CN101275954A

CN101275954A - Breast cancer early warning chip for easily rapid measuring human I type thymidine kinase gene protein

Info

Publication number: CN101275954A
Application number: CNA2008100312811A
Authority: CN
Inventors: 丁克祥; 刘卫国; 费定宇; 刘莉; 丁宇
Original assignee: Individual
Current assignee: Individual
Priority date: 2008-05-09
Filing date: 2008-05-09
Publication date: 2008-10-01
Anticipated expiration: 2028-05-09
Also published as: CN101275954B

Abstract

The present invention relates to a breast cancer early warning chip for measuring I type theymine deoxyriboside kinase gene protein (hTK1) easily and rapidly. The invention is characterized in that the breast cancer early warning chip is composed of the following components: a strip fiber chromatography solid phase carrier NC film which is encapsulated with an anti-hTK1 monoclone antibody (number for 2A6-McAb) that is expressed and prepared by eukaryon at one end and is encapsulated with rabbit anti-mouse IgG monoclone antibody at the other end, a filtering sample paper, a water-absorbing glass fiber paper, and a polyester diaphragm which is marked with colloidal gold and secondary antibody hTK1 monoclone antibody (number for 3E8-McAb) bonder and the like components which are adhibited on a white PVC plastic piece. The invention has the characteristics and advantages of trace quantity, high speed, sensibility, accuracy, easiness, good stability, high repeatability, strong specificity, without special equipment or device, low detecting cost, measurement to single share or a plurality shares of specimen respectively, convenient carrying and convenience for site or field general investigation or physical examination.

Description

A kind of breast cancer early warning chip of easy fast measuring human I type thymidine kinase gene protein

Technical field

The present invention relates to the breast cancer early warning chip of a kind of easy fast measuring I type thymidine kinase gene protein (hTK1); adopt interdisciplinary science technology such as gene engineering method and molecular immune learn a skill development; be mainly used in the risk early warning that women's breast cancer takes place or develops; simultaneously, the new method of a kind of health care that also can be used for prompting of women's health protectiveness and early-stage breast cancer clinical diagnosis.

Background technology

About breast cancer and diagnostic techniques background thereof: breast cancer is human common and multiple malignant tumours, is a kind of malignant tumour of epithelial origin, also is a kind of complex disease of general.Early stage modal clinical sign and performance comprise mainly that painless property lump, udder shape or big or small change, depressed nipple, nipple discharge, skin of breast " dimple sample " depression or the epidermis of breast and armpit are that " tangerine peel sample " changes, the eczema sample such as oozes out at clinical sign around the nipple, have 10% patient that in various degree secret anguish can be arranged approximately, its age of onset is in the majority with 40～60 years old women.Its generation development is the result that multiple factor is regulated and control altogether in the body, particularly change with hormone and spiritual psychological factor that too nervous life brings and women previously mammary gland disease history etc. be easily to suffer from the strongest hazards of breast cancer effect.And estrogen exposure duration prolongation, as maiden's menarche in advance, middle-aged women menopause is delayed, and the postponement of fertility of child bearing age women time of nursing or minimizing then increase the breast cancer occurrence risk.In addition, the life style in west and postmenopausal women use the hormone replacement method for a long time, fat and high protein take in to increase etc. and can cause that all estrogen level improves in the diet, thereby danger that increase to suffer from breast cancer, and environmental factor is to the influence of breast cancer morbidity even surpass the influence of inherent cause.

The incidence of disease of mammary gland disease rises year by year, and in the U.S., breast cancer incidence accounts for 100 people/100,000 people, and China is 20 people/100,000 people, and the coastland is up to 30-40 people/100,000 people.Look back the diagnostic history of breast cancer, it is its initial means that breast cancer is checked oneself in the hand touch, but this method positive rate is low, and clinical examination is is at present seldom accepted and believed; The method that breast cancer is checked in the X photography that occur the seventies is except that existing the low problem of positive rate equally, and spinoff is greatly its critical defect; Liquid crystal type and thermal imaging system are to utilize human heat's principle diagnosing mammary cancer, because of being subjected to the influence of room temperature, and are difficult for preserving, and effect can not make the doctor satisfied; The cold light source transilluminator of releasing the beginning of the eighties be owing to can not show and shortcoming such as document image, and is not easy to make a definite diagnosis in early days ablactation gland cancer; The infrared breast diagnosis instrument that occur the beginning of the nineties at the end of the eighties is with infrared light transillumination breast tissue, with observe absorbed by haemoglobin what and in image, demonstrate the different gray scales of lump, the principle that the blood vessel shadow changes detects the mammary gland innocent and malignant tumour, in a single day but could show under the situation that has tumour to exist because of only checking, lose the opportunity of early treatment and show that breast cancer reaches an advanced stage; The method that occurs various inspection breast cancers after the nineties clinically successively, as improved X line mammography for diagnosing, colored B ultrasonic, thermal map diagnosis, computer body-layer scanning, nuclear magnetic resonance, diaphany, diagnosis of exfoliative cytology, tumor markers inspection etc. significant role is played in the early detection of breast cancer really, but also have different aspect and deficiency in various degree.

By description to the breast cancer diagnosis technical background, can therefrom find the relative deficiency of existing early diagnosis technology of breast cancer and instrument, conclude to get up mainly to show as: 1. hand touch mammary gland self-examination influence factor is a lot, the method leak is very big, self-assessment is difficult to, and the positive rate of breast cancer early diagnosis is very low; Even experienced doctor, diameter＜1cm breast lump also is difficult to be found, and is prone to and fails to pinpoint a disease in diagnosis or mistaken diagnosis, can't realize breast cancer early diagnosis truly; 2. the mammary X-ray Photographic technique is except that existing positive rate than low, and spinoffs such as its radiocontamination and damage are its major defects, and the accuracy rate of early diagnosis is lower; The susceptibility of improved X line mammography for diagnosing diagnosis is still not high enough, especially develops not good enough to some dense form mammary gland; The check rate and the biopsy rate of full visual field digitizing mammography machine are still lower; The each inspection of xerography imaging still makes the mammary gland X-ray receiving amount excessive, and influences picture quality and easily cause reinspection; 3. liquid crystal type and thermal imaging system are to utilize human heat's principle to carry out breast cancer diagnosis, are subject to surrounding environment and room temperature influence, and clinical error is bigger; The cold light source transilluminator is not easy to breast cancer make a definite diagnosis disconnected in early days because of not showing and shortcomings such as document image; 4. ultrasonic technique right＜1.0cm breast lump specificity is poor, can't accomplish the video picture of full milk gland, it is bigger that diagnostic accuracy is influenced by the operator; And B ultrasonic can not show the microcalciffcation kitchen range, to diameter＜0.5cm focus diagnosis rate only 44%, even the many array element ultrasonic transducer technology after improving, digital ultrasound scan conversion technology, ultrasonic Doppler detection technique, digital sound beam shaping technology etc. also are difficult to realize early detection; 5. fiber latex dust introscope technology just is found after forming the carninomatosis kitchen range, is difficult to early diagnosis; Nipple discharge smear cytolgical examination positive rate is lower; Area of computer aided 3 D stereo location and seal wire guiding excision biopsy technology are because of this apparatus expensive, and technical requirement is very high, is difficult to popularization and application; The fine needle aspiration suction of cells is checked because of the inaccurate delay treatment of easily failing to pinpoint a disease in diagnosis that punctures, sometimes still need append the histology biopsy and confirm its conclusion, and be difficult to distinguish infiltrating cancer and carcinoma in situ, to some cellular morphology unusually but do not reach cancer standard focus as yet, can't make and clarifying a diagnosis; 6. CT apparatus expensive, and scanning expense is higher, and X line amount is bigger, and inconvenience is as the routine inspection method; Mammary gland nuclear magnetic resonance check spatial resolution is low, and swollen object detail is clear not as the photography of X line, can not show microcalciffcation, has only enhanced ct scans to help to improve the accuracy of diagnosis and differential diagnosis, adds to cost an arm and a leg, only can be as the auxiliary examination method.PET-CT costs an arm and a leg surprisingly, and complex operation, and mechanism is complicated, and the result judges necessary experienced specialist, and detection can only just can be carried out in ultra-large type general hospital, and general hospital and health organ can't use at all; 7. also there be problem and doubtful point in various degree in more existing tumor markerses or tumour related substances and gene protein etc. as the breast cancer examination time, be not the specificity marker thing of breast cancer early diagnosis as carcinomebryonic antigen (CEA); The positive rate of breast cancer related antigen 225, I II phase breast cancer only are 8-22%, and early diagnosis is worth little; CA15-3 only is 0-25% to the positive rate I phase of breast cancer, and the II phase also has only 8-42%, and the 20-30% metastatic breast cancer do not raise, thus can not be separately as the index of monitoring breast cancer; CA27-29 (as pregnant preceding 3 months) under some benign tumour or physiological conditions also raises, and therefore detects to be difficult to make a definite diagnosis, and early diagnosis also is difficult to realize.The NCC-ST-439 positive is except that seeing breast cancer, cancer of pancreas, liver cancer, cholangiocarcinoma, colorectal cancer, lung cancer, cervical carcinoma etc. also may be positive, and also can raise before pancreatitis, chronic hepatitis, the amenorrhoea, its positive rate in breast cancer is I, II phase only to be 25-30%, III, IV phase also have only 40-42%, and recurrent cases is 55%, and specificity is lower; Tissue polypeptides specific antigen (TPA) has more after the present metastasis of cancer, and certain limitation is arranged aspect early diagnosing mammary cancer.The positive rate that breast cancer p53 gene mutation is expressed only is 20-60%, and also there is dispute in particularly present prognostic value for p53; Urokinase type plasminogen activator (uPA) mainly is an index of judging Prognosis in Breast Cancer, and is worth limited to early diagnosing mammary cancer; Cathepsin D is one of sign of Metastasis in Breast Cancer, and its many prompting patient with breast cancer poor prognosis that raise for the tendency of recurrence, also be the Prognosis in Breast Cancer factor, and aspect the early diagnosis of breast cancer, its value are still limited also.Telomerase all has positive expression in other malignant tumour, does not have obvious specificity.In a word, at present any independent application-aware in the breast cancer related neoplasms mark of finding is all lower, I, II phase positive rate all＜30%, the easy omission of early stage patient.8. direct determined dna sequence; denaturing gradient gel electrophoresis; heteroduple analysis; fluorescently-labeled mispairing analysis and protein truncation analysis; real-time fluorescence PCR technology etc.; single-strand conformation polymorphism analysis; multiple heteroduple analysis and order-checking and fluorophore double chain probe method etc. be mostly by patent protection, and because of some method and technology difficulty is big, the equipment requirements height; operation steps is loaded down with trivial details; poor specificity, the expense costliness is polluted the false-positive characteristics that cause easily; general hospital laboratory is difficult to use, and also is not suitable for large-scale examination.9. aspect cancer early warning instrument development, wrist formula cancer early-warning apparatus, hand held cancer cell detector, mammary tumor detector, dual wavelength tissue oxygenation detection technique (being called for short TBO)--breast tumor detecting instrument, the infrared mammary gland detector of computer etc., positive rate, early diagnostic rate, specificity, purchasing price etc. are all unsatisfactory though have.10. several relevant tumor markerses of TSGF tumour related substances combined detection kit (original name malignant tumour specificity growth factor quick detection kit) energy joint-detection are to improve lesion detection sensitivity, but step, method difference that various marks are measured, the determining instrument complexity, complex operation, various in style, time-consuming bothersome, the expense costliness.

