CN102608322A - Suspension chip detection kit used for diagnosing preoperative ovarian cancer pelvic external transfer and preparation method thereof - Google Patents
Suspension chip detection kit used for diagnosing preoperative ovarian cancer pelvic external transfer and preparation method thereof Download PDFInfo
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- CN102608322A CN102608322A CN2012100809375A CN201210080937A CN102608322A CN 102608322 A CN102608322 A CN 102608322A CN 2012100809375 A CN2012100809375 A CN 2012100809375A CN 201210080937 A CN201210080937 A CN 201210080937A CN 102608322 A CN102608322 A CN 102608322A
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Abstract
The invention discloses a suspension chip detection kit used for diagnosing preoperative ovarian cancer pelvic external transfer and a preparation method thereof. The kit mainly comprises three kinds of coupling hydroxyl fluorescent microspheres and three kinds of goat anti-human polyclonal antibodies, wherein the three kinds of coupling hydroxyl fluorescent microspheres respectively coat three kinds of murine anti-human monoclonal antibodies of full-length cDNA (complementary DNA) coding protein, i.e., MMP9 (matrix metalloproteinase-9), Hpa1 and CL; and the three kinds of goat anti-human polyclonal antibodies respectively are streptavidin and mycin-phycoerythrobilin affinity coupling MMP9, Hpa1 and CL goat anti-human polyclonal antibodies. The kit adopts a suspension chip technology to realize joint detection of the content of three kinds of extracellular matrix proteases MMP9, Hpa1 and CL. The kit can be used for diagnosing preoperative pelvic external transfer of ovarian cancer patients and contributes to determining the best therapeutic schedule and judging the prognosis condition and the like.
Description
Technical field
The present invention relates to the suspending chip detection kit in medical biotechnology field, especially a kind of outer suspending chip detection kit that shifts of the preceding oophoroma pelvic cavity of diagnostics and preparation method thereof that is used for.
Background technology
Malignant tumor of ovary is at present domestic and international common gynecologic malignant tumor, and mortality ratio is in the gynecologic malignant tumor first place, and five year survival rate is merely about 30%.Causing one of high major reason of its mortality ratio is that the early infiltrate transfer very easily takes place oophoroma, has been late period when 70% patient finds.For the treatment of oophoroma is the supplemental treatment after factors such as character, histological type, operation-pathological staging and patient's age according to tumour determine whether undergoing surgery.The most of patient of the early stage epithelial ovarian cancer postoperative of the outer transfer of anovaria cancer does not need further treatment, and 90% above patient can not have the knurl survival for a long time; In all early ovarian cancer patients with high risk factors; 30%-40% has risk of relapse; 25%-30% perform the operation first dead in back 5 years; These patients after comprehensively operation finishes by stages, the comprehensive chemotherapy regimen of 4-6 the course of treatment then also need carrying out being basis with the platinum class, and in early days epithelial ovarian cancer with recur relevant high risk factor in just comprise that oophoroma shifts outward.For the outer advanced epithelial ovarian carcinoma standard care pattern that shifts of pelvic cavity takes place be: the tumour cell that the patient should be satisfied with at the very start subtracts the art of going out, and as much as possible makes tumors remaining less than 2cm (size of postoperative residue tumour is directly relevant with prognosis of patients); Satisfied tumour cell subtracts the patient of the postoperative that goes out should first-selected platinum medicine (cis-platinum or carboplatin) and the combined chemotherapy of taxol, at least 6 courses of treatment; In addition, quite a lot of if the patient subtracts the postoperative residue tumour quantity of going out at tumour cell first, can give the new adjuvant chemotherapy of 2-3 the course of treatment, and then interline property tumour cell subtracts the art of going out, and postoperative gives the chemotherapy of 3-6 the course of treatment again.Therefore the outer transfer of CT oophoroma pelvic cavity will help to improve accuracy rate and definite patient's therapeutic regimen of operation.
