healthy controls change of serum C RT.We find, the CRT level in affinity antibody to SpA is apparently higher than normal control.Therefore, our reported first successfully establishes the ELISA kit of the CRT detection being directed to diagnosing tumor.
Summary of the invention
The object of this invention is to provide the enzyme-linked immunosorbent assay kit of a kind of cancer diagnosis or evaluation risk of cancer, described kit comprises: the specific antibody of calprotectin (CRT), and described specific antibody is by taking calprotectin as the monoclonal antibody that obtains of immunogene and/or polyclonal antibody.
Wherein said monoclonal antibody is mouse resource monoclonal antibody, and its preparation method is as follows: use the calprotectin of purifying to carry out immunity as immunogene to Balb/c mouse; After bioactivity is up to standard, carry out Fusion of Cells; Obtain the hybridoma of secrete monoclonal antibody through screening and cloning, through hybridoma cell strain secretion, obtain monoclonal antibody.Wherein, preferably use mouse restructuring calprotectin (i.e. CRT antigen) of purifying as immunogene, the recombinant plasmid of application build prepares mouse recombinant C RT albumen (its amino acid sequence is as shown in SEQ ID NO:1) by prokaryotic expression system.
The step preferably preparing monoclonal antibody is: before immune Balb/c mouse, first obtains serum-20 DEG C preservation of about 100 μ l, after be mixed and made into emulsion with the CRT antigen of 50 μ g purifying and Freund's complete adjuvant, through intracutaneous back, multi-point injection carries out immunity.After 1 month, add incomplete Freund's adjuvant with the antigen of 25 μ g and carry out booster immunization totally 3 times through intracutaneous injection, every minor tick 2 weeks.After bioactivity is up to standard, the mouse boosting cell obtain immunity and SP2/0 myeloma cell carry out Fusion of Cells, and feeder cells is Turnover of Mouse Peritoneal Macrophages, and merging reagent is the PEG4000 of 50%, the ratio of splenocyte and myeloma cell is 5: 1, is placed in 5%CO
2cultivate in 37 DEG C of incubators of saturated humidity, 3rd, the complete RPMI-1640 containing HAT within 6th, 9,10, is changed to, with limiting dilution assay, clone is diluted in 5 piece of 96 orifice plate respectively, rear absorption supernatant is grown up to hybridoma, supernatant antibody titer is detected with ELISA, filter out positive hybridoma cell (namely secrete monoclonal antibody is many, and the good hybridoma of cell growth state).Again through the hybridoma cell strain secretion that screening obtains, after purifying, obtain monoclonal antibody.
Polyclonal antibody in described kit is rabbit source polyclonal antibody; Its preparation method is as follows: first obtain immunize New Zealand White Rabbit serum before about 5ml injections of antigens, after be mixed and made into emulsion with 500 μ g CRT antigens and Freund's complete adjuvant, through intracutaneous back, multi-point injection carries out immunity.After 1 month, add incomplete Freund's adjuvant with the antigen of 250 μ g and carry out booster immunization totally 3 times through intracutaneous injection, every minor tick 2 weeks.After mensuration is tired, arteria carotis is got blood collected after centrifugation serum and add isopyknic glycerine-20 DEG C preservation after saturated ammonium sulphate method purifying.
Described kit also comprises: enzyme labelled antibody, is coated with the microwell plate of described monoclonal antibody, the goat-anti rabbit enzyme labelled antibody of HRP mark, standard positive control calprotectin, standard negative control Escherichia coli BL-21 mycoprotein, confining liquid, sample diluting liquid, cleansing solution, stop buffer, substrate nitrite ion.
Confining liquid is made up of 1%BSA liquid and 1% blood of goats clear liquid, and BSA liquid and preparation method thereof is as follows: sodium chloride 8g, potassium chloride 0.2g, sodium hydrogen phosphate 1.44g, and potassium dihydrogen phosphate 0.27g, 10g BSA powder is dissolved in ddH
2in O, adjust pH to be 7.4, be settled to 1L, be 1%BSA, filter packing and be stored in-20 DEG C.Lowlenthal serum is added, make it that concentration is confining liquid 1% when closed.