About hTK1 and detection technique background thereof: people's thymidine kinases I type, Chinese is called for short thymidine kinase I type (Human thymidine kinase, hTK1) be and closely-related kinases of cell proliferation, differentiation and cell cycle, the key enzyme that is considered to synthetic DNA, it mainly contains two kinds of isodynamic enzymes, hTK1 and hTK2.HTK1 mainly is present in the tenuigenin, and hTK2 then mainly is present in the mitochondria of subcellular structure.And wherein closely-related with the cell proliferation relation be hTK1.Closely related just because of hTK1 and cell division, lower at cell division G1 phase content, raise gradually to the S after date, reach the highest to the G2 phase, therefore, the mRNA of coding hTK1 and the mark that expressed protein also just becomes hyperplasia thereof.

HTK1 is that thymidine participates in the synthetic key enzyme of DNA as a kind of kinases, the conversion between its energy reversibility catalysis thymidine and the phosphoric acid deoxyribonucleoside, and it can truly and objectively reflect the propagation situation of cell.Existing document shows, greater than 95% of total amount, and TK2 is lower than 5% to the activity of TK1 at tumour cell, detects very lowly maybe cannot detect in the serum of normal healthy people, therefore adopts and detects the grade malignancy that serum T K activity will be assessed tumor proliferation.HTK1 content or activity when the cellular abnormality hyperplasia increase, and the excision of particularly more notable increasing in the body fluid of the vigorous early stage malignant tumor patient of hyperplasia or blood, and the height of hTK1 level and malignant tumour and recurrence are and reduce and trend of rising.There is the scholar to increase and adopts 12 kinds of human cancer tissues to carry out immunohistochemical analysis, find that TK1 all presents positive findings, this has proved that tentatively the cancer patient increases relevant with TK1 vigor or content, and further test also proves, along with tumor cell proliferation speed obviously improves, the TK1 vigor strengthens, and the DNA aggregate velocity is accelerated, thereby promoted the further propagation of tumour cell, formed the vicious cycle that the state of an illness constantly increases the weight of.Therefore, the scholar is abroad arranged is that denier is low according to normal person's TK1 level or may detect hardly and experiment that malignant tumor patient increases unusually repeatedly proposes, adopt serum T K1 activity test method and TK1 antibody mediated immunity detection method to be suitable for mass survey, be used for the infantile tumour pre-detection.A large amount of clinical testings confirm that hTK1 not only can be used as a kind of mark of malignant tumour, but also can be by detecting the level of hTK1, judge malignant tumour generation and the rule of development or curative effect, lapse to and the judgement of prognosis.The U.S. has a patent just to introduce the active anticancer of TK1 specially.Moreover, the declarers of some authoritative magazines and famous experts and scholars and patent of the present invention find in nearest 5 years thematic scientific research also pleasantly surprisedly both at home and abroad, in some common malignant tumours, the variation of hTK1 content or activity and breast cancer and liver cancer are closely related, be that the expression of hTK1 in breast cancer and liver cancer is maximum, show also more obvious, and research is also found, a limited number of early stage when breast cancer or hepatoma carcinoma cell, have the expression of hTK1 gene protein and the change of content among its cancerous issue or even the blood equally.

The content of hTK1 is extremely low in the normal cell, just just high level can occur under the paraplasm situation of cell transition.There is scholar's result of study to show, the hTK1 level is apparently higher than the mammary gland benign tumor patient in patient with breast cancer's serum, and apparently higher than the normal healthy controls crowd, and hTK1 level among the mammary gland benign tumor patients serum and health check-up healthy person do not have significant difference (P＞0.05).Also have the scholar to extract the cytoplasm fraction of cell from 1692 breast cancer patient tissues, the activity of TK1 detects demonstration, and high TK1 is active closely related with the tumor proliferation degree, and high TK1 active indication endocrine drug therapy patient's poor prognosis and life span weak point; And born of the same parents breed in the prominent thyroid adenoma histocyte of low palilate TK1 active with normal structure relatively, the 2-4 that only raises is doubly.The present inventor once adopted the research of immunosensor method to cross activity and the content of hTK1 in present common eight kinds of malignant tumour serum, found that patient with breast cancer hTK1 level obviously increases than other malignant tumours.There are some researches show that also the half life period of breast cancer serum T K1 is about 30 days.Source about hTK1 in patient with breast cancer's serum has the scholar to analyze, and this is likely that tumor cell secretion or tumour cell abnormality proliferation cause dissolving the condensate that discharges thymidine kinase.And for the patient with breast cancer that lymph node does not shift, the hTK1 level is obvious downward trend in the back serum of performing the operation.The hTK1 level dropped to 50% in back one month in operation among the patients serum who does not shift behind the full resection operation of breast cancer, and perform the operation back 3 months (90 days) will drop to normal person's level.Therefore, the hTK1 level not only can be used as a susceptibility index of breast cancer diagnosis in the serum, and helps the observation of curative effect of breast cancer.For this reason, the warning index that hTK1 is diagnosed as women's early-stage breast cancer has certain theory and experimental basis, also has positive meaning aspect clinical generaI investigation and the early diagnosis.

In the clinical and research work in the past, be used to detect or monitor hTK1 content or active method mainly contains: radioimmunology (RIA), euzymelinked immunosorbent assay (ELISA), chemoluminescence method, SABC method, western blotting method, some Western blotting chemoluminescence method, ELISA method, immunostimulant chemoluminescence method, piezoelectric quartz wafer immunosensor method etc.Though, these breadboard detection methods all can determine content or the activity of hTK1 in cancerous issue or body fluid and the blood (serum or blood plasma) comparatively accurately at present, but the past, the main radioimmunology (RlA) that adopts detected the enzymatic activity of serum hTK1, promptly can be by labelled with radioisotope ³Total amount TK activity is represented the serum T K1 activity of (being called for short STK1) in the method detection serum of H thymidine, but the sensitivity of this method is not high, does not fit into the detection of TK1 in the clinical serum; Developed again later on the thymidine analog- ¹²⁵I-5-iodo-2-BrdU is set up the higher serum T K1 of a kind of sensitivity (STK) radioactive isotope detection method as substrate, is applied to human serum and learns the tumor cell proliferation markers tests.Its method is summarized as follows: add 20 microlitre blood serum samples in the cleaning micro tube, add respectively simultaneously and contain 20 microlitres and contain 40,10,5 and TK-REA standard to the cleaning micro tube of 2.5U/L.Every pipe is added the radioactive isotope that 500 microlitres are provided by reagent suppliers ¹²⁵I-5-iodo-2-BrdU substrate, after 37 degree reactions reaction in 4 hours is finished, separate with excessive substrate in order to make reaction product, also to add solid phase and separate the tablet adsorbed product, jolt reaction 15 minutes, the wash solution that provides with reagent suppliers is 4 times again, and this contains the activity that reaction product tablet precipitation is sent into γ-scintiloscope counting TK.According to standard relatively,, enzymatic activity is the active exceptional value of TK1 greater than normal healthy people standard threshold (2.4U/L), this shows, adopts ¹²⁵I labeled dideoxynucleotide uracil is the half life period short (only at 4 time-of-weeks) not only, and has radioactive contamination, cause easily tester's health damage and environmental pollution in the course of time, and the thymidine analog- ¹²⁵I-5-iodo-2-BrdU is not to be the selectivity substrate of TK1 activity analysis, and in the determination of activity of serum T K, very responsive to pH and temperature, the inaccurate result of normal generation, and need to use valuable detecting instrument (γ scintiloscope), this detection needs special equipment and those of skill in the art's operation, therefore, this detection technique has significant limitation in clinical practice, also be difficult in clinically and extensively promote the use of.Afterwards, had the scholar to adopt enzyme process and sandwich ELISA method to make serology TK1 again and identified that not finding had significant difference between healthy human serum TK1 and the cancer patient's serum T K1, think sandwich ELISA method insensitive due to.Report is abroad arranged later on, use content or activity that WesternBlotting detects hTK1 in the serum, and can carry out quantitative test, mix method aspect the assessment of tumour cell, being far superior to thymine, because thymidine kinase 1 is measured the thymidine kinase of directly representing in this tumour 1 and is measured and the not interference of the outer testing conditions of acceptor, also exempted and influence exogenous thymine and mix influence in the DNA, but in clinical practice and inapplicable; Though some Western blotting luminescence method has utilized the high sensitivity of luminescence technology, and expose by X-ray film, avoided use luminous detection instrument, but and long preservation experimental result, but need by setting up standard control, and carry out quantitative test, but, also be difficult to popularization and application clinically because of these method complex operations according to the depth of spot.And strengthen luminous-immune dot blot main reference blotting principle, and its concrete grammar is: gets person under inspection's blood sample on an empty stomach, leaves standstill more than 1 hour, then, place hydro-extractor, and with 4000 rev/mins, centrifugal 8-10 minute, separation of serum.The accurate point sample of the blood serum sample of 3 microlitres to nitrocellulose filter, simultaneously, is done the standard items point sample with the variable concentrations people TK1 (rhTK1 40.5,13.5,4.5 and 1.5pM) that recombinates.Air-dry 30 minutes, jolt washing 3 times (10 minutes/2 times, 5 minutes/1 time) with TBS pH7.5 (20mmol/L Tris, 0.15mol/L NaCl).Add 10% skimmed milk power with the configuration of TBS damping fluid, room temperature was blocked 2/4 hour, removed blockade reagent, added the TK1 antibody with the best dilute concentration of TBS configuration, immune response 2 hours or in refrigerator overnight.After reaction is finished,, jolt washing 3 times (10 minutes/2 times, 5 minutes/1 time) with the rapid rinsing of TBST (TBST adds 0.1% polysorbas20) 2 times.Add biotinylation second antibody (by the optium concentration dilution) again, jolt reaction 40 minutes under the room temperature.After reaction is finished, after the same method washing, add Avidin-horseradish peroxidase.After the same method washing, accurately reacted 1 minute with the ECL luminescence reagent, after the drying, this film is sealed in bag film, puts into the CCD detecting instrument, the power of this luminous signal will be by CCD imaging system analyser lock-on signal and scanning quantitative test, or film is put into film holder and X-ray film clamp, sensitization after 2～5 minutes in the darkroom is developed, photographic fixing immediately, with Western blot atlas analysis instrument, analyze the signal of printing and dyeing on egative film.And calculate this pM concentration for examination person's TK1 value according to the typical curve of rhTK1.According to standard relatively, the concentration as TK1 is exceptional value greater than normal healthy people level threshold value (2.0pM).Adopt this typical curve, though the convenient level of calculating cancer patient's TK1 in the treatment changes, with relation corresponding to the growth rate of patient's tumour cell, and adopt the detection method of antibody mediated immunity combination can reach enough low detection limit, particularly can detect the difference of the level of the healthy people TK1 of Different Individual, the lowest detection amount can reach below the 0.3pM, but we are not difficult to find out, how complicated and loaded down with trivial details this method operation is, not only take length, need special good and highly sensitive anti-TK1 antibody as the probe of surveying the TK1 molecule of trace in serum or the tissue, and a large amount of valuable instrument and equipment and the veteran professional operating personnel of needs, not only can't also be difficult to clinical practice in large-scale synthesis hospital in the different medical unit popularization and application.Even studying successful piezoelectric quartz wafer immunosensor method not long ago, patented claim person of the present invention has than the said method more superiority, but also exist and grow (40-60 minute), detection difficulty detection time and more greatly, particularly can only measure a sample at every turn, working sample etc. simultaneously in batches, this uses almost very difficulty clinically.The method of particularly above-mentioned all detection TK1 all needs comparatively expensive and special instrument and equipment and matched reagent basically, adds that operating process is loaded down with trivial details, detection time is longer, the technical requirement height needs certain instrument etc., can't be in different medical unit or family's popularization and application.But if high specificity, two kinds of good and highly sensitive TK1 antibody of affinity are arranged, immune sandwich method is still up-and-coming method, and this also is that the present invention adopts one of foundation of maximizing favourable factors and minimizing unfavourable ones.

