CN104422770A - Up-conversion luminescence-based chip detection system and reagent for gynecological tumor markers CA125, CA153, SCCA, HE4 - Google Patents
Up-conversion luminescence-based chip detection system and reagent for gynecological tumor markers CA125, CA153, SCCA, HE4 Download PDFInfo
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- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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Abstract
The invention relates to an up-conversion luminescence-based detection system for four gynecological tumor markers (CA125, CA153, SCCA, HE4), and the detection is performed by using a reaction cup detection system. The reaction cup detection system comprises a carrier plate, a reaction detection cup, and a fluorescence detector. The reaction detection cup is composed of a reaction detection cup and a test tube cover. A sample is dropwise added into the reaction detection cup for detection; after reaction for 5-15 min, the carrier plate is taken out and inserted into a slot of the fluorescence detector for value reading. The invention is characterized in that: the detection of four gynecological tumor markers CA125, CA153, SCCA, HE4 is carried out simultaneously by combining with an up-conversion luminescence detection method. The background value is small, and the measurement is rapid, accurate, and simple in operation. Data are transmitted to a doctor through wifi so as to obtain the detection results rapidly, and thus the purpose of rapid diagnosis is realized.
Description
Technical field
The present invention relates to a kind of immune detection system and reagent detection gynecological tumor CA125, CA153, SCCA, HE4 tetra-marks, are a kind of devices being detected gynecological tumor four marks by up-conversion luminescence method, belong to medical product field.
Background technology
The gynecological tumor diseases such as breast cancer, oophoroma, carcinoma of endometrium, SCC, cervix cancer are the major obstacles of the health affecting numerous women.Cervical carcinoma is the modal tumour of female genital tract, the incidence of disease is had to raise in recent years, the feature of morbidity rejuvenation, in gynecological cancer in the world, cervical carcinoma causes main causes of death, incidence is 493243 examples/year, mortality ratio 273505 examples/year, and the mortality ratio in global range is 55%.Gynecological tumor causes the extensive concern of various circles of society and medical domain.Gynecological tumor scholar is devoted to the research of gynecological tumor method of early diagnosis, namely finds a kind of simple, tumor markers that Sensitivity Specificity is high.Specific serum detects the discovery of thing, has saved the life much suffering from patients with gynecologic malignancies.The state of an illness can be monitored over the course for the treatment of by mark.
Conventional gynecological tumor mark as: alpha-fetoprotein (AFP), carcinomebryonic antigen (CEA), human chorionic gonadtropin (hCG), CA125, sugar antigen CA199, sugar antigen CA724 etc., they play an important role to state of illness monitoring.
CA125 has large quantifier elimination in gynecological tumor mark, to be a kind of molecular weight be 200000 glycoprotein.Bast etc. with ovary serous bursa adenocarcinoma cell immune mouse, and hybridize with myeloma cell the monoclonal antibody obtained in nineteen eighty-three.As the important symbol thing of monitoring ovarian epithelial carcinoma, it is a kind of conventional noninvasive method distinguishing benign and malignant diseases.About the experiment of CA125 and clinical research article nearly more than 2000 sections in nearly more than 10 years.By finding that to the research of CA125 half life period it is obviously relevant to the life cycle of ovarian cancer patients.If the research such as Gadducci prove Patients with Advanced Ovarian Carcinoma CA125 half life period≤25 days, the probability that chemotherapy reaches pathology complete incidence graph (CR) after cytoreductive surgery is high 3.585 times compared with >'s 25 days, half life period, elder showed poor prognosis, should give to strengthen treatment.The CA125 half life period be chemotherapy early stage time an independently prognostic indicator, can it can point out patient reach the life cycle of pathology CR and patient.CA125 is except the reference index as diagnosis clinically, also can be used as and follows up a case by regular visits to and the important references index of state of illness monitoring.After treatment, decline the rapidly prognosis of then patient of CA125 value is often better.CA125 reaches normal value and raises later again, often indicates tumor recurrence.The rising of CA125 can be checked through tumour more clinically and take early 3 ~ 6 months.The rising of CA125 value prior to clinical manifestation, but not yet to be affirmed by the value that it is used as oophoroma examination in most ovarian cancer patients.Although CA125 is the mark of good epithelial ovarian cancer, main positive rate in ovarian serous carcinoma and undifferentiated carcinoma is higher, and in mucus cancer, then positive rate is lower.The rising also having CA125 in some benign diseases is also pointed out in much research, as benign ovarian tumor, pelvic inflammatory disease and cirrhosis etc.In the epithelium that CA125 is also present in many organs or mesothelium, as fallopian tubal, uterus, peritonaeum, pleura and pericardium etc., but little at the lifting range of non-tumour person, should take in before making clinical judgment.CA125 is an important mark as the auxiliary diagnosis of oophoroma, relevant with the course of disease.