At present, the outer antidiastole of shifting of oophoroma pelvic cavity mainly relies on the iconography means.Ultrasonic examination can clear demonstration pelvic organ and the image of pathology, can find oophoropathy in early days according to size, form, blood flow and the vascular distribution of survey ovary.CT examination can clear demonstration pelvic organ and the image of pathology, the position and the character of lump, can also find surrounding wetting focus and lymphatic metastasis.CT is flat to be swept the good pernicious etiologic diagnosis accuracy rate of ovarian neoplasm is 70%～80% not wait, and strengthens CT ovary innocent and malignant tumour qualitative diagnostic accurate rate is reached 91.4%.Therefore, CT can be used as the trial inspection method of confirming to have or not big focus, but is difficult to find less than the focus of 2cm to being positioned at peritonaeum, mesenterium, omentum majus and diameter.The typical oophoroma of CT diagnosis performance is easy; Antidiastole CT performance is still had any problem with similar other pathologies of oophoroma; Like chronic inflammation or proliferative tuberculosis enclosed mass etc., both misdiagnosis rates reach 30% clinically, and its reason is that the form and the oophoroma of this type pathology is closely similar.In addition, the atypical oophoroma of CT diagnosis performance also is not easy.Magnetic resonance imaging (MRI) is a kind of imaging diagnosis method that grows up the eighties, and it has characteristics such as good spatial resolution to human body soft tissue, good contrast and non-invasive radiationless and imaging plane be flexible.The MRI enhancing is very pernicious to the identification of ovarian tumour; Enhanced ct scans has been improved the demonstration of inside tumor structure and the relation of tumour and surrounding tissue simultaneously; More can clearly show the peculiar sign of malignant ovarian lump; And improve recall rate to plantation METs such as less pelvic cavity, peritonaeums, to the diagnosis of malignant tumour and higher directive significance is arranged by stages.Yet MRI is still not ideal enough to detecting of minimal disease, the qualitative difficulty of some pathology, even the minority inflammatory mass is difficult to differentiate with tumour.
Existing clinical research confirmation, oophoroma is planted with abdominopelvic cavity, directly soaks into to be diffused as the master, secondly is lymphatic metastasis, and bloody path shifts few relatively.And the ability of tumor cell invasion and transfer and generation or induce the ability of the proteinase that produces the degradation of cell epimatrix closely related.The proteolytic enzyme of having found that impels extracellular matrix degradation comprise matrix metalloproteinase family (matrix metalloproteinases, MMPS), cathepsin (Cathepsins, CPS), heparitinase (heparanase, Hpa) etc.ELISA (ELISA) detects and has confirmed that the protease content of extracellular matrix in the tumor patient serum and transfer are proportionate.The assay method of the haemocyanin factor mainly contains radiommunoassay, EIA enzyme immunoassay, chemiluminescence immune assay, electrochemiluminescence immunoassay etc. at present; These methods are the single index detection method, and monofactor detects the process and the level that can not reflect the oophoroma infiltration metastasis fully.If want to avoid this situation every part of sample is carried out very costliness of many index analysis expense, and need serum amount bigger.In light of this situation, need to set up the technology platform that can simultaneously, fast, accurately detect multiple serum factor.The suspending chip of exploitation organically combines Flow Cytometry and enzyme marking detecting technology in recent years; Utilize 100 kinds of different iridescent target microballoons to be reaction carriers; The part of mark capturing different target molecule detects each microballoon through red, green two bundle different wave length laser, through Computer Analysis and typical curve match; Can realize the fast detecting that diversification, high flux, single-factor are quantitative respectively, have characteristics such as efficient, quick, responsive, special, low cost.
Summary of the invention
The technical matters that the present invention will solve provide a kind of efficient, fast, accurately, do not have wound, responsive, special, be used for outer suspending chip detection kit that shifts of oophoroma pelvic cavity and preparation method thereof before the diagnostics cheaply, the many deficiencies that exist with the recall rate that overcomes plantation METs such as having pelvic cavity before the oophoroma art, peritonaeum now.