In sum, one-sidedness result (false negative or false positive) all appears in the single diagnostic method of above-mentioned any breast cancer and the resulting diagnosis of detection technique or testing result unavoidably, even may occur and the clinical mistake result who does not conform to, even adopt clinical and experiment several methods use in conjunction, replenish mutually and confirmation, make gained diagnosis or testing result comparatively correct reliable and reference value arranged, but in case this diagnostic result is positive, basically the occupancy focus that has occurred breast cancer, in fact also non-substantial early diagnosis, and bring challenges to later treatment or more.Modern oncology studies shows, breast cancer from from the beginning of the division growth of a cancer cell, to the little lump that develops into the clinical about 1cm of diameter that can detect, need 30 multiplications approximately, its growth period, oneself exceeded 3 years at least, provided time enough to cancer metastasis, so, even the subclinical transfer of whole body when its diameter of tumor＞1cm, just might take place in the I primary breast cancer of Que Rening clinically; And comprised in the II phase case that axillary lymphatic metastasis is arranged, even the nodi lymphatici axillares negative patient, the primary tumo(u)r diameter oneself surpass the subclinical transfer of 2cm can be up to 25%-30%.So, from the histopathology angle, I, among the II phase patient oneself considerable part is arranged not is early stage.

Even adopt some the most modern advanced diagnostic method and detection techniques at present, certain foundation is provided for the early diagnosis of clinical breast cancer, but because of these diagnostic methods and detection technique need specific condition, need just can carry out in some large-scale synthesis hospitals, and, these diagnostic methods and detection technique, most detectable specialty, the assay method complexity, detecting instrument is various, complex operation step, Laboratory Request is special, and time-consuming bothersome effort, mensuration expense costliness makes a lot of people, particularly some people at highest risk often are difficult to and in time go to hospital to check or generally investigate, thereby, missed manyly to early diagnosing mammary cancer, the opportune moment of early detection is to such an extent as to cause lifelong sorry and painful.

This shows, invent a kind of science more, advanced, innovation, practical, and have highly sensitive, high specificity, good stability, the accuracy height, use quick, easy and simple to handle, convenient sources, good and cheap, popularize easily, be easy to check at any time, the breast cancer inspection of applying, check oneself or census method, for early detection truly, early diagnosis, the early treatment breast cancer, perhaps strive for obtaining best treatment opportunity, seek best therapeutic scheme, reach best result of treatment, to reduce the patient suffering, prolong patient's life, raising patient life quality etc. has the clinical value and the practical significance of particular importance.

Particularly by setting up this easy, quick, Yi Xueyi meeting, being easy to check oneself or generally investigate or regularly regular breast cancer is checked means, be devoted to set up a perfect breast cancer examination system, be devoted to set up standard examination breast cancer more, have the scientific consciousness of early diagnosis in mind, and then, improve constantly the normalized active inspection of the whole people or check oneself consciousness, and be fully recognized that by regular examination early detection and early diagnosis breast cancer are how important; Simultaneously, also should have according to self risk and accept corresponding examination and find the consciousness that suspicious sign is in time sought medical advice.Also only in this way, could real reduce effectively owing to despise or the mistake that is ignorant of professional and the people at highest risk who causes lack of standardization is examined, failed to pinpoint a disease in diagnosis, mistaken diagnosis.

Summary of the invention

The objective of the invention is to be improved, innovate, a kind of breast cancer early warning chip of easy fast measuring human I type thymidine kinase gene protein is provided at shortcoming that exists in the background technology and problem.

Technical scheme of the present invention is: described breast cancer early warning chip is that (code name is 2A to an anti-hTK1 monoclonal antibody that is prepared by eukaryotic expression by an end bag ₆-McAb) and the other end then wrap by the fibre strip chromatography solid phase carrier NC film of rabbit anti-mouse igg polyclonal antibody, filter sample paper, water adsorption glass fibrous paper and be marked with collaurum and two anti-hTK1 monoclonal antibodies (code name is 3E ₈-McAb) polyester film sheet etc. of bond is pasted on white PVC plastic sheet according to this and forms.

The preparation method of the breast cancer early warning chip of easy fast measuring human I type thymidine kinase gene protein of the present invention may further comprise the steps:

One, preparation contains anti-hTK1 monoclonal antibody 2A ₆Cellulose nitrate miillpore filter sheet with the rabbit anti-mouse igg polyclonal antibody:

1) will resist hTK1 monoclonal antibody 2A ₆Use 10-20mmol/L respectively with the rabbit anti-mouse igg polyclonal antibody, the phosphate of pH7.2-7.4 (PBS) damping fluid is diluted to 0.8-1.6g/L;

2) they are wrapped respectively by on the cellulose nitrate miillpore filter by machine with bag;

3) will be coated with hTK1 monoclonal antibody 2A ₆With place 10-20mmol/L on the cellulose nitrate miillpore filter of rabbit anti-mouse igg polyclonal antibody, in the phosphate of pH7.2-7.4 (PBS) damping fluid (1-2%BSA), 37 ℃ of constant temperature soak 60min;

4) use 10-20mmol/L again, the phosphate of pH7.2-7.4 (PBS) damping fluid washing places after room temperature or the vacuum drying standby;

Two, carry out the assembling of hTK1 gene protein breast cancer early warning chip:

Well 3 with positive bar mid point is a starting mark, and sample paper, water adsorption glass fibrous paper, polyester film sheet (collaurum-hTK1 monoclonal antibody 3E will be filtered successively with hot imperial glue respectively in the left side and the right ₈Bond), be coated with anti-hTK1 monoclonal antibody 2A ₆Be pasted on the PVC plastic sheet of white with the cellulose nitrate miillpore filter and the absorbent filter of rabbit anti-mouse igg polyclonal antibody, and on this diaphragm, cover the effective diaphragm of one deck; Can carry out vacuum drying after finishing pad pasting; At last, the batten that all posts is cut into the strip diaphragm of certain specification on cutter special, in the plastic casing of packing into, adds the drying agent sealing and preserve, breast cancer gene albumen early warning chip 4.

Ultimate principle of the present invention: be a kind of solid phase labelling and double-antibody sandwich immuno-chromatographic assay technology that several different methods such as analytical technology organically combine that technique for gene engineering, colloid gold label developing technology, specific immunity detection technique and ply of paper are analysed.The immune colloidal gold technique of the gene protein chip application of the present invention's preparation is a kind of novel immunolabelling technique that is applied to detect new breast cancer mark people thymidine kinases (hTK1) antigen with collaurum as the mark of spike or colour developing.Its ultimate principle is: collaurum is by gold chloride (HAuCl ₄) in reductive agent trisodium citrate (citric acid trisodium) effect down, polymerization becomes specific and even big or small gold grain is the collaurum state because of its electrostatic interaction becomes a kind of stable colloidal state.Collaurum is electronegative under the weak base environment, can form firm combining with the positive charge group of protein molecule, and this combination is the static combination, so do not influence the biological nature of protein.Its chemical nature of people's thymidine kinases monoclonal antibody (hTK1 McAb) also is protein (Immunoglobulin IgG), and therefore, it possesses fully with collaurum static and combines the biological nature that does not influence this antibody activity.And colloid gold particle has the characteristic of high electron density, and at gold mark protein combination place, at the visible pitchy particle of microscopically, when these colloid gold label things were assembled in a large number at corresponding part place, naked eyes are red color visible or pink spot then.Utilization collaurum itself has this characteristics of chromogenic reaction, chromogenic reaction that can be by visual marking thing collaurum is clear, intuitively and exactly judge and detect the experimental result that whether contains breast cancer mark people thymidine kinases (hTK1) antigen in the sample.Thereby the present invention can be used as the qualitative or semiquantitative easy tachysynthesis detection method of early-stage breast cancer.

Know-why of the present invention: be to be that 45-50 moves the cellulose nitrate miillpore filter (NC) of 1cm and glass fibre second as solid phase carrier with fibre strip chromatographic material-5 μ m aperture, liquid translational speed, (an anti-code name is 2A with one of them monoclonal antibody specific ₆)-people thymidine kinases (hTK1) monoclonal antibody (McAb) is fixed on the appropriate location of cellulose nitrate miillpore filter (NC); And another monoclonal antibody specific (two anti-code names are 3E ₈)-people thymidine kinases monoclonal antibody (hTK1 McAb) then is marked on the uniform nanoscale colloid gold particle (particle diameter 20-25nm) and is fixed on the specific polyester film.When actual the detection, because ply of paper is analysed the natural capillary action in the process, carrying out property is mobile naturally forward along this fibre strip chromatographic material-cellulose nitrate miillpore filter (NC), polyester film and glass fibre to make detected sample-blood or serum or blood plasma; At this moment, if detect in sample-blood or serum or the blood plasma and contain corresponding determinand-hTK1 antigen, then this hTK1 antigen is moving to certain position, just therewith fibre strip chromatographic material-cellulose nitrate miillpore filter (NC) and, the immune association reaction of high specific, high-affinity promptly takes place at the corresponding monoclonal antibody-hTK1 McAb of this determinand hTK1 antigen on the polyester film.Since the immune complex of the antigen-antibody combination in this specific immunity chromatography process by enrichment constantly or be trapped in this fibre strip chromatographic material a certain zone, and formed a coloured detection band (being positive findings) naturally.But, if detect in sample-blood or serum or the blood plasma and do not contain corresponding determinand-hTK1 antigen, then this detects sample-blood or serum or blood plasma and moves to any position, not therewith fibre strip chromatographic material-cellulose nitrate miillpore filter (NC) and, on the polyester film at corresponding monoclonal antibody-hTK1 McAb generation high specific of this determinand hTK1 antigen, the immune association reaction of high-affinity.In the chromatography process, there is not this specific antigen-antibody immune complex formation just because of detecting sample yet, some free labels are then crossed the detection band of this fibre strip chromatographic material, automatically separate with binding label, any color reaction does not appear, promptly, can not form a coloured detection band (being negative findings) in a certain zone of cellulose nitrate miillpore filter.