CA153 is found in breast cancer cell the earliest, it is the mucin-like glycoprotein that a kind of molecular weight be positioned on cell membrane is larger, relative molecular mass 300000-450000, comprise a Ge Mo district, an intracellular region and one are rich in the cell outskirt of glycosyl, identified by anti-human butterfat ball membrane antibody 115D8 and DF3, be present in multiple gland cancer, as breast cancer, lung cancer, oophoroma and cancer of pancreas.When cell carcinogenesis, because glycosyl invertase is activated, proteinase and sialidase activity on cell membrane is caused to increase, cytoskeletal disruption, CA sugar antigen increases and separates from cancer cell membrane, discharge in blood, in recent years release as markers for breast cancer, normal < 40U/ml women breast-feeding their children or benign breast tumor are all lower than this value.In breast cancer late period 100%, other this value of phase 75% obviously raises.Can be used as the auxiliary diagnosis that tumor markers is applied to tumour, the judgement of curative effect monitoring and transfer and relapse.To the dynamic tracing of breast cancer, judge that relapse and metastasis has certain values.
SCCA (squamous cell carcinoma antigen) is a kind of protein extracted from the hepatic metastases stove of cervical squamous cancer in 1977, and its molecular weight is about 48000.This antigen is high expressed in the tumor tissues such as cervix cancer, lung cancer, cutaneum carcinoma, the cancer of the esophagus, laryngocarcinoma, is one of Cervical Tumor mark few in number at present.SCCA is mainly present in the cervical carcinoma containing squama cancer composition, and its critical value is that the positive rate of 1.5ng/ml, SCCA is respectively 0 phase 12.4%, I phase 30.0%-40.0%, II phase 60.0%-70.0%, III phase and IV phase 80.0%-90.0%.Recent research shows, whether the clinical stages of preoperative serum SCCA level and cervical carcinoma, tumor focus size, interstitial invasive depth, lymph node are accumulated etc. because have correlativity.The change of SCCA value is consistent with the change of progression of disease or improvement.If treatment with chemotherapy, the state of an illness after chemotherapy changes also consistent with the change of SCCA value.If the SCCA persistent levels of 2-3 month raises and means that treatment is imitated unsuccessfully after treatment.SCCA finds recurrent focus in individual prior to clinical 2-6 month.Even if SCCA is negative before treatment, when cancer knurl recurs also still likely there is positive reaction in it.Therefore SCCA is a good mark to cervical carcinoma state of illness monitoring, provides significant information to the determination of patient treatment protocol.
Susceptibility in the oophoroma of HE4 (I phase) in early days and detect the total susceptibility of oophoroma all higher than CA125, is the responsive and special mark of oophoroma, can be used for the treatment situation to oophoroma early diagnosis and auxiliary monitoring ovarian cancer patients.One of Britain research to 200000 women with 30 kinds of tumors labels individually or combine with CA125 and carry out oophoroma examination, HE4 can make susceptibility and the specificity raising of diagnosis of ovarian cancer.HE4 antigen is high expressed in serosity, endometrial-like and transparence cellularity oophoroma.Do not break up with transitional cell carcinoma in have expression.Express hardly in mucus, reproduction cell and sex cords oophoroma.
The advantage that tumor markers detects is:
(1) diagnosing tumour can be assisted to originate.