For solving the problems of the technologies described above, the present invention adopts following technical scheme: be used for the outer suspending chip detection kit that shifts of the preceding oophoroma pelvic cavity of diagnostics, mainly comprise three kinds of coupling hydroxyl fluorescent microspheres and three kinds of goat-anti people polyclonal antibodies; Three kinds of coupling hydroxyl fluorescent microspheres encapsulate the mouse-anti human monoclonal antibodies of MMP9, Hpa1, three kinds of full-length cDNA encoding proteins of CL respectively; Three kinds of goat-anti people polyclonal antibodies are respectively MMP9, Hpa, the CL goat-anti people polyclonal antibody of the affine mycin of chain-rhodophyll affinity coupling.
The above-mentioned preparation method who is used for the outer suspending chip detection kit that shifts of the preceding oophoroma pelvic cavity of diagnostics: get the mouse-anti human monoclonal antibodies that three kinds of coupling hydroxyl fluorescent microspheres encapsulate MMP9, Hpa1, three kinds of full-length cDNA encoding proteins of CL respectively; Affine mycin-the rhodophyll of chain and MMP9, Hpa, CL goat-anti people polyclonal antibody are carried out affinity coupling.
This method may further comprise the steps:
< 1>fluorescent microsphere encapsulates the mouse-anti human monoclonal antibodies
Get three kinds of coupling hydroxyl fluorescent microspheres, three kinds of each corresponding a kind of coupling hydroxyl fluorescent microspheres of mouse-anti human monoclonal antibodies; To contain 1.0 * 10 respectively
5Three kinds of coupling hydroxyls of 8 μ l fluorescent microsphere of individual microballoon is resuspended in the 250 μ l dilutions, and after adding 10ul concentration was the EDC of 50mg/ml, adding 10ul concentration rapidly was the S-NHS activation microballoon of 50mg/ml, and the room temperature lucifuge is hatched 20min, and is centrifugal, abandons supernatant; With the resuspended microballoon of 250ul dilution, adding concentration respectively is the mouse-anti human monoclonal antibodies 250 μ l of 1ug/mL, mixing; The room temperature lucifuge is hatched 30min, and is centrifugal, abandons supernatant; Clean crosslinked good microballoon twice with dilution; Be settled to 200 microballoons of final concentration/μ l at last, room temperature keeps in Dark Place, and promptly gets three kinds of coupling hydroxyl fluorescent microsphere solution of the mouse-anti human monoclonal antibodies that encapsulates MMP9, Hpa1, three kinds of full-length cDNA encoding proteins of CL respectively;
< 2>the affine mycin of chain-rhodophyll affinity coupling goat-anti people polyclonal antibody
Use dilution compound concentration respectively is three kinds of goat-anti people polyclonal antibody solution of 4 μ g/ml; Use the ultrapure water compound concentration to be the affine mycin of the chain of 10mmol/L-phycoerythrin solution; The affine mycin of isopyknic chain-phycoerythrin solution is joined in the goat-anti people polyclonal antibody solution; After jolting 2h on ice, obtaining concentration is the affine mycin of chain-rhodophyll affinity coupling goat-anti people polyclonal antibody solution of 2ug/mL, be sub-packed in-20 ℃ frozen subsequent use.
Dilution is the 10mM phosphate buffer that contains 1% bovine serum albumin pH 7.4.