Major technique design of the present invention:

The technology of the present invention design is divided into three parts: first: the hTK1 Monoclonal Antibody of high specific and high-affinity.Mainly finish: 1. adopt technique for gene engineering and molecular biology and molecular immune to learn a skill, will with the closely-related gene of breast cancer-I type people thymidine kinases, also abbreviating I type thymidine kinase (human thymidine kinase I is called for short hTK1) gene as clones; 2. utilize eucaryon excretion vector this hTK1 gene of recombinating; 3. adopt the autarcetic method of gene to prepare the hTK1 monoclonal antibody of high specific and high-affinity.Second portion: the preparation of collaurum and mark.Mainly finish: the 1. preparation of collaurum: at first adopt trisodium citrate reduction method to carry out the preparation of collaurum; 2. the preparation of collaurum-anti-hTK1 monoclonal antibody bond: (code name is 3E with another monoclonal antibody specific to adopt bSA (BSA) method ₈)-people thymidine kinases monoclonal antibody (hTK1 McAb) is marked at (particle diameter 20-25nm) on the uniform nanoscale colloid gold particle; 3. preparation contains collaurum-hTK1 monoclonal antibody 3E ₈The polyester film sheet of bond.Third part: I type thymidine kinase (hTK1) gene protein breast cancer early warning chip preparation.Mainly finish: 1. preparation contains anti-hTK1 monoclonal antibody 2A ₆Cellulose nitrate miillpore filter sheet with the rabbit anti-mouse igg polyclonal antibody; 2. carry out the assembling of breast cancer gene albumen early warning chip.

The main thought of technical solution of the present invention:

First: hTK1 Monoclonal Antibody: at first use Trizol reagent (a kind of kit) to extract the total RNA (ribonucleic acid) of handling through methotrexate 48 hours of cell (RNA), handle water-soluble separating with pyrocarbonic acid diethyl ester (DEPC) behind the purifying and precipitate gained liquid 30 μ l from 5,000,000 Hela cells (HeLa cell is a kind of heLa cell) from total RNA of extraction such as zooblast and tissue, bacterium, fungies.This liquid can amplify hTK1cDNA (cDNA is meant with mRNA to be template, the complementary DNA that forms) through reverse transcription PCR (in external a kind of method by the synthetic specific DNA fragment of enzymatic reaction) under the effect of reverse transcriptase; (promptly utilize the function of the terminal ligase that the Taq polymerase has simultaneously TA clone, 3 '-A jag of the automatic interpolation of 3 ' end at every pcr amplification product) can carry out dna sequence analysis after, whether correct to identify through formed hTK1 gene order behind the pcr amplification; Then, insert between the HindIII (restriction enzyme site) of pSecTag2/Hygro B carrier (eucaryon excretion vector) and XhoI (restriction enzyme site) site (pSecTag/TK1) by subclone, the protein of insertion fragment expression can be secreted into outside the born of the same parents from eukaryotic.Directly with pSecTag/TK1 recombinant plasmid DNA (deoxyribonucleic acid) (DNA) immune mouse; Merge the preparation hybridoma with myeloma cell and splenocyte again; Screen, match from many strain of hybridoma strain of preparing, it is higher and can produce the hybridoma cell strain (called after DZ1 and DZ2) of anti-TK1 monoclonal antibody to filter out two strain affinity at last; This selected two strains affinity is higher and can produce the hybridoma cell strain DZ1 and the DZ2 of anti-hTK1 monoclonal antibody, be 5X10 according to quantity respectively ⁵Individual hybridoma directly injects respectively and prepares the ascites that contains the hTK1 monoclonal antibody specific in two groups of mouse peritoneals; After treating that these are tried ascites in the mouse peritoneal and acquire a certain degree, can be respectively extract the mouse ascites of two kinds of anti-TK1 monoclonal antibodies of the high and high specificity of affinity that this two strain of hybridoma produced out with injection needle; With these mouse ascites of collecting with high-speed refrigerated centrifuges with greater than the centrifugal 5-10 of the speed more than the 10000rpm/min minute, the reject precipitation, its supernatant adopts caprylic acid one sulfuric acid amine method to extract respectively, and with Protein G post affinitive layer purification, (DZ1, the secreted monoclonal antibody of DZ2 hybridoma are respectively hTK1 monoclonal antibody 2A can to obtain the monoclonal antibody of purified two kinds of TK1 ₆With hTK1 monoclonal antibody 3E ₈); Two kinds of hTK1 monoclonal antibodies of behind the purifying this are used quantitatively antibody of protein quantification reagent (BCA) respectively, and are last, detect its antibody purity through sodium dodecylsulphonate (SDS)-PAGE (polyacrylamide) gel electrophoresis; Simultaneously, carry out the IgG type of antibody, subgroup identification, and the binding site of antibody and affinity mensuration etc.; Afterwards, placing low temperature refrigerator to preserve, can be subsequent applications fully.

Second portion: at first will prepare certain density gold chloride and use liquid 100ml with pure distilled water, after being heated to boiling with electric furnace, add certain density trisodium citrate (citric acid trisodium) rapidly and use liquid 2ml, after continuing heated and boiled 10min, through after the reduction reaction, after it is progressively cooled off, can form near red collaurum and use liquid.Whether then, utilize automatic spectrophotometer that this collaurum is used liquid and carry out autoscan in visible-range (400-600nm), it is even to analyze prepared colloid gold particle.When wavelength was the 520-521nm place, this collaurum is used liquid can maximum absorption band, and the main peak width is narrower, and this confirms that prepared colloid gold particle is even fully, and granularity is at 20-25nm.Be reduced into after the suitable and uniform collaurum of size distribution of grain size uses liquid determining to finish gold chloride, can adopt this collaurum to use the mark that liquid carries out people's thymidine kinases (hTK1) monoclonal antibody (McAb), the i.e. preparation of collaurum-hTK1 monoclonal antibody bond.When carrying out the preparation of collaurum-hTK1 monoclonal antibody bond, should be earlier with hTK1 monoclonal antibody 3E ₈Concentration adjustment is to suitable level (800-1600 μ g/mL); Then, get the colloidal gold solution that dosage is 100mL, and be the sal tartari (K of 0.1mol/mL with concentration ₂CO ₃) collaurum is used liquid transfer to PH 8.2-9.0 a little more than hTK1 monoclonal antibody isoelectric point; At this moment, in constantly stirring fast, slowly adding 1mL adjusted is the hTK1 monoclonal antibody 3E of 800-1600 μ g/mL to concentration ₈, then its labelled protein amount is to contain 8-16 μ g hTK1 monoclonal antibody 3E in every milliliter of collaurum application liquid ₈After continuing to stir 15min, add the calf serum (BSA) of 100-200mg; After stirring through 10-20min again, place refrigerated centrifuge low-speed centrifugal (1000-1500rpm/min) 10-20min.Reject precipitates carefully; After getting its supernatant and carrying out high speed centrifugation (10000-15000rpm/min) 20-30min again, careful again the suction removed supernatant, keeps precipitation, and this is collaurum one anti-hTK1 monoclonal antibody 3E ₈Bond.And then, can prepare and contain collaurum-hTK1 monoclonal antibody 3E ₈The polyester film sheet of bond.At first, (1-2%BSA pH8.2) has precipitated good collaurum-hTK1 monoclonal antibody 3E with above-mentioned can to adopt the Tris-HCl damping fluid of suitable concn ₈Bond is diluted to, and it is evenly distributed on the polyester film sheet of certain area, and is fixing and carry out vacuum drying, can obtain containing collaurum-hTK1 monoclonal antibody 3E ₈The dry polyester film sheet of bond.

Third part: I type thymidine kinase gene protein breast cancer early warning chip preparation.Mainly finish: 1. preparation contains anti-hTK1 monoclonal antibody 2A ₆Cellulose nitrate miillpore filter sheet with the rabbit anti-mouse igg polyclonal antibody; 2. carry out the assembling of hTK1 gene protein breast cancer early warning chip.At first, preparation contains anti-hTK1 monoclonal antibody 2A ₆With the cellulose nitrate miillpore filter sheet (NC) of rabbit anti-mouse igg polyclonal antibody, be about to anti-hTK1 monoclonal antibody 2A ₆Use 10-20mmol/L respectively with the rabbit anti-mouse igg polyclonal antibody, the phosphate of pH7.2-7.4 (PBS) damping fluid is diluted to 0.8-1.6g/L, after carrying out mark, they is wrapped respectively by on the appropriate location of cellulose nitrate miillpore filter by machine with bag; And then will be coated with hTK1 monoclonal antibody 2A ₆With place 10-20mmol/L on the cellulose nitrate miillpore filter of rabbit anti-mouse igg polyclonal antibody, in the phosphate of pH7.2-7.4 (PBS) damping fluid (1-2%BSA), 37 ℃ of constant temperature soak 60min; At last, use 10-20mmol/L again, the phosphate of pH7.2-7.4 (PBS) damping fluid washing 1-2 time; Take out subsequently, suck dry moisture places room temperature or vacuum drying standby as far as possible.Contained hTK1 monoclonal antibody 2A finishing bag ₆Behind the cellulose nitrate miillpore filter of rabbit anti-mouse igg polyclonal antibody, can carry out the assembling of hTK1 gene protein breast cancer early warning chip: before formally carrying out the chip assembling, must carry out the pre-service of diaphragm earlier, then, paste multiple film again: the well 3 with positive bar mid point is a starting mark, and sample paper, water adsorption glass fibrous paper, polyester film sheet (collaurum-hTK1 monoclonal antibody 3E will be filtered successively with hot imperial glue respectively in the left side and the right ₈Bond), be coated with anti-hTK1 monoclonal antibody 2A ₆Be pasted on the PVC plastic sheet of white with the cellulose nitrate miillpore filter and the absorbent filter of rabbit anti-mouse igg polyclonal antibody, and on this diaphragm, cover the effective diaphragm of one deck.Wherein, filter sample paper (separation of whole blood film) and water adsorption glass fibrous paper be all through the damping fluid processing and be attached to below the well 3, and successively with polyester film sheet (collaurum-hTK1 monoclonal antibody 3E ₈Bond) connect, and polyester film sheet (collaurum-hTK1 monoclonal antibody 3E ₈Bond) then be coated with anti-hTK1 monoclonal antibody 2A ₆Be connected with the cellulose nitrate miillpore filter of rabbit anti-mouse igg polyclonal antibody; Cellulose nitrate miillpore filter afterbody links to each other with absorbent filter.Can carry out vacuum drying after finishing pad pasting; At last, the batten that all posts is cut into the strip diaphragm of certain specification on cutter special, in the plastic casing of packing into, adds the drying agent sealing and preserve, breast cancer gene albumen early warning chip.