(2) not damaged inspection is belonged to, can repeatedly rechecking.
(3) method is easy, and basic hospital also can detect.
(4) objective and accurate numerical value can be obtained.
But existing clinical marker thing specificity is poor.In view of the susceptibility of current most tumors mark and specificity still undesirable, false positive and false negative happen occasionally.For improving the accuracy that these tumor markerses detect, the tumor markers with different characteristics can be combined joint-detection.Joint-detection is better than only detecting single any one tumor markers, which increases diagnostic value.The CA125 such as Woolas distinguishes the optimum and Malignant mass of pelvic cavity as mark, and its susceptibility and specificity are respectively 78.1% and 76.8%.If by 5 kinds of mark (CA125, OVX1, LASA, CA153 and CA724) joint-detection, and through statistical treatment, then make its susceptibility and specificity bring up to 90.6% and 93.2% respectively, substantially increase the accuracy of judgement.Therefore by CA125, CA153, SCCA, HE4 tetra-kinds of antigen combinations, joint-detection gynecological tumor, to improve detection accuracy and specificity.
Up-conversion luminescence particle UCP has tremendous potential in marker detection field.Up-conversion luminescence has unique optical characteristics, and it has following characteristics:
1) sensitivity is high: not by extraneous background interference.
2) stability is high: the quenching phenomenon avoiding sample corrosion or self decay initiation.
3) dirigibility is high: can combine diversified spectrum.
4) security is high: be all safe from harm for tester, detection sample and environment.
5) facilitate simple and easy: can to blood, urine, the samples such as saliva directly detect, without the need to special processing.
Above feature determines that its application prospect in Biomarker assay is more wide.Particularly at the other detection field of bed.
Immune detection is the sensitive method measuring a kind of differential protein.Typical immunologic detection method comprises, and enzyme linked immunosorbent assay (elisa) and Western blot detect (western blot), and the conventional program of immune detection is tediously long time-consuming, not portable and cost is higher.Therefore the other trend of the times detected in area of medical diagnostics of bed, its feature is quick, is convenient for carrying, simple to operate.Not only can use in hospital, can also use in basic unit.Therefore a kind of rapid detection system of detection gynecological tumor four marks based on up-conversion luminescence and combined detection reagent have important using value in clinical detection field.
Summary of the invention
For above Problems existing, the invention provides a kind of immune detection system and reagent detection gynecological tumor CA125, CA153, SCCA, HE4 tetra-marks, feature of the present invention is: detect gynecological tumor four marks in conjunction with up-conversion luminescence method simultaneously.Utilize four kinds of label joint inspections, its accuracy and specificity higher; Utilize up-conversion luminescence method to measure, its background value is little, and it is convenient, accurate, simple to operate to measure.
In one of them preferred embodiment: based on the upper reaction cup system transforming luminous detection gynecological tumor four marks, comprising: carrier board, reaction detection cup, fluorescence detector.Carrier board is coated with simultaneously antibody mouse-anti people CA125, CA153, SCCA, HE4 of four kinds of antigens to be detected.100 microlitres will be added in reaction cup marked CA125, CA153, SCCA, HE4 tetra-kinds of antibody mixed liquors (concentration is 1mg/ml) of UCP particle, foraminate lid in the middle of test tube cap be covered in reaction cup, freeze-drying in freeze dryer.Carrier board intercalation reaction is detected in cup, drip 100 HL serum or body fluid, tissue fluid equal samples detects, sample can directly and primary antibodie and freeze-drying liquid generation single step reaction, carrier board is taken out reading numerical values in the draw-in groove inserting fluorescence detector by reaction 5-15min, is brought in typical curve by numerical value and calculates concentration of specimens.Direct printing testing result, or testing result is transferred to Data Centre in Hospital by wifi and allows doctor directly have access to testing result, arrives and detects fast, the object of quick diagnosis.And this detecting instrument is light, less than 1Kg; Easy and simple to handle, a key goes out result.Be widely used in health check-up, 120 emergency treatments, the environment such as field detection.