The inventor finds under study for action and single extracellular matrix protein enzyme detects process and the level can not reflect the oophoroma infiltration metastasis fully, and three kinds of extracellular matrix protein enzyme MMP9, Hpa1, CL joint-detection then can effectively remedy this defective.Yet, every part of sample is carried out three kinds of time-consuming length of extracellular matrix protein EIA enzyme immunoassay respectively, and needs serum amount bigger.For this reason, the present invention carries out organic combination with Flow Cytometry and enzyme marking detecting technology, but has set up the multicolor fluorescence microballoon suspending chip detection kit of three kinds of extracellular matrix protein enzymes of joint-detection MMP9, Hpa1, CL content.This kit combines to excavate number diagnostic model and can be used for the outer diagnosis of shifting of pelvic cavity before the ovarian cancer patients art, helps to confirm therapeutic regimen and judging prognosis situation etc., has characteristics such as efficient, quick, responsive, special, low cost.It compared with prior art has following beneficial effect the present invention:
< 1>but still do not have the outer serum reagent box that shifts of pelvic cavity before the efficient diagnosis oophoroma art on the high market of accuracy rate, and kit susceptibility of the present invention and specificity are above 90%;
< 2>big this kit of flux can detect three kinds of extracellular matrix protein enzyme content in the same sample simultaneously;
< 3>required sample only needs the serum sample of 1 μ l can accomplish detection less;
< 4>simple fast detecting operation need not cyclic washing, can accomplish less than 2 hours, and experimental result is read by corresponding analyser automatically, has got rid of subjectivity.
Description of drawings
Fig. 1 uses suspending chip detection kit of the present invention to detect serum MMP9, Hpa1, CL content judgement metastases diagnostic model dendrogram.
Fig. 2 uses the typical curve that suspending chip detection kit of the present invention detects serum MMP9, Hpa1, CL, among the figure: 1 cathepsin L, 2 heparitinases, 3 matrix metalloproteinases.
Fig. 3 is that application suspending chip detection kit of the present invention and diagnostic model judge that ovarian epithelial carcinogenesis pelvic cavity shifts the result outward, among the figure:
*The total case load of n=(be pelvic cavity shift the positive+pelvic cavity outward shift feminine gender outward).
Embodiment
Be used for the outer suspending chip detection kit that shifts of the preceding oophoroma pelvic cavity of diagnostics and preparation method thereof research
One, experiment material
Matrix Metalloproteinases 9 (MMP9), Heparanases 1 (Hpa1), cathepsin L (CL) three kinds of total length coded amino acids mouse-anti human monoclonal antibodies and Matrix Metalloproteinases 9 (MMP9; C-terminus), Heparanases 1 (Hpa1; C-terminus), three kinds of goat-anti people polyclonal antibodies of cathepsin L (CL C-terminus) are all available from Santa Cruz Biotechnology, Inc.
Used polychrome microballoon is the coupling hydroxyl fluorescent microsphere of Luminex company.
Affine mycin-the rhodophyll of chain (Streptavidin-PE) is from Britain Innovabiosciences company.
Two, the preparation of kit
Preparation fluorescent microsphere suspending chip
Get the mouse-anti human monoclonal antibodies that three kinds of coupling hydroxyl fluorescent microspheres encapsulate MMP9, Hpa1, three kinds of full-length cDNA encoding proteins of CL respectively; Affine mycin-the rhodophyll of chain and MMP9, Hpa, CL goat-anti people polyclonal antibody are carried out affinity coupling.Concrete steps are following:
< 1>fluorescent microsphere encapsulates the mouse-anti human monoclonal antibodies
Get three kinds of coupling hydroxyl fluorescent microspheres, three kinds of each corresponding a kind of coupling hydroxyl fluorescent microspheres of mouse-anti human monoclonal antibodies; To contain 1.0 * 10 respectively
5Three kinds of coupling hydroxyls of 8 μ l fluorescent microsphere of individual microballoon is resuspended in 250 μ l dilution (the 10mM phosphate buffers that contain 1% bovine serum albumin; PH 7.4) in; After adding 10ul EDC (50mg/ml), add 10ul S-NHS (50mg/ml) activation microballoon rapidly, the room temperature lucifuge is hatched 20min; Centrifugal, abandon supernatant; With the resuspended microballoon of 250ul dilution, adding concentration respectively is the mouse-anti human monoclonal antibodies 250 μ l of 1ug/mL, mixing; The room temperature lucifuge is hatched 30min, and is centrifugal, abandons supernatant; Clean crosslinked good microballoon twice with dilution; Be settled to 200 microballoons of final concentration/μ l at last, room temperature keeps in Dark Place, and promptly gets three kinds of coupling hydroxyl fluorescent microsphere solution of the mouse-anti human monoclonal antibodies that encapsulates MMP9, Hpa1, three kinds of full-length cDNA encoding proteins of CL respectively;
< 2>the affine mycin of chain-rhodophyll affinity coupling goat-anti people polyclonal antibody
Use dilution compound concentration respectively is three kinds of goat-anti people polyclonal antibody solution of 4 μ g/ml; Use the ultrapure water compound concentration to be the affine mycin of the chain of 10mmol/L-phycoerythrin solution; The affine mycin of isopyknic chain-phycoerythrin solution is joined in the goat-anti people polyclonal antibody solution; After jolting 2h on ice, obtaining concentration is the affine mycin of chain-rhodophyll affinity coupling goat-anti people polyclonal antibody solution of 2ug/mL, be sub-packed in-20 ℃ frozen subsequent use.