Technology operation method of the present invention:

With this breast cancer gene albumen early warning chip, can directly insert in blood or serum or the blood plasma, moments later, directly be placed on the table top 3-5min of level, can detect breast cancer gene albumen hTK1 fast and accurately, thereby realize early warning diagnosis early-stage breast cancer; Perhaps this breast cancer gene albumen early warning chip level is assemblied in the specific model (breast cancer gene albumen early warning chip fully supporting reactive tank and test card) therewith, and drawing sample that 20-50 μ l peripheral blood or vein serum or plasma sample directly be added drop-wise to this breast cancer gene albumen early warning chip with the micropipet that contains the anticoagulant heparin agent or dropper, to add end (too dense thick as blood preparation, then available PBS damping fluid dilutes simultaneously), the serum in the visible whole blood sample is divided a word with a hyphen at the end of a line forward by chromatography (or kapillary) effect.In the process of dividing a word with a hyphen at the end of a line forward, as there being hTK1 in the blood, then this antigen earlier with the colloid gold label thing-hTK1 monoclonal antibody 3E that is fixed on the glass fibre ₈The combination of Fab terminal specific, and carrying colour developing label-collaurum and continuing to divide a word with a hyphen at the end of a line forward, constantly with the cellulose nitrate miillpore filter on contain anti-hTK1 monoclonal antibody 2A ₆Assemble with rabbit anti-mouse igg polyclonal antibody generation antigen-antibody binding reaction, produce macroscopic two red stripes, be positive findings.This detects only needs 3-20min, and 3-5min can go out the result when measuring hTK1 strong positive sample; But 5-10min goes out the result when measuring the weak positive sample of hTK1; Then must go out the result by 15-20min when measuring hTK1 feminine gender sample.In addition, judge more accurately as need, the hTK1 standard antigen can be added to respectively in healthy anti-freezing vein whole blood or serum or the plasma sample according to various dose, make it to form the concentration gradient 0.5 μ g/L of a blank determination, 1 μ g/L, 2 μ g/L, 3 μ g/L, 4 μ g/L, 5 μ g/L, 6 μ g/L are added drop-wise to respectively on this breast cancer gene albumen early warning chip then, with quadrat method test and carry out the result and judge.If occur less than the then negative result of 0.5ng/mL; If appearance is approaching or equal 1-2ng/mL standard items p-wire, then can be judged to the weak positive; If appearance is approaching or equal 3-4ng/mL standard items p-wire, then can be judged to the positive; If occur near or equal or be deeper than 5-6ng/mL standard items p-wire, then can be judged to strong positive; If nature controlling line 1 and p-wire all do not occur, then show this breast cancer gene albumen early warning chip test invalidation.

Major technique characteristics of the present invention: this breast cancer early warning method of the present invention and analytical technology have trace, quick, sensitive, accurate, easy, stable, high specificity, need not specific installation or instrument, and can single part mensuration, detect characteristics and advantages such as cost is low, be suitable for scientific research and prevention unit and hospital at different levels and carry out uses such as medical treatment, health care, health check-up, also can be used for the preventative generaI investigation that individual, kinsfolk and relatives carry out breast cancer, so that early detection and early diagnosis.

Major technique innovative point of the present invention: 1. adopt the eucaryon secretion type expression hTK1 and the innate immunity thereof to prepare the hTK1 monoclonal antibody method, and with this with the hTK1 monoclonal antibody of the hTK1 of eukaryotic expression and two different affinity of innate immunity preparation thereof, different binding sites and to be prepared into breast cancer gene albumen early warning chip according to double antibody sandwich method be that the present invention initiates; 2. in the process of preparation breast cancer early warning chip, use concentration that the innate immunity prepares the hTK1 monoclonal antibody or dosage and reaction conditions (particularly pH value 8.8) and initiate for the present invention; 3. in the process of preparation breast cancer early warning chip, use the hTK1 monoclonal antibody of two high specifics and high-affinity innate immunity preparation simultaneously and adopt double antibody sandwich method promptly can measure in whole blood (end is blood slightly) or vein serum or the blood plasma hTK1 and initiate for the present invention; 4. the hTK1 Monoclonal Antibody breast cancer early warning chip with this innate immunity preparation both can be used for end blood slightly, can detect venous blood (comprising serum or blood plasma) hTK1 again, particularly design increased complete blood cell and separating plasma diaphragm for fundamentally solve micro-end slightly whole blood hTK1 mensuration lay a good foundation; 5. adopt a whole blood or serum or blood plasma only 1 time or the step result that can measure twice breast cancer gene albumen hTK1 simultaneously initiate for the present invention more accurately and reliably; 6. for preventing that whole blood from solidifying or dense thick and serum is difficult for diffusion, when measuring, design matching used anticoagulant heparin suction pipe or dropper, be determined as the present invention's initiative again after perhaps diluting blood sample according to blood viscosity; 7. this breast cancer gene albumen early warning chip appearance design form and change 3 designs of micro chip and well into and can make more convenient to operate and simple and direct and practical at the diaphragm center; 8. having increased a magnification eyepiece in supporting fully reactive tank of the novel albumen of breast cancer gene therewith early warning chip and the test card can make the colour developing result amplify and improve the accuracy of observing; 9. hTK1 gene protein breast cancer early warning chip initial end polyester film (on collaurum and hTK1 monoclonal antibody 3E are arranged ₈Bond) glass fibre diaphragm of being installed under and filter sample paper were handled especially through damping fluid, and the influence and the maintenance testing result that can make detection sample or liquid not changed by pH value stabilize to characteristic of the present invention more; 10. sheep anti-mouse igg polyclonal antibody, hTK1 monoclonal antibody 2A ₆And hTK1 monoclonal antibody 3E ₈By the determining of concentration, and last testing result is negative less than 0.5ng/ml, is the weak positive near 1-2ng/ml Deng best bag, near the positive result of 3-4ng/ml, initiates for strong positive result waits to the present invention near 5-6ng/ml.

Technical advantage of the present invention and characteristics mainly show as: this breast cancer gene albumen early warning chip and analytical approach thereof, from gene clone, eukaryotic expression and the Monoclonal Antibody of hTK1 and chip assembling thereof etc., source technology and raw material are inventor oneself preparation, industrialization rapidly; And technique for gene engineering makes this breast cancer gene albumen early warning chip thoroughly solve " nuclear core-hTK1 monoclonal antibody " problem, and this should use not only convenient sources, low price and easily-learned easily mastered from now on, be easy to popularize and apply.The technical method and the product that form of the present invention particularly, has trace, fast, sensitive, accurately, easy, good stability, repeatability is high, high specificity, need not specific installation or instrument, the detection cost is low, can single part or many parts of samples measure respectively, and it is easy to carry, be easy to scene or characteristics and advantages such as field reconnaissance or health check-up, can be suitable for scientific research and prevention unit and hospital at different levels and carry out medical treatment, health care, uses such as health check-up, also can be widely used in the individual, kinsfolk and relatives carry out the preventative of breast cancer and check oneself or generally investigate, so that early detection and early diagnosis.

Specifically, the technical advantage of patent of the present invention and characteristics mainly show as:

1. micro-: as to adopt the present invention to carry out the closely related mark TK1 of breast cancer gene protein and detect, common required test sample-Mo blood or serum or blood plasma 1 (approximately 20-50 μ l) slightly gets final product), even test sample (serum or blood plasma) has only 5-10 μ l still can finish detection.And some other can be used for the immunochemiluminescence method, ELISA method, little balance quality testing method of hTK1 content detection etc. and needs about at least 2 milliliters of blood specimens, and the sample consumption surpasses more than 20 times.

2. quick: the present invention has that translational speed is fast, the reaction time is short, detect characteristics such as rapid, moves from dripping test sample, hair and blood effect, finishes, judges at last the result to chromogenic reaction and all finished within 3-20 minute.The sample detection of some very typical case (being that hTK1 content is higher), its result are judged even were only needed get final product all in 3-5 minute; And some other can be used for the method for hTK1 content detection, can detect hTK1 though comprise immunochemiluminescence method, ELISA method, little balance quality testing method etc., need that special instruments and equipment and complicated operation, flow process are loaded down with trivial details, that the time is grown (needing at least more than 1-2 hour), some even time is longer.

3. sensitive: as, can to detect the closely related mark TK1 of breast cancer of ng level level because of the present invention adopts the design of antigen and antibody specific combination principle.As the technology of the present invention is to adopt double antibody sandwich method, its sensitivity even can be up to 1ng/ml, can be in every milliliter of whole blood or serum or plasma sample, can detect the closely related mark TK1 of 1ng and breast cancer, as seen high, one of the method for a kind of highly sensitive breast cancer early detection and early warning diagnosis of can yet be regarded as of its sensitivity.

4. accurate: adopt the present invention to adopt a whole blood or serum or plasma specimen replicate determination twice simultaneously, so accuracy is higher, it is more accurate to judge.Once 30 parts of typical patients with mastocarcinoma serum specimens were measured in this way, wherein the positive early warning result of testing result all appears in 28 parts of serum, and disposable recall rate is greater than 93%.Simultaneously, compare with method of the present invention and the enzyme immunoassay of classics or up-to-date little balance mass method, it closes rate, susceptibility, specificity etc. relatively all more than 90%.This explanation this method a kind of relatively accurate and reliable breast cancer early warning method of can yet be regarded as.

5. easy: the present invention's operation is very easy, not carried out at any time by condition restriction, and need not any detecting instrument and equipment, as long as the tester is to specifications, a whole blood or serum or plasma specimen are added on the good breast cancer gene albumen early warning chip of prepared beforehand of the present invention, lie in a table top in room temperature water, wait for 3-5 minute then, last visual inspection result gets final product.Because collaurum itself has chromogenic reaction clearly, steps such as agent of interpolation reaction and display and stop buffer have been omitted than ELISA, thereby operation steps is simplified greatly, both be suitable for going out health check-up, generaI investigation or patient bedside rig-site utilization, and be specially adapted to family and the individual uses or check in emergency circumstances or auxiliary diagnosis are used.

6. safety: the present invention all adopts is nontoxic, radiationless, no inflammable, explosive, perishable, easy stimulation, no carcinogenic, teratogenesis, cause the starting material of gene mutation, entire reaction is not added objectionable impuritiess such as any radioactive isotope, pungent and o-phenylenediamine yet, so when practical application, neither can damage tester or testee's health, also not pollute the environment.Therefore, the present invention have such as detection methods such as radioactive isotope or enzyme mark and other harmful chemical method characteristics such as incomparable security and clean environment firendly.

7. stable: the present invention adopts the design of antigen and antibody specific combination principle, reaction is disturbed by other influence factor seldom, influenced not quite by extraneous factors such as environment temperature, humidity, illumination, oxygen content, add that colloid gold label hTK1 monoclonal antibody is a simple physics course of reaction, and antibodies is firm relatively, substantially can not cause the bioactive change of hTK1 monoclonal antibody, so detection method is very stable.

8. good reproducibility: measure same standard control sample with 12 breast cancer gene albumen early warning chips of a collection of preparation with the inventive method, perhaps measure same standard control sample each 12 times with the breast cancer gene albumen early warning chip of 3 different lot numbers of the technology of the present invention preparation, the developing time of its nature controlling line 1 and detection line 2, shade and net result are judged basic identical, illustrate in its batch and batch between repeatability all better.Simultaneously, adopt the inventive method to measure simultaneously with a typical patients with mastocarcinoma serum specimen, all occur the positive early warning result of testing result at every turn with 12 breast cancer gene albumen early warning chips of a collection of preparation; And, adopt the breast cancer gene albumen early warning chip of the inventive method with a collection of preparation, every day 1 time, continuous 12 days are to measuring with a typical patients with mastocarcinoma serum specimen, the result also shows each all positive early warning result that all occurs detecting, this shows that the repeatability of the breast cancer gene albumen early warning chip of employing the inventive method preparation better.

9. high specificity: the present invention adopts the TK1 antigen in TK1 monoclonal and polyclonal antibody specific detection patients with mastocarcinoma serum or the blood plasma, because the high degree of specificity and the compatibility of this antigen and antibodies make the technology of the present invention have the characteristics of high specificity.Association reaction all appears in the standard TK1 antigen that adopts the present invention to detect the variable concentrations dilution; Do not occur and all have the specificity association reaction with the cancer antigen of other type.