Sample diluting liquid is 1 × PBS, and its preparation method is: sodium chloride 8g, potassium chloride 0.2g, sodium hydrogen phosphate 1.44g, and potassium dihydrogen phosphate 0.27g, is dissolved in ddH
2in O, adjust pH to be 7.4, be settled to 1L, filter, 4 DEG C of preservations.
Cleansing solution be containing concentration be the PBS of 0.05%Tween-20, preparation method is: in 1L1 × PBS, add Tween-20500 μ l, 4 DEG C of preservations.
Stop buffer is the concentrated sulphuric acid of 2M, the distilled water of 178.3ml is dropwise added the concentrated sulphuric acid (98%) of 21.7ml.
Substrate nitrite ion comprises A liquid and B liquid, and wherein the preparation process of A liquid is: urea peroxide 1g, sodium hydrogen phosphate 35.8g, and citric acid 10.2g, Tween-20100 μ l, adds ddH by former three
2o lucifuge adds Tween-20 after dissolving and is settled to 1L, and 4 DEG C keep in Dark Place; The preparation process of B liquid is TMB0.7g, citric acid 10.3g, DMSO40ml, adds ddH after first dissolving the above two by DMSO lucifuge
2o is settled to 1L, and 4 DEG C keep in Dark Place; Use front equal-volume mixing A liquid and B liquid, lucifuge develops the color.
By the mensuration of square formation titrimetry, the monoclonal antibody bag when CRT concentration be 5ng/ml, OD value is 1.0 is optium concentration by concentration and many anti-bindings concentration, the anti-concentration of goat-anti rabbit-horseradish peroxidase two.Be respectively monoclonal antibody 800 times dilution, polyclonal antibody 4500 times dilution, goat-anti rabbit-HRP two anti-(i.e. the goat-anti rabbit enzyme labelled antibody of HRP mark) 3000 times of dilutions, negative control 0.108.
Kit of the present invention, can be used for detecting cancer, is preferably lung cancer.
The present invention also provides a kind of ELISA detection method of calprotectin, and its step comprises:
1) with carbonate buffer solution (0.05M Na
2cO
3/ NaHCO
3, pH9.6)) wrap by the CRT monoclonal antibody prepared by above-mentioned preparation method of 1: 800 gradient dilution, every hole 100 μ l, 4 DEG C of bags are washed elisa plate 1 time by 16h, PBST cleansing solution;
2) add 200 μ l confining liquids (1%BSA and 1% lowlenthal serum PBS), 37 DEG C of closed 1h, wash 1 time;
3) add CRT antigen (0.5 μ g/ml, 100 μ l/ holes) 37 DEG C of 1h that 100 μ l1%BSA dilute, wash 3 times by PBST cleansing solution;
4) add 100 μ l CRT many anti-(4500 ×), 37 DEG C hatch 1h after wash 3 times with PBST cleansing solution;
5) the goat-anti rabbit enzyme labelled antibody (3000 ×) 37 DEG C adding 100 μ l HRP marks hatches 1h, washs 3 times with PBST cleansing solution;
6) 100 μ l/ hole substrate nitrite ion A+B liquid chamber temperature lucifuge colour developing 15min;
7) the 2mol/L H in 50 μ l/ holes is added
2sO
4(stop buffer) cessation reaction;
8) in 450nm wavelength microplate reader, absorbance is measured.
Accompanying drawing explanation
Figure 1 shows that the expression of pET28a (+)-CRT in BL21 (DE3) bacterium and the purifying of CRT albumen, wherein: M is Unstained Protein Molecular Weight Marker (SM0431); 1 BL21 (DE3) bacterium transformed for pET28a (+)-CRT before induction, 2 BL21 (DE3) bacterium that are pET28a (+)-CRT conversion after IPTG induction, 3 is the CRT albumen of purifying.
Figure 2 shows that ELISA method measures CRT antibody titer result, A figure is depicted as: after the CRT monoclonal antibody of preparation carries out gradient dilution, be combined, read absorbance (OD450) with the CRT of bag quilt; B figure is depicted as: after the CRT polyclonal antibody of preparation carries out gradient dilution, be combined with the CRT of bag quilt, reads absorbance (OD450).