Three kinds of final concentration are encapsulated microspheres solution and three kinds of polyclonal antibody solution are packed separately, be assembled into detection kit then, subsequent use.
Three, the application of kit
Using this kit, to detect MMP9 in the serum, Hpa1, CL step following: get MMP9, Hpa1, CL monoclonal antibody fluorescent microsphere solution 25 μ l respectively; Add the affine mycin of isopyknic chain-rhodophyll affinity coupling goat-anti people's polyclonal antibody solution and test serum 1 μ l respectively; Replenish dilution to total reaction volume 100 μ l; The whirlpool mixing, lucifuge, 1h is hatched in the room temperature vibration; After the 100 μ l dilution washed twice, after three kinds of fluorescent microspheres mixing, be settled to 100 μ l with dilution at last, 100 type streaming luminoscopes detection in Luminex company.
Judge that oophoroma generation pelvic cavity shifts outward: get the Santa Cruz MMP9 of company, Hpa1, CL standard items, be diluted to 8,16,32 respectively; 64,128,256,512; 1024,2056,4128U/ml is respectively the concentration of three kinds of enzymes with horizontal ordinate, and ordinate is fluoroscopic examination value (FI) production standard curve.Blank is a sample diluting liquid.Each typical curve highest detection limit is that the concentration of analyte is reached capacity; LDL (LOD value) is inferred from the match typical curve, is the pairing detection substrate concentration of 3 times of sums that the average fluorescent strength of analyte is equivalent to blank background and standard deviation.With as MMP9＞52.5U/ml, judge that oophoroma generation pelvic cavity shifts outward when and Hpa1＞66U/ml or CL＞201U/ml.Suspending chip detects the excavation of oophoroma serum MMP9, Hpa1, the foundation of CL content and counts the following Fig. 1 of diagnostic model.
Serum is gathered: patient's back untreated of being admitted to hospital extracts 10ml venous blood on an empty stomach, after 4 ℃ of refrigerators are placed natural coagulation in 2 hours, 4 ℃ of centrifugal 10min of 3000r/, get the upper serum packing after-80 ℃ of refrigerators preserve.
To epithelial ovarian cancer 83 examples (being divided into pelvic cavity with pathological diagnosis as " goldstandard " shifts positive 56 examples outward and shift negative 27 examples outward with pelvic cavity) patient with suspending chip kit detection MMP9, Hpa1, CL serum content; Typical curve according to standard items foundation; The LOD value that detects MMP9 is 6.2U/ml; The LOD value of Hpa1 is 5.8U/ml, and the LOD value of CL is 7.4U/ml, and the dynamic detection range of three kinds of enzymes is in 7～2056U/ml (see figure 2).According to typical curve, the coefficient of variation that suspending chip detects MMP9, Hpa, CL value in the 24 routine samples is 3.39～6.15%, in the trusted interval range.