10. it is low to detect cost: TK1 monoclonal antibody that the present invention uses and polyclonal antibody all adopt the preparation of Protocols in Molecular Biology innate immunity animal and get, and the real cost of batch process is very low; And solid phase carrier of the present invention is domestic and international general cellulose nitrate miillpore filter, and collaurum also is that gold chloride makes by the sodium citrate reduction, and not only steady quality, and price is also all very cheap; Add required reagent of actual detected and sample consumption all seldom, and can carry out single part of sample at any time and detect that this all makes detection cost and applied cost decline to a great extent.

In addition, quality with the breast cancer gene albumen early warning chip of the inventive method preparation is also comparatively stable, as randomly draw 12 breast cancer gene albumen early warning chips, add respectively with a standard control sample, pick up counting from the beginning application of sample, obvious color writing time occurs to nature controlling line 1, its sample translational speed and chromogenic reaction time etc. are basically identical all, illustrate that the seepage velocity and the homogeneity of the breast cancer gene albumen early warning chip for preparing with the inventive method is better.

Description of drawings

Fig. 1 is an appearance assumption diagram of the present invention

Embodiment

(1) main raw material(s): 1. anti-hTK1 monoclonal antibody 3E ₈: its purity is 96.7%, and the affinity coefficient is 1.8X10 ⁹, this raw material is inventor's self-control; 2. anti-hTK1 monoclonal antibody 3E ₈: its purity is 96.1%, and the affinity coefficient is 1.2X10 ⁹, this raw material is inventor's self-control; 3. hTK1 standard items: this raw material during use, is diluted to 10 μ g/L, 5 μ g/L, 6 μ g/L, 3 μ g/L, 8 μ g/L, 4 μ g/L, 2 μ g/L, 1 μ g/L, 0.5 μ g/L for inventor's self-control respectively, and is after the rower of going forward side by side is fixed, standby after the freeze-drying; 4. gold chloride (AuCl ₃.HCl.4H ₂O): U.S. Sigma company product; 5. sheep anti-mouse igg polyclonal antibody: ancient cooking vessel state biological products company; 6. calf serum: Pierce company product; 7. glass fibre: U.S. Sigma company product; 8. cellulose nitrate miillpore filter (NC film): U.S. genome company product, aperture are 5 μ m, and the about 50s of liquid translational speed moves 1cm; 9. chemical reagent commonly used: sal tartari (K ₂CO ₃) solution, dipotassium hydrogen phosphate (K ₂HPO ₄) and potassium dihydrogen phosphate (KH ₂PO ₄) damping fluid, tris-HCl damping fluid, homemade analysis pure (AR), chemical reagents corporation buys the back and prepares according to conventional method; Distilled water: laboratory self-control.10. Polyvinylchloride (PVC) material: Shanghai Sheng Hui Plastic Products Co., Ltd product.And polyester film, filter paper, plastic plate, colour atla etc. are Hangzhou Ai Kang company product.

(2) key instrument and equipment: 1. high speed freezing centrifuge; 2. vacuum drier; 3. automatic spectrophotometer; 4. liquid flash mixer; 5. adjustable electric furnace; 6. constant temperature oven; 7. dedicated scan pen; 8. golden mark machine; 9. put film machine and lath cutter automatically; 10. freeze drier.

(3) specific implementation method:

1. the hTK1 gene is cloned and sequential analysis:

1) reverse transcription PCR amplification TK1 cDNA: with can from 5,000,000 Hela cells, extracting RNA from the Trizol reagent kit of the total total RNA (ribonucleic acid) of extraction of extractions (RNA) such as zooblast and tissue, bacterium, fungi, handled 48 hours through methotrexate, and use again after purified a kind of efficient alkylating agent-pyrocarbonic acid diethyl ester (DEPC) handle water-soluble separate precipitate 30 μ l.Because DEPC can destroy ribonuclease (RNase) activity, the therefore pollution that can remove RNase.

When carrying out reverse transcription, add 5 times of damping fluids: the deoxynucleoside triphosphate (dNTP) of 4 μ l, primer Oligo-dT (0.5 μ g/1 μ l) 1 μ l, 10mMol/L concentration: 2 μ l, RNA:10 μ l, deionized water water: 2 μ l, abundant mixing, and place 70 ℃ of waters bath with thermostatic control 10 minutes; Then, place 2 minutes more on ice; Add 1 μ l reverse transcriptase (50U, Roche), mixing places 42 ℃ of water-baths 90 minutes again, its reverse transcription volume totally 20 μ l.

Design a pair of primer according to people TK1 gene order:

Primer 1. 5-AGCTAAGCTTAGCTGCATTAACCTGCCCAC-3

Primer 2 5-TACTCTCGAGGTTGGCAGGGCTGCATTGCAGAATC-3

Primer 1 is introduced the HindIII enzyme and is cut sequence; Primer 2 is introduced the XhoI enzyme and is cut sequence

Carry out pcr amplification again, promptly add 10 times of damping fluids: 5 μ l, dNTP (2.5mMol/L): 4 μ l, primer 1:1 μ l, primer 2: 1 μ l, cDNA:5 μ l, Taq enzyme: 1 μ l, deionized water: 33 μ l; The long-pending totally 50 μ l of pcr amplification liquid.Mixing, put on the PCR instrument and increase: 94 ℃ 60 seconds, 55 ℃ 60 seconds, 72 ℃ 120 seconds, carry out 30 circulations altogether, add at last 72 ℃ 5 minutes.Then, get 10 μ l PCR products electrophoresis on 2% Ago-Gel, downcut the DNA band of 700bp position, reclaim kit recovery dna fragmentation with gel and be used for the TA clone.

2) TA clone and dna sequence analysis: the PCR product of purifying and pMD-18T carrier are heavily changed the Escherichia coli group, change JM109, coat on the LB agar medium that contains ampicillin, 37 ℃ of overnight incubation, single bacterium colony enlarged culture of 10 whites of picking next day is used the plasmid extraction kit plasmid purification, and double digestion (HindIII and XhoI) is identified and inserted fragment, in selected 10 clones the insertion fragment is arranged all, clip size is about 700bp.TK1 reorganization TA clone (TA9) uses T7 and the two-way order-checking of SP6 primer, and the TK1 sequence of result and GenBank proves that relatively the cDNA of this time cloning is a people TK1 gene, 702 bases of total length.

2. TK gene subclone is gone into the eucaryon secreted expression carrier

Adopt eucaryon secreted expression carrier pSecTag2/Hygro B (being called for short pSecTag2), it can arrive the extracellular with the protein excretion of recombinant gene expression, because TK1 does not have signal peptide, is the method an of the best so select excretion vector.

With HindIII and XhoI respectively enzyme cut TK1/TA cloned plasmids and pSecTag2 plasmid, reclaim the reagent dna fragmentation of purifying TK1 and pSecTag2 respectively with gel electrophoresis and glue, carry out coupled reaction then.The coupled reaction volume is totally 15 μ l, i.e. 10 times connection damping fluid: 1.5 μ l, TK1 dna fragmentation: 8.0 μ l, PSecTag2 DNA:4.5 μ l, T4 dna ligase 1.0 μ l.Mixing is put 4 ℃ of refrigerator overnight; Add the competent e. coli jm109 of 300 μ l, put in the ice bath 1 hour, 42 ℃ of water-baths 90 seconds, in ice-water bath 5 minutes again; Add LB nutrient culture media 1ml, mixing is put 37 ℃ and was cultivated 1 hour, and centrifugal collecting precipitation is applied to the thalline that precipitates on the LB agar medium that contains ampicillin (50 μ g/ml), in 37 ℃ of incubated overnight.10 bacterium colony enlarged culture of picking, for a short time put forward plasmid DNA with kit, identify recombinant plasmid with restriction endonuclease and electrophoresis again, 10 plasmids being selected all contain the TK1 gene, electrophoresis showed can be downcut the dna fragmentation of about 700bp, and a SfI restriction enzyme site is arranged in the TK1 fragment simultaneously.TK1 recombinant plasmid called after pSecTag/TK1.

3. a large amount of preparations of PSecTag/TK1 DNA

PSecTag/TK1 reorganization bacterium spends the night in 37 ℃ of activation culture, is inoculated in then in the LB nutrient culture media of 50ml (containing ampicillin 50 μ g/ml), and 37 ℃ of shaken cultivation 5 hours change over to and continue in the 500mL nutrient culture media to cultivate centrifugal recovery thalline 5 hours; Press the kit program and extract plasmid DNA; In the end select sterile distilled water during wash-out for use.The plasmid DNA of purifying can be extracted 400 μ g plasmid DNA with the thalline of UV spectrophotometer measuring purity and quantitatively .500ml cultivation, and is frozen standby in the refrigerator.

4. genetic immunization prepares the TK1 monoclonal antibody

1) TK1 genetic immunization Balb/c mouse: immunologic process: get 43 of Balb/c pure lines female mices in age in week (wherein in contrast not immune), every mouse both sides quadriceps muscle of thigh injects 50 μ g pSecTag/TK1DNA (every side 50 μ l) respectively; The contrast mouse is injected the physiological saline of equivalent; Immunity in per 15 days once immune 5 times altogether, immune back 10 days of the second time began to detect antibody in the serum; A week is carried out Fusion of Cells after the last immunity.

2) TK1 genetic immunization mouse boosting cell and murine myeloma cell merge: A: the feeder cells preparation: the Balb/C mouse draws neck to put to death, and gets its thymus gland, places the 10ml nutrient solution, grinds cell dispersion on steel mesh, abandons bulk tissue, with cell furnishing 5.0X10 ⁶/ ml, the every hole 100ul of 96 well culture plates.B: the myeloma cell prepares: murine myeloma cell is the NS1 cell line, cell well-grown before merging, and cell concentration is 5X10 ⁶/ ml.C Fusion of Cells: 2X10 ⁷Individual myeloma cell and 2X10 ⁸Individual cell mixing, centrifugal, abandon supernatant, slight vibration mixing in 37 ℃ of water-baths, dripped the PEG of 1ml 50% in 90 seconds, drip the 20ml serum free medium then, centrifugal, abandon supernatant, wash again once, the nutrient culture media 20ml (containing HAT) that adds 15% serum, mixing, every hole add 100 μ l (feeder cells of original 100 μ l), are provided with independent splenocyte and independent myeloma control wells (12 holes/each) simultaneously.Change liquid after 4 days; Sucking-off 100 μ l add 100 μ l and contain 15% serum HAT nutrient culture media; It when changing liquid for the third time the HT nutrient culture media; Every day, the clone appearred in the observation of cell growth conditions about 10 days.

3) hybridoma culture supernatant TK1 detection of antibodies: with the antibody in the direct ELISA detection hybridoma culture supernatant, with the antigen coated elisa plate of TK1, add culture supernatant, the sheep anti-mouse igg that adds horseradish peroxidase-labeled after the flushing, after incubating bath and flushing, add chromogenic substrate OPD, survey the optical density of 492nm then, to be higher than negative control optical density positive clone more than 2 times.There are 28 holes to show the TK1 antibody positive in the culture supernatant in result's two weeks after fusion.Cloning is carried out in selection three the high holes of tiring.