Figure 3 shows that Western blot identifies monoclonal antibody specificity; Upper figure: using monoclonal antibody measures the endogenic CRT of eukaryotic Cos-7 and A549; Figure below: the CRT(rCRT by restructuring brachymemma) transfectional cell, using monoclonal antibody carries out the expression measuring CRT.
Figure 4 shows that Western blot identifies many anti-specific results, wherein: 1, the CRT albumen of purifying; The CRT protein expression of 2, IPTG induction; 3, the contrast before induction.
Figure 5 shows that the ELISA of foundation measures the typical curve of CRT.
Figure 6 shows that the result measuring CRT protein content in clinical serum sample.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all to arrange three times or more and repeats experiment, results averaged.
Main material and reagent
1, material
Culture flask, plate, ELISA check-out console (Costar, the U.S.); Experimental apparatus: Gel Logic-200 gel imaging system (Kodak company of the U.S.), the rectilinear protein electrophorese system of Mini type, protein transferring system (Bio-Rad company of the U.S.), TS100 type inverted fluorescence microscope (Japanese Nikon company), Multifunction fluorescent microplate reader (Tecan company of Switzerland), 2MX-988B automatic plate washer (Beijing's celestial stone medical supplies make institute), CR21GII series of high speed refrigerated centrifuge (HIT).
2, reagent
Recombinant plasmid pET-28a (+)-CRT built for this seminar all; The recombinant plasmid of application build prepares mouse CRT recombinant protein (its amino acid sequence is as shown in SEQ ID NO:1) (shown in Fig. 1) by prokaryotic expression system; By preparation method of the present invention, immune mouse and rabbit, prepared mouse source CRT monoclonal antibody and rabbit source CRT polyclonal antibody respectively.
Streptavidin-HRP and substrate TMB (Sigma, St Louis, MO, the U.S.); RPMI-1640 nutrient solution (Invitrogen company); Bovine serum albumin (BSA, the magnificent company in Beijing); 10 × PBS solution (NaCl80.0g, Na
2hPO
414.2g, KH
2pO
42.72g, KCl2.0g add distilled water dissolve and constant volume to 1000ml, be diluted to 1 × PBS during use, tune pH be 7.4); Lowlenthal serum (Gibco company); Skimmed milk power (Inner Mongolia Yili Industry Group Co., Ltd); PEG4000 (Amresco company).
Preparation of reagents:
Confining liquid is 1%BSA liquid+1% blood of goats clear liquid; Preparation method: sodium chloride 8g, potassium chloride 0.2g, sodium hydrogen phosphate 1.44g, potassium dihydrogen phosphate 0.27g, 10g BSA powder is dissolved in ddH
2in O, adjust pH is 7.4, is settled to 1L, is 1%BSA, filters packing and is stored in-20 DEG C.When closed, add lowlenthal serum, make it at 1% of confining liquid.
Sample diluting liquid is 1 × PBS, preparation method: sodium chloride 8g, potassium chloride 0.2g, sodium hydrogen phosphate 1.44g, and potassium dihydrogen phosphate 0.27g, is dissolved in ddH
2in O, adjust pH is 7.4, is settled to 1L, filters, 4 DEG C of preservations.
Cleansing solution (PBST) is contain the PBS that concentration is the Tween-20 of 0.05%, and preparation method is: in 1L1 × PBS, add Tween-20500 μ l, 4 DEG C of preservations.
Stop buffer is the concentrated sulphuric acid of 2M, is dropwise added by the distilled water of 178.3ml in the concentrated sulphuric acid (98%) of 21.7ml.