Content input diagnostic model with 83 routine patients serum MMP9, Hpa1, CL; Be used to judge whether tumour pelvic cavity takes place shift outward; The result shows (see figure 3), and the sensitivity of model is 91.1% (51/56), specificity 92.6% (25/27); Positive predictive value 87.9% (51/58), negative predictive value 68.7% (22/32).
Claims (4)
1. one kind is used for the outer suspending chip detection kit that shifts of the preceding oophoroma pelvic cavity of diagnostics, it is characterized in that mainly comprising three kinds of coupling hydroxyl fluorescent microspheres and three kinds of goat-anti people polyclonal antibodies; Said three kinds of coupling hydroxyl fluorescent microspheres encapsulate the mouse-anti human monoclonal antibodies of MMP9, Hpa1, three kinds of full-length cDNA encoding proteins of CL respectively; Said three kinds of goat-anti people polyclonal antibodies are respectively MMP9, Hpa, the CL goat-anti people polyclonal antibody of the affine mycin of chain-rhodophyll affinity coupling.
2. according to the said preparation method who is used for the outer suspending chip detection kit that shifts of oophoroma pelvic cavity before the diagnostics of claim 1, it is characterized in that: get the mouse-anti human monoclonal antibodies that three kinds of coupling hydroxyl fluorescent microspheres encapsulate MMP9, Hpa1, three kinds of full-length cDNA encoding proteins of CL respectively; Affine mycin-the rhodophyll of chain and MMP9, Hpa, CL goat-anti people polyclonal antibody are carried out affinity coupling.
3. according to the said preparation method who is used for the outer suspending chip detection kit that shifts of oophoroma pelvic cavity before the diagnostics of claim 2, it is characterized in that this method may further comprise the steps:
< 1>fluorescent microsphere encapsulates the mouse-anti human monoclonal antibodies
Get three kinds of coupling hydroxyl fluorescent microspheres, three kinds of each corresponding a kind of coupling hydroxyl fluorescent microspheres of mouse-anti human monoclonal antibodies; To contain 1.0 * 10 respectively
5Three kinds of coupling hydroxyls of 8 μ l fluorescent microsphere of individual microballoon is resuspended in the 250 μ l dilutions, and after adding 10ul concentration was the EDC of 50mg/ml, adding 10ul concentration rapidly was the S-NHS activation microballoon of 50mg/ml, and the room temperature lucifuge is hatched 20min, and is centrifugal, abandons supernatant; With the resuspended microballoon of 250ul dilution, adding concentration respectively is the said mouse-anti human monoclonal antibodies 250 μ l of 1ug/mL, mixing; The room temperature lucifuge is hatched 30min, and is centrifugal, abandons supernatant; Clean crosslinked good microballoon twice with dilution; Be settled to 200 microballoons of final concentration/μ l at last, room temperature keeps in Dark Place, and promptly gets three kinds of coupling hydroxyl fluorescent microsphere solution of the mouse-anti human monoclonal antibodies that encapsulates MMP9, Hpa1, three kinds of full-length cDNA encoding proteins of CL respectively;
< 2>the affine mycin of chain-rhodophyll affinity coupling goat-anti people polyclonal antibody
Use dilution compound concentration respectively is three kinds of goat-anti people polyclonal antibody solution of 4 μ g/ml; Use the ultrapure water compound concentration to be the affine mycin of the chain of 10mmol/L-phycoerythrin solution; The affine mycin of isopyknic chain-phycoerythrin solution is joined in the goat-anti people polyclonal antibody solution; After jolting 2h on ice, obtaining concentration is the affine mycin of chain-rhodophyll affinity coupling goat-anti people polyclonal antibody solution of 2ug/mL, be sub-packed in-20 ℃ frozen subsequent use.
4. according to the said preparation method who is used for the outer suspending chip detection kit that shifts of oophoroma pelvic cavity before the diagnostics of claim 3, it is characterized in that: said dilution is the 10mM phosphate buffer that contains 1% bovine serum albumin pH 7.4.
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Application publication date: 20120725 |