4) cloning and enlarged culture: the cell in the polyclone culture hole adopts limiting dilution assay to carry out cloning, with the medium preparation mouse chest cell that contains HAT as feeder cells, cell in the polyclone hole is diluted to every hole and contains 100,10 and 0 cells (100 μ l), cultivate and observe, the culture supernatant of having only a clone hybridization knurl is detected.Select positive colony to change over to and carry out enlarged culture in 24 well culture plates, every hole 1ml, cultivate after 5 days, add the 4ml nutrient solution, mixing divides to go in 4 culture hole, continue to cultivate 5 days, change over to then and carry out enlarged culture in the culture flask, select antibody positive, and the stable clone that tires enlarges further with frozen.The result obtains the hybridoma of two strain binding site differences and stably excreting TK1 antibody, called after: DZ1 and DZ2.

5) a large amount of preparations of TK1 monoclonal antibody and purifying: the Balb/C mouse peritoneal injects the 0.5ml whiteruss, and every the mouse peritoneal in a week back injects 5X10 ⁵Individual hybridoma, when obvious ascites formed, ascites was put in the syringe needle puncture then.Centrifugal 5 minutes of the ascites 12000rpm that collects abandons precipitation, supernatant Protein G post affinitive layer purification.With the quantitative antibody of the TK1 monoclonal anti body and function protein quantification reagent (BCA) behind the purifying, packing is preserved.

6) evaluation of TK1 monoclonal antibody: the specific evaluation of monoclonal antibody: A: directly the ELISA:TK1 bag is added TK1 ascites, again antibody and enzyme labelled antibody by solid phase.B: double-antibody sandwich elisa: with the ascites bag by solid phase (1: 200); The TK1 that adds variable concentrations adds the IgY of the anti-TK1 of alkali phosphatase enzyme mark then, with the alkaline phosphatase substrate colour developing, surveys the optical density of 570nm.C: SABC: preparation HeLa cell smear, be divided into two groups: one group is the HeLa cell; Another group is induced 48 hours HeLa cell for aminopterin-induced syndrome, and EDTA digests cultured cells, makes its dispersion, washes once with PBS, is diluted to 5X10 ⁵It a little is 30 μ l that/ml, each cell are coated with; Drying at room temperature, acetone fixed 5 minutes was handled 30 minutes with 1% hydrogen peroxide then; Again with 3% BSA sealing 30 minutes.Add antibody (dilution in 1: 400, culture supernatant stoste) then, incubate and bathe and flushing, add enzyme labelled antibody, add the colour developing of DAB substrate after the flushing, observations under the light microscopic is carried out with the commercially available reagent box contrast simultaneously.The result shows that the antibody of DZ1, the secretion of DZ2 hybridoma is the special monoclonal antibody 2A of TK1 ₆The monoclonal antibody 3E special with TK1 ₈At last, detect its antibody purity through sodium dodecylsulphonate (SDS)-PAGE (polyacrylamide) gel electrophoresis; Simultaneously, carry out the binding site of IgG type, subgroup identification and antibody of antibody and affinity mensuration etc.Afterwards, placing low temperature refrigerator to preserve, can be subsequent applications fully.

5. the preparation of label-collaurum develops the color:

1) gold chloride is used the preparation of liquid and trisodium citrate application liquid: accurately take by weighing high-quality gold chloride 10mg, place the clean beaker of 100ml immediately; Adding the dual distilled water of 100ml again, then, open the liquid flash mixer again, constantly stir, make it to dissolve fully, is 0.25X10 and obtain concentration ^-3The chlorauric acid solution 100ml of mol/L (0.01%); Simultaneously, accurately take by weighing trisodium citrate 1.0g again, place another clean 100ml beaker, add the dual distilled water of 100ml again; Equally, with the continuous stirring and dissolving of liquid flash mixer, make it to dissolve fully, obtaining concentration is 34X10 ^-3The citric acid three sodium solution 100ml of mol/L (1%).

2) collaurum is used the preparation of liquid: with the above-mentioned concentration that has prepared is 0.25X10 ^-3The chlorauric acid solution 100ml of mol/L (0.01%) place be lined with progressively be heated to boiling on the adjustable electric furnace of heat insulation asbestos gauge after, rapidly the concentration that prepared of adding is 34X10 ^-3The trisodium citrate of mol/L (1%) (citric acid trisodium) solution 2ml, after continuing to boil heating 10-15min, examine, after the color of collaurum application liquid becomes claret or aubergine, close adjustable electric furnace, it is progressively cooled off after, can form needed colloidal gold solution, after the cooling, it is standby to place 4 ℃ of refrigerators to preserve.

3) colloid gold particle size and uniformity coefficient are identified: above-mentioned colloidal gold solution is scanned in visible-range (400-600nm) with automatic spectrophotometer, find that it is when wavelength is the 520-521nm place, this colloidal gold solution has maximum absorption band, and the main peak width is narrower, confirm that prepared collaurum granularity at 20-25nm, meets the requirement of follow-up use.

6. collaurum-hTK1 monoclonal antibody 3E ₈The dry polyester film sheet preparation of bond: after determining that finishing gold chloride is reduced into the uniform collaurum of particle size distribution, can carry out colloid gold label hTK1 monoclonal antibody 3E ₈Preparation.

1) alkalescent collaurum filtrate and hTK1 monoclonal antibody 3E ₈Use the preparation of liquid: get the colloidal gold solution that dosage is 100mL, place among the clean beaker of 250mL; Then, be the sal tartari (K of 0.1mol/mL with the concentration that has prepared in advance ₂CO ₃) solution progressively transfers to PH8.8 with the potential of hydrogen of this colloidal gold solution, then, is the filter membrane negative pressure filtration of 0.45 μ m with the aperture again, can obtain alkalescent collaurum filtrate filtered fluid.And then, get the good hTK1 monoclonal antibody 3E of aforementioned purifying ₈And contain hTK1 monoclonal antibody 3E in being mixed with every milliliter with dual distilled water ₈The solution of 1500 μ g.

2) collaurum-hTK1 monoclonal antibody 3E ₈The preparation of bond: get above-mentioned alkalescent collaurum filtrate filtered fluid 100mL, in constantly stirring fast, slowly adding 1mL adjusted is the hTK1 monoclonal antibody 3E of 1500 μ g/mL to concentration ₈, then its labelled protein amount is to add 15 μ g hTK1 monoclonal antibody 3E in every milliliter of collaurum ₈After continuing to stir 15-20min, it is 20% bSA (BSA) 1mL (200mg) of 20mmol/L tris-HCl damping fluid preparation that pH8.2, concentration are used in adding; After stirring through 10-15min again, place refrigerated centrifuge to carry out low temperature (0-4 ℃) low-speed centrifugal (1500rpm/min) 10min.Careful reject precipitation; After getting supernatant and carrying out low temperature (0-4 ℃) high speed centrifugation (12000rpm/min) 20-30min again, careful again the suction removed supernatant, keeps precipitation, and this is collaurum one anti-hTK1 monoclonal antibody 3E ₈Bond.

3) collaurum-anti-hTK1 monoclonal antibody 3E ₈The preparation of the polyester film sheet of bond: at first, compound concentration be the 0.01mol/LTris-HCl damping fluid (1%BSA, pH8.2); Then, precipitated good collaurum-hTK1 monoclonal antibody 3E with this damping fluid with above-mentioned ₈Bond is diluted to 10mL; At last, this is diluted good collaurum-hTK1 monoclonal antibody 3E ₈Bond is evenly distributed on polyester film with golden mark machine with it according to the dosage of 0.8ml/ bar (0.4cmX20cm/ bar) and inhales on the diaphragm and carry out mark, carry out vacuum freeze drying then, perhaps place 37 ℃ of thermostatic drying chamber dried overnight of having sterilized, can obtain containing collaurum-anti-hTK1 monoclonal antibody 3E ₈The dry polyester film diaphragm of bond, the sealing back is standby.

7. hTK1 gene protein breast cancer early warning chip preparation

1) preparation contains hTK1 monoclonal antibody 2A ₆Cellulose nitrate miillpore filter sheet with the sheep anti-mouse igg polyclonal antibody: respectively with hTK1 monoclonal antibody 2A ₆Use 10mmol/L respectively with the sheep anti-mouse igg polyclonal antibody, the phosphate of pH 7.4 (PBS) damping fluid is diluted to 1500mg/L (1.5mg/ml).Wherein, the former is a detection line 2, and the latter is quality control line (nature controlling line 1).These two kinds of antibody are respectively charged in the dedicated scan pen of two cleanings, and detection line 2 (contains hTK1 monoclonal antibody 2A ₆) and all wide 2mm of band line of nature controlling line 1 (containing the sheep anti-mouse igg polyclonal antibody), but between the two at a distance of 8mm.Program design is imported some film machine automatically in the back well, and quantity for spray is 1.0 μ L.Then, open some film machine switch automatically, promptly enter duty, at this moment, automatically some film machine wraps these two kinds of antibody respectively respectively by on cellulose nitrate miillpore filter (NC film) on the position that is positioned at a left side, cellulose nitrate miillpore filter (NC film) middle part 1/2, carry out mark respectively, promptly detection line 2 (contains hTK1 monoclonal antibody 2A ₆) and nature controlling line 1 (containing the sheep anti-mouse igg polyclonal antibody); Equally, some film machine wraps these two kinds of antibody respectively respectively by on cellulose nitrate miillpore filter (NC film) automatically on the position that is positioned at cellulose nitrate miillpore filter (NC film) middle part right 1/2, carries out mark respectively, and promptly detection line 2 (contains hTK1 monoclonal antibody 2A ₆) and nature controlling line 1 (containing the sheep anti-mouse igg polyclonal antibody).Simultaneously, at mid point (50mm and 2mm) the design one circular well 3 of the middle of this NC film (long 100mm, wide 4mm) and carry out mark.Wherein, make this two hTK1 monoclonal antibody 2A ₆Lay respectively at the position of this well 3 each 25mm of the right and left; The sheep anti-mouse igg polyclonal antibody then lays respectively at the position of this well 3 each 33mm of the right and left.And then, will be coated with hTK1 monoclonal antibody 2A ₆With place 10mmol/L on the cellulose nitrate miillpore filter of sheep anti-mouse igg polyclonal antibody, in the phosphate of pH7.4 (PBS) damping fluid (containing 1%BSA), 37 ℃ of constant temperature soak 60min; At last, use 10mmol/L again, the phosphate of pH7.4 (PBS) damping fluid washing 1-2 time; Take out subsequently, suck dry moisture as far as possible places room temperature or 37 ℃ of constant temperature oven dryings or vacuum drying, promptly gets and contains hTK1 monoclonal antibody 2A ₆With the cellulose nitrate miillpore filter sheet of sheep anti-mouse igg polyclonal antibody, sealing, standby.