Described carrier board is black polystyrene plate.Length and width are 6mmX10mm.It has 3X4 point, dot spacing is 1.5mm, and spot diameter is 1.5mm.It is coated with mouse-anti people CA125, CA153, SCCA, HE4 tetra-kinds of antibody.
In the preferred embodiment of another kind, immuno-chromatographic test paper strip detection system is utilized to detect.Analyzing film is nitrocellulose filter, wraps by 4 kinds of antibody by four regions on film simultaneously, dries; Wrap by the anti-sheep IgG (1mg/ml) of rabbit as Quality Control point in test strips front end; Utilize UCP to mark CA125, CA153, SCCA, HE4 tetra-kinds of antibody, the four kinds of antibody marked by UCP infiltrate on fiberglass packing, dry.Be cut into rectangular after oven dry.Nitrocellulose filter, thieving paper, pad, sample pad are attached on base plate.Sample adds on application of sample pad by sample application zone by pad, and result interpretation place correspond to detection zone and the quality control band of analyzing film.
Described fluorescence detector size only has 12cmX6cmX5.5cm, can arbitrarily carry.Handled easily.
Accompanying drawing explanation
Fig. 1 is test tube stereographic map, and be rectangular parallelepiped, length, width and height are followed successively by 10mmX4mmX40mm.There is negative pressure device bottom.
Fig. 2 is tube facing side figure, and internal diameter is 8mm.This side pipe body is sanded transparent face apart from bottom 10mm.
Fig. 3 is test tube outboard profile, and internal diameter is 2mm.
Fig. 4 is inserted with the lid of plastic bar, lid is rectangle, there is rectangle sheet handle upper end, facilitate lid folding, lid central authorities lower end is connected with wide about 1-3mm pole, pole lower end is connected with the polystyrene board of the black height adhesion protein that can freely take off, and this plate is rectangular parallelepiped, and length, width and height are followed successively by 6mmX0.5-1mmX10mm.
Fig. 5 is the foraminate lid of central zone, and lid is rectangle, and central authorities leave the aperture that diameter is 1mm, after building lid, liquid reagent can be added in test tube and carry out freeze-drying in freeze dryer.
Fig. 6 is overally compressed to A4 paper size when being the production of black polystyrene plate, and each small pieces can remove easily from whole plate.There is 3X4 or 4X5 concave surface aperture in black polystyrene plate front, and aperture is 1mm or 0.5mm.
Fig. 7 has a buttonhole after the upper end of plate, designs little button with pole lower end, and both can arbitrarily fasten and be separated.A is outboard profile, and B is front view (FV).
Fig. 8 A is fluorescence detection device, and B is screen, leaves draw-in groove, as Fig. 9 in laser lamp side.
Figure 10 can be placed on clamp after polystyrene board is taken off, and then clamp is put into draw-in groove and carries out reading.
Figure 11 forwards light immuno-chromatographic test paper strip detection system structural representation, wherein 1 is sample pad; 2 is pad; 3 is analyzing film; 4 is adsorptive pads; 5 is the low lining of viscosity; 6 is 4 kinds of proteoglycan link protein antibody-UCP bonds; 7 is labelled protein 1; 8 is labelled protein 2; 9 is labelled protein 3; 10 is labelled protein 4.
Sample adds in the reaction cup of four kinds of freeze proof stem body mixed liquors that UCP particle marker is housed by Figure 12.
Figure 13 is by carrier board intercalation reaction cup, and the freeze-drying mixed liquor of sample and four kinds of antibody is wherein housed.
Figure 14 is reaction bonded schematic diagram, the antigen in sample and the antibody on carrier board and marked the antibody generation single step reaction of UCP particle.
Embodiment
Case study on implementation one: reaction cup detection system
1, as added CA125, CA153, SCCA, HE4 protein solution totally 100 microlitres (concentration is 1mg/ml) of UCP particle marker in Fig. 1 body, covering as round-meshed lid in the middle of Fig. 5, putting into freeze dryer freeze-drying under-50 DEG C of conditions.