Substrate nitrite ion comprises A liquid and B liquid, and wherein the preparation process of A liquid is: urea peroxide 1g, sodium hydrogen phosphate 35.8g, and citric acid 10.2g, Tween-20100 μ l, adds ddH by former three
2o lucifuge adds Tween-20 after dissolving and is settled to 1L, and 4 DEG C keep in Dark Place; The preparation process of B liquid is TMB0.7g, citric acid 10.3g, DMSO40ml, adds ddH after first dissolving the above two by DMSO lucifuge
2o is settled to 1L, and 4 DEG C keep in Dark Place; Use front equal-volume mixing A liquid and B liquid, lucifuge develops the color.
the preparation of embodiment 1, CRT monoclonal antibody and qualification
1, the preparation of monoclonal antibody
Before immunity Balb/c mouse, the serum-20 first obtaining about 100 μ l is spent and is preserved, after be mixed and made into emulsion with the CRT antigen of 50 μ g purifying and Freund's complete adjuvant, through intracutaneous back, multi-point injection carries out immunity.After 1 month, add incomplete Freund's adjuvant with the antigen of 25 μ g and carry out booster immunization totally 3 times through intracutaneous injection, every minor tick 2 weeks.After bioactivity is up to standard, the mouse boosting cell obtain immunity and SP2/0 myeloma cell carry out Fusion of Cells, and feeder cells is Turnover of Mouse Peritoneal Macrophages, and merging reagent is the PEG4000 of 50%, the ratio of splenocyte and myeloma cell is 5: 1, is placed in 5%CO
2cultivate in 37 DEG C of incubators of saturated humidity, 3rd, the complete RPMI-1640 containing HAT within 6th, 9,10, is changed to, with limiting dilution assay, clone is diluted in 5 piece of 96 orifice plate respectively, supernatant is drawn after hybridoma is grown, detect supernatant antibody titer with ELISA, filter out positive hybridoma cell (namely secrete monoclonal antibody is many, and the good hybridoma of cell growth state).
Then, the hybridoma of secrete monoclonal antibody is filtered out.Again through the hybridoma cell strain secretion that screening obtains, after purifying, obtain monoclonal antibody.
2, ELISA method measures antibody titer
Use carbonate buffer solution bag by CRT antigen (concentration is 1 μ g/ml, every hole 100 μ 1) 4 DEG C of bags by 16h;
Use PBST cleansing solution washing elisa plate 1 time, 200 μ l confining liquids (1%BSA and 1% lowlenthal serum PBS), 37 DEG C of closed 1h;
The five times gradient dilution antibody of CRT antibody using 1/100,1/500,1/2500,1/12500,1/62500,1/312500,1/1562500 are as primary antibodie, and 100 μ l add elisa plate, wherein replace CRT antibody as negative control using PBST.37 DEG C hatch 1h after, use PBST cleansing solution washing elisa plate 3 times;
Add the sheep anti-mouse igg (1 ﹕ 3000) that 100 μ l HRP mark to resist as two, 37 DEG C hatch 1h after, same washing 3 times;
100 μ l TMB lucifuge colour developing 15min;
50 μ l2mol/L H
2sO
4cessation reaction, 450nm wavelength microplate reader measures absorbance.
By the CRT albumen bag of purifying by elisa plate, after purifying, monoclonal antibody carries out gradient dilution, and detect antibody through ELISA and have very high tiring, monoclonal antibody can reach 1: 31 ten thousand times, has good dose-effect relationship (Fig. 2 A).Negative control hole A value is 0.116.
3, monoclonal antibody specificity identification
Collect and grow to Cos-7, A549 of 80%-90% and transfection pcDNA3.1 (+)-CRT
⊿cell, with cell pyrolysis liquid cracking on ice 30 minutes, centrifugal rear acquisition supernatant added 5 × sds gel sample-loading buffer, and 100 DEG C are boiled 5min, ice bath 4min, and centrifugal under room temperature (12000 × g) 1min gets 20 μ l and carries out SDS-PAGE electrophoresis.Nitrocellulose filter is shifted after SDS-PAGE electrophoresis, close with 5% skimmed milk power, the primary antibodie added respectively is monoclonal antibody (1 ﹕ 3000) and the β-Actin antibody (1 ﹕ 3000) of CRT, after TBST washs 3 times, add sheep anti-mouse igg (1 ﹕ 3000) the room temperature reaction 1h of HRP mark respectively, the same washing; ECL develops the color.
Collect and grow to Cos-7, A549 of 80%-90% and transfection pcDNA3.1 (+)-CRT
⊿cell pyrolysis liquid, nitrocellulose filter is shifted after SDS-PAGE electrophoresis, western blot detects the expression of endogenous CRT and recombinant C RT, and result can detect the recombinant C RT(rCRT of CRT and 30KD of 36KD under being presented at the consistent condition of β-actin).Prompting CRT monoclonal antibody has good specificity (Fig. 3).
the preparation that how anti-embodiment 2, CRT be and qualification
1, how anti-preparation
First obtain immunize New Zealand White Rabbit serum before about 5ml injections of antigens, after be mixed and made into emulsion with 500 μ g CRT antigens and Freund's complete adjuvant, through intracutaneous back, multi-point injection carries out immunity.After 1 month, add incomplete Freund's adjuvant with the antigen of 250 μ g and carry out booster immunization totally 3 times through intracutaneous injection, every minor tick 2 weeks.After mensuration is tired, arteria carotis is got blood collected after centrifugation serum and add isopyknic glycerine-20 DEG C preservation after saturated ammonium sulphate method purifying.
2, ELISA method measures antibody titer
Use carbonate buffer solution bag by CRT antigen (concentration is 1 μ g/ml, every hole 100 μ 1) 4 DEG C of bags by 16h;
Use PBST cleansing solution washing elisa plate 1 time, 200 μ l confining liquids (1%BSA and 1% lowlenthal serum PBS), 37 DEG C of closed 1h;
CRT polyclonal antibody respectively using 1/100,1/500,1/2500,1/12500,1/62500,1/312500,1/1562500 5 times of gradient dilution antibody as primary antibodie, 100 μ l add elisa plate, wherein replace CRT antibody as negative control using PBST.37 DEG C hatch 1h after, use PBST cleansing solution washing elisa plate 3 times;
Add 100 μ l HRP mark goat anti-rabbit igg (1: 3000) as two resist, 37 DEG C hatch 1h after, wash equally;
100 μ l TMB lucifuge colour developing 15min;
50 μ l2mol/L H
2sO
4cessation reaction, the microplate reader of 450nm wavelength measures absorbance.
By the CRT albumen bag of purifying by elisa plate, after purifying, polyclonal antibody carries out gradient dilution, and detect antibody through ELISA and have higher tiring, polyclonal antibody can reach 1: 31 ten thousand times, has good dose-effect relationship (Fig. 2 B).Negative control hole A value is 0.116.
3, how anti-specific detection
Taking out from-80 degree recombinant plasmid pET28a (+)-CRT built is transformed into Host Strains BL21 (DE3), through IPTG (final concentration 1.0mmol/L) abduction delivering, the bacterium of collecting adds 5 × sds gel sample-loading buffer, 100 DEG C are boiled 5min, ice bath 4min, centrifugal under room temperature (12000 × g) 1min, gets 20 μ l and carries out SDS-PAGE electrophoresis.Coomassie brilliant blue staining, is measured by gel imaging system and expresses output.Western blot detects restructuring pET28a (+)-CRT albumen, nitrocellulose filter is shifted after SDS-PAGE electrophoresis, close with 5% skimmed milk power, the primary antibodie added is many anti-(1:5000) of CRT, TBST adds goat anti-rabbit igg (1: 3000) the room temperature reaction 1h of HRP mark after washing 3 times, the same washing; ECL develops the color.
By recombinant plasmid pET28a (+)-CRT Plastid transformation in e. coli bl21 (DE3) competent cell, after 1.0mmol/L IPTG induces 4 ~ 6h, expressing protein is transferred to nitrocellulose film bar through 10%SDS-PAGE electrophoretic separation albumen.Resist for primary antibodie with CRT more, HRP mark sheep anti-mouse antibody be two resist, can see the CRT albumen of the bacterium liquid after induction and purifying about 32kD place occur specific albumen, without induce bacterium liquid and negative control all without respective strap (Fig. 4).