2, as Fig. 6 black polystyrene being fixed with the albumen of 12-20 point, be added in the concave surface aperture as Fig. 6, often some 0.1-0.5 microlitre.Dot spacing 0.5-1mm.
3, coating protein, in thin polystyrene sheet, wraps by 4 kinds of albumen mouse-anti people CA125, CA153, SCCA, HE4, makes 4 kinds of albumen on the straight line that parallel with reaction cup base, above detection albumen, wrap by the anti-sheep IgG (1mg/ml) of rabbit as Quality Control point.Be fixed on as on Fig. 4 handle frame.Put into the test tube as Fig. 1, put into isothermal vibration shaking table and react 10 minutes under 37 DEG C of conditions.
4, after completely reacted, pipe is put into the liquid of negative pressure leaching device extraction, the handle frame taking out band black polystyrene thin slice faces up the draw-in groove put into as Figure 10, inserts in the square opening of detector side and detects.
5, obtain reacting fluorescent value, by the concentration of typical curve calculation sample by fluorescence detector reading.
Case study on implementation two: immuno-chromatographic test paper strip detects
1, bag quilt: anti-for rabbit sheep IgG is diluted to 1mg/ml, as C line coating buffer, by mouse-anti people CA125, CA153, SCCA, HE4 tetra-kinds of monoclonal antibody (concentration is 1mg/ml) mixing, as T line.By Membrane jetter, by T line coating buffer and C line coating buffer bag by nitrocellulose filter, dry, the tested bar of monoclonal antibody bag can be obtained.
2, utilize UCP to mark CA125, CA153, SCCA, HE4 tetra-kinds of antibody for subsequent use.
3, the preparation of pad: the above-mentioned four kinds of antibody utilizing UCP to mark are infiltrated on fiberglass packing, toasts 2h at putting into 37 DEG C, baking oven.Be cut into rectangular after drying.
4, assembling test strips: nitrocellulose filter, thieving paper, pad, sample pad are attached on base plate.
5, be added to by sample in sample pad, after 5-15min, test strips inserted fluorescence detector, at analyzing film result viewing, place carries out numerical value reading.
Claims (4)
1. based on gynecological tumor mark CA125, CA153, SCCA, HE4 chip detecting system and the reagent of up-conversion luminescence, to it is characterized in that: up-conversion luminescence UCP particle marker CA125, CA153, SCCA, HE4 tetra-kinds of antibody.
2. gynecological tumor mark CA125, CA153, SCCA, HE4 chip detecting system based on up-conversion luminescence and reagent, it is characterized in that: the carrier board in reaction cup detection system is black polystyrene, be coated with mouse-anti people CA125, CA153, SCCA, HE4 tetra-kinds of antibody above.
3. gynecological tumor mark CA125, CA153, SCCA, HE4 chip detecting system based on up-conversion luminescence and reagent, it is characterized in that: sample drop is added in reaction detection cup and detects, after reaction 5-15min, card is taken out carrier board and insert reading numerical values in the draw-in groove of fluorescence detector, numerical value is brought in typical curve concentration of specimens is calculated.
4. as claimed in claim 3, detecting sample is serum, body fluid, tissue fluid.
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| CN105242051A (en) * | 2015-09-23 | 2016-01-13 | 上海凯璟生物科技有限公司 | Up-conversion luminescence-based human insulin-like growth factor binding protein-1 (IGFBP-1) chip detection system and reagent |
| CN111247432A (en) * | 2017-10-16 | 2020-06-05 | 亨利技术有限公司 | Breast cancer detection kit and breast cancer detection method using saliva, and fertile period detection kit and fertile period detection method using saliva |
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| CN101930007A (en) * | 2010-07-30 | 2010-12-29 | 北京热景生物技术有限公司 | Up-converting fast quantification kit for preterm detection of pregnant woman |
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| CN105242051A (en) * | 2015-09-23 | 2016-01-13 | 上海凯璟生物科技有限公司 | Up-conversion luminescence-based human insulin-like growth factor binding protein-1 (IGFBP-1) chip detection system and reagent |
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Application publication date: 20150318 |