embodiment 3, set up double crush syndrome detection method
1, the basic step of double crush syndrome detection method
With carbonate buffer solution (0.05MNa
2cO
3/ NaHCO
3, pH9.6)) bag is by 1: 800CRT monoclonal antibody, and every hole 100 μ l4 DEG C of bag is washed 1 time by 16h, PBST;
Add 200 μ l confining liquids (1%BSA and 1% lowlenthal serum PBS), 37 DEG C of closed 1h, wash 1 time;
Add CRT antigen (0.5 μ g/ml, 100 μ l/ holes) 37 DEG C of 1h that 100 μ l1%BSA dilute, wash 3 times by PBST;
Add 100 μ l CRT many anti-(4500 ×), 37 DEG C hatch 1h after wash 3 times with PBST;
The goat anti-rabbit antibody (3000 ×) 37 DEG C adding 100 μ l HRP marks hatches 1h, washs 3 times with PBST;
100 μ l/ hole A+B nitrite ion room temperature lucifuge colour developing 15min;
Add the 2mol/LH in 50 μ l/ holes
2sO
4cessation reaction, 450nm wavelength microplate reader measures absorbance.
2, square formation titration measuring CRT protein content
The monoclonal antibody of this experimental applications purifying is as coated antibody, and polyclonal antibody, as binding antibody, selects the suitable anti-concentration of HRP-goat-anti rabbit two, for measuring the content of CRT albumen.
By the mensuration of square formation titrimetry, the monoclonal antibody bag when CRT concentration be 5ng/ml, OD value is 1.0 is optium concentration by concentration and many anti-bindings concentration, the anti-concentration of goat-anti rabbit-horseradish peroxidase two.Be respectively monoclonal antibody 800 times dilution, many anti-4500 times of dilutions, the anti-3000 times of dilutions of goat-anti rabbit-HRP two, negative control 0.108(is in table 1).
Table 1 square formation titration measuring CRT protein content
3, ELISA measures the typical curve foundation of CRT
CRT albumen 1%BSA exact dilution is become 40,20,10,5,2.5,1.25,0.625, the standard items of 0.3125ng/mL, with the OD value of CRT protein standard substance for ordinate, corresponding log concentration is horizontal ordinate, Criterion curve (Fig. 5), can calculate the CRT protein content in testing sample according to linear regression equation.
embodiment 4, detect CRT content in sample with kit of the present invention
1, the collection of serum and storage, use
49 routine Serum of Cancer Patients (man 34, female 15) are collected and are derived from affiliated hospital of SanXia University first oncology, and 53 routine normal controls (man 33, female 20) come from this Hospital Physical Examination section.Diagnosing tumor standard is according to clinical criteria in the past, radiation and endoscopy, is finally determined by pathology detection, put into after serum collection-80 DEG C for subsequent use.In use, doubly dilute with 1 × PBS5, measure CRT content in serum by ELISA method.
2, clinical tumor patients serum CRT measures
By clinical sample 4 DEG C of thawings, with sample diluting liquid 5 times dilution lung cancer and normal human serum as double antibodies sandwich detectable antigens, according to the OD value measured, application standard curve, calculate CRT protein content in each sample, be multiplied by extension rate, obtain the actual CRT protein content (see Fig. 6) of sample.Result display Normal group change of serum C RT content is 7.728 ± 1.613, lung cancer patient be 11.775 ± 4.902.Adopt SPSS13.0 software analysis F=8.757, there were significant differences for P=0.004, has statistical significance (P<0.01).
Sequence table
SEQUENCE LISTING
<110> SanXia University
The preparations and applicatio of a <120> tumor markers calprotectin detection kit
<130>2013
<160>1
<170>PatentIn version3.3
<210>1
<211>253
<212>PRT
<213> recombined small-mouse calprotectin
<400>1
Ser Lys His Lys Ser Asp Phe Gly Lys Phe Val Leu Ser Ser Gly Lys
151015
Phe Tyr Gly Asp Leu Glu Lys Asp Lys Gly Leu Gln Thr Ser Gln Asp
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Ala Arg Phe Tyr Ala Leu Ser Ala Lys Phe Glu Pro Phe Ser Asn Lys
354045
Gly Gln Thr Leu Val Val Gln Phe Thr Val Lys His Glu Gln Asn Ile
505560
Asp Cys Gly Gly Gly Tyr Val Lys Leu Phe Pro Ser Gly Leu Asp Gln
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Lys Asp Met His GlyAsp Ser Glu Tyr Asn Ile Met Phe GlyPro Asp
859095
Ile Cys Gly Pro Gly Thr Lys Lys Val His Val Ile Phe Asn Tyr Lys
100105110
Gly Lys Asn Val Leu Ile Asn Lys Asp Ile ATg Cys Lys Asp Asp Glu
115120125
Phe ThT His Leu Tyr ThT Leu Ile Val ATg Pro Asp Asn ThT Tyr Glu
130135140
Val Lys Ile Asp Asn Ser Gln Val Glu Ser Gly Ser Leu Glu Asp Asp
145150155160
TrpAsp Phe Leu Pro Pro Lys Lys Ile Lys Asp Pro Asp Ala Ala Lys
165170175
Pro Glu Asp TrpAsp Glu Arg Ala Lys Ile Asp Asp Pro Thr Asp Ser
180185190
Lys Pro Glu Asp TrpAsp Lys Pro Glu His Ile Pro Asp Pro Asp Ala
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Lys Lys Pro Glu Asp Trp Asp Glu Glu Met Asp Gly Glu Trp Glu Pro
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Pro Val Ile Gln Asn Pro Glu Tyr Lys Gly Glu Trp Lys Pro ATg Gln
225230235240
Ile Asp Asn Pro Asp Tyr Lys Gly Thr TrpIle His Pro
245250
Claims (5)
1. the enzyme-linked immunosorbent assay kit of a cancer diagnosis or evaluation risk of cancer, it comprises: the specific antibody of calprotectin (CRT), described specific antibody is by taking calprotectin as the monoclonal antibody that obtains of immunogene and/or polyclonal antibody, the amino acid sequence of described calprotectin as shown in SEQ ID NO:1, to diagnose or the cancer of estimated risk is lung cancer.
2. kit according to claim 1, wherein said monoclonal antibody is mouse resource monoclonal antibody, and its preparation method is as follows: use the calprotectin of purifying to carry out immunity as immunogene to Balb/c mouse; After bioactivity is up to standard, carry out Fusion of Cells; Obtain the hybridoma of secrete monoclonal antibody through screening and cloning, through hybridoma cell strain secretion, obtain monoclonal antibody.
3. kit according to claim 1, wherein: described polyclonal antibody is rabbit source polyclonal antibody.
4. the arbitrary described kit of claim 1-3, it also comprises: enzyme labelled antibody, be coated with the microwell plate of described monoclonal antibody, the goat-anti rabbit enzyme labelled antibody of HRP mark, standard positive control calprotectin, standard negative control Escherichia coli BL-21 mycoprotein, confining liquid, sample diluting liquid, cleansing solution, stop buffer, substrate nitrite ion.
5. kit according to claim 4, wherein said confining liquid, sample diluting liquid, cleansing solution, stop buffer, component and the proportioning of substrate nitrite ion are as follows:
1) confining liquid is made up of 1%BSA liquid and 1% blood of goats clear liquid, and lowlenthal serum adds when closed, and make it that concentration is confining liquid 1%;
2) sample diluting liquid is 1 × PBS;
3) cleansing solution be containing concentration be the PBS of 0.05%Tween-20;
4) stop buffer is the sulfuric acid of 2M;
5) substrate nitrite ion comprises A liquid and B liquid, and the preparation process of A liquid is: urea peroxide 1g, sodium hydrogen phosphate 35.8g, and citric acid 10.2g, Tween-20 100 μ l, adds ddH by former three
2o lucifuge adds Tween-20 after dissolving and is settled to 1L, and 4 DEG C keep in Dark Place; The preparation process of B liquid is TMB 0.7g, citric acid 10.3g, DMSO 40ml, after first dissolving TMB and citric acid by DMSO lucifuge, adds ddH
2o is settled to 1L, and 4 DEG C keep in Dark Place; Use front equal-volume mixing A liquid and B liquid, lucifuge develops the color.
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