2) hTK1 gene protein breast cancer early warning chip assembling: contained hTK1 monoclonal antibody 2A finishing bag ₆Behind the cellulose nitrate miillpore filter of rabbit anti-mouse igg polyclonal antibody, can carry out the assembling of breast cancer early warning chip.At first, carry out the preceding pre-service of diaphragm assembling: getting width is white Polyvinylchloride (PVC) plate of 80mm, before the use, after the clean repeatedly processing of pure water, soaks or flushing with distilled water, and places natural drying at room temperature.And then, paste multiple film: the well 3 with positive bar mid point is a starting mark, and sample paper, water adsorption glass fibrous paper, polyester film sheet (collaurum-hTK1 monoclonal antibody 3E will be filtered successively with hot imperial glue respectively in the left side and the right ₈Bond), be coated with anti-hTK1 monoclonal antibody 2A ₆Be pasted on the PVC plastic sheet of white with the cellulose nitrate miillpore filter and the absorbent filter of rabbit anti-mouse igg polyclonal antibody, and on this diaphragm, cover the effective diaphragm of one deck.Wherein, filter sample paper (separation of whole blood film) and water adsorption glass fibrous paper be all through the damping fluid processing and be attached to below the well 3, and successively with polyester film sheet (collaurum-hTK1 monoclonal antibody 3E ₈Bond) connect, and polyester film sheet (collaurum-hTK1 monoclonal antibody 3E ₈Bond) then be coated with anti-hTK1 monoclonal antibody 2A ₆Be connected with the cellulose nitrate miillpore filter of rabbit anti-mouse igg polyclonal antibody; Cellulose nitrate miillpore filter afterbody links to each other with absorbent filter.After finishing pad pasting, can carry out vacuum drying; At last, the batten that all posts is cut into the strip diaphragm that a specification is wide 4mm, long 100mm on cutter special, in the plastic casing of packing into, adds the drying agent sealing and preserve, breast cancer gene albumen early warning chip.

8. the method for testing of hTK1 gene protein breast cancer early warning chip

1) unpacks, get one of this breast cancer early warning chip rapidly, and, cover the clamp lid immediately with pack into relevant position in the special supporting clamp of its smooth and standard ground, directly be placed on the table top (* annotates: the special supporting clamp that test is used is declared utility model patent with other) of level.

2) put finger or the ear-lobe of wiping away the person under inspection on the skin with preprepared ethanol for disinfection cotton balls, treat that alcohol is done after, puncture with the blood sampling three-edged needle of having sterilized immediately, extruding goes out blood flow gently; Then, suck 50 μ L whole bloods with the micropipet that contains the anticoagulant heparin agent and directly be added to the circular port place that the clamp mid point indicates well 3.As find end blood thickness comparatively slightly, and the available micropipet that contains the anticoagulant heparin agent sucks 25 μ L whole bloods and 25 μ L 10mmol/L, and the phosphate of pH7.4 (PBS) damping fluid after the mixing, directly is added to the circular port place that the clamp mid point indicates well 3.

3) at this moment, blood plasma in this anticoagulation or the blood plasma in the blood dilution liquid are owing to capillary action, and move naturally both sides to the left and right respectively.As containing hTK1 gene protein (antigen) in the blood plasma, then this antigen earlier the glass fibre by the left and right sides chromatography effect nature to direction separately move ahead and with left and right sides polyester film sheet on the hTK1 monoclonal antibody 3E of colloid gold label ₈Specificity is in conjunction with forming antigen antibody complex.

4) simultaneously, because of capillary continuation, the antigen antibody complex that the left and right sides has a colloid gold label continues to travel to both sides NC film to the place ahead separately, and immediately with the hTK1 monoclonal antibody 2A that is coated on the NC film of both sides ₆The specificity association reaction takes place, and forms the antigen antibody complex of double-antibody sandwich.At this moment, do not continued to pass through the hTK1 monoclonal antibody 3E of left and right sides polyester film sheet and colloid gold label in the blood plasma by the hTK1 antigen of combination fully ₈Specificity continues to move ahead to both sides after forming antigen antibody complex, and combines with sheep anti-mouse igg polyclonal antibody on being coated on both sides NC film.The about 3-20min of Total Test process.

5) since collaurum constantly by enrichment, should respectively present two range estimation visible redness or aubergine bands (whole clamp on should be four) at the left and right sides NC film this moment.

6) result judges: feminine gender: all do not develop the color near the detection band that adds sampling point on the NC diaphragm of the left and right sides, quality control band on the NC diaphragm of the left and right sides then develops the color, be that the left and right sides 1 red line (should be two on the whole clamp) respectively occurs away from the position that adds sampling point, expression detects in the sample and does not contain the hTK1 gene protein; Positive: detection band and quality control band on the NC diaphragm of the left and right sides all develop the color, and promptly left and right sides appearance 2 red lines (should be four on the whole clamp) then contain the hTK1 gene protein in the expression detection sample; Invalid: as if calibration tape and quality control band on the NC diaphragm of the left and right sides all do not develop the color, all not have red line on the promptly whole clamp and occur, then show test invalidation, represent that this breast cancer early warning chip lost efficacy.

Points for attention of the invention process:

First, nucleus-hTK1 the monoclonal antibody that is used to prepare this breast cancer gene albumen early warning chip must be carried out specific evaluation earlier, comprising and adopt direct ELISA method, double-antibody sandwich elisa method, SABC method etc., is the specific monoclonal antibody of TK1 with the secreted antibody of the selected hybridoma of definite confirmation.

Second, after the selected secreted antibody of hybridoma of definite confirmation is the hTK1 monoclonal antibody, also need simultaneously this hTK1 monoclonal antibody to be carried out affinity and measure, all must be with the affinity coefficient of two selected specificity hTK1 monoclonal antibodies of definite confirmation greater than 1X10 ⁶More than, and preferably at 1X10 ⁹More than.

The 3rd, (code name is 2A to two specificity hTK1 of selected this monoclonal antibody ₆And 3E ₈) at process affinity coefficient determination, and after determining its affinity with height, the mouse ascites process antibody purification process that also must will contain the hTK1 monoclonal antibody earlier is in order to avoid because this antibody purity influences inadequately or the sensitivity and the accuracy of Interference Detection.

The 4th, collaurum also is the key factor that the present invention successfully prepares breast cancer gene albumen early warning chip as the important marker of hTK1 monoclonal antibody.Therefore, successfully preparing the high-quality collaurum, is the important step of finishing qualified collaurum-hTK1 monoclonal antibody bond preparation process, must pay much attention to, conscientiously operation.When carrying out Preparation of Colloidal Gold, need add when reducing trisodium citrate (citric acid trisodium) solution of suitable concn except gold chloride, also need to control well pH value, time and the temperature of entire reaction.In order to ensure the quality of collaurum, in case of necessity, must scan in visible-range (400-600nm) this collaurum with automatic spectrophotometer, whether analyze prepared colloid gold particle even.It is emphasized that the particle of collaurum is advisable with 20-25nm if use competition law; And adopt the particle of the collaurum of double-antibody sandwich rule employing to be advisable with 25-30nm.

The 5th, when preparation high-quality collaurum, used glassware must thoroughly clean according to normalized operation; Preferably adopt glassware when having ready conditions through silicidation; Perhaps adopt and last time prepared the glassware of qualified collaurum, but before reusing, just can re-use after must washing repeatedly with distilled water, in order to avoid influence the hTK1 monoclonal antibody combined and activated the back colloid gold particle with colloid gold particle stability.

The 6th, the size of colloid gold particle and homogeneity thereof are very important, and the colloid gold particle that can not obtain 20-40nm will influence itself and hTK1 monoclonal antibody adhesion and stability.Therefore, all reagent all must keep its purity during requirement preparation collaurum, i.e. all necessary operational analysis of all reagent is pure or top grade is pure, comprises the trisodium citrate (citric acid trisodium), gold chloride, the sal tartari (K that use in the preparation ₂CO ₃) and the reagent etc. of preparation damping fluid.Particularly used gold chloride quality must meet the requirements, i.e. purity height, and impurity is few, and preferably the top grade of import is pure.Simultaneously, employed bSA (BSA) must fresh, totally not have any rotten and pollution.The used pure water of preparation mentioned reagent must just can be used for preparation behind the deionization, and is facing with preceding with the reagent process ultrafiltration or miillpore filter (the 0.22 μ m) filtration that prepare, to remove polymkeric substance and other impurity that may sneak into wherein fully.Words with good conditionsi, pure water are preferably used dual distilled water or triple distillation water.And, also must be when reality is prepared in strict accordance with the dependency rule of laboratory preparation preparation.As the pH for preparing colloidal gold solution is better with neutral (pH7.2), adopts the preparation of dual distilled water or triple distillation water usually.

The 7th, during actual the use, the application concentration of gold chloride is 0.01% aqueous solution usually, but aqueous solution of chloraurate concentration can keep the several months to stablize constant under 4 ℃ of conditions at 1.0% o'clock.Because gold chloride is easy to the loss that deliquescence causes, when reality is prepared, can place 4 ℃ of refrigerators to preserve the aqueous solution that is mixed with 1.0% after the disposable dissolving of gold chloride behind the Kaifeng for avoiding.

The 8th, after determining that finishing gold chloride is reduced into the uniform collaurum of particle size distribution, can carry out colloid gold label people thymidine kinases (hTK1) monoclonal antibody (McAb), i.e. the preparation of collaurum-hTK1 McAb bond.The label collaurum depends primarily on the pH value for the adsorption effect of the hTK1 monoclonal antibody that is labeled, and under the condition near the isoelectric point of hTK1 monoclonal antibody or inclined to one side slightly alkali, collaurum and hTK1 monoclonal antibody more easily form firm bond.When if the pH value of collaurum is lower than the isoelectric point of hTK1 monoclonal antibody during actual fabrication, then can be very fast generation assemble and lose binding ability.Detect through the isoelectric focusing immunoelectrophoresis, the hTK1 isoelectric point is 8.3-8.5, and therefore, we determine that adjusting the pH value with solution of potassium carbonate is 8.6-9.0 (the actual pH value that adopts is 8.8).In addition, the size of colloid gold particle, ionic strength, degree of uniformity and hTK1 monoclonal antibody working concentration and molecular weight etc. also all may influence the ability that collaurum combines with it.In this PH scope, PH is higher, the electronegative increase of collaurum, the positively charged increase of hTK1 monoclonal antibody, helping collaurum and hTK1 monoclonal antibody bond forms to greatest extent and combines firmly and stablize, but PH is higher, may cause that then the yield of collaurum and hTK1 monoclonal antibody bond reduces; And PH is on the low side, improves though help the yield of collaurum and hTK1 monoclonal antibody bond, and PH is low excessively, and then the possibility of colloidal gold aggregation increases, and instability strengthens.Therefore, the pH value of determining at last also answer strictness by experiment the result determine.

The 9th, carrying out collaurum-hTK1 monoclonal antibody 3E ₈When bond prepares, must earlier hTK1 monoclonal antibody concentration adjustment be arrived suitable level.In colloidal gold solution, adding hTK1 monoclonal antibody 3E ₈Before, must with solution of potassium carbonate the pH value of colloidal gold solution be adjusted to 8.6-9.0 earlier, and add bSA, the effect of mark can be better like this.Because calf serum can be filled up the defective of collaurum and hTK1 monoclonal antibody, play good sealing effect and fixation; And contain anti-hTK1 monoclonal antibody 2A in preparation ₆During with the cellulose nitrate miillpore filter sheet (NC) of rabbit anti-mouse igg polyclonal antibody, then must dilute with phosphate (PBS) damping fluid of pH7.2-7.4, contain bSA equally, they are wrapped respectively by on cellulose nitrate miillpore filter sheet by machine with bag then, and at 37 ℃ of constant temperature immersion 60min, like this, they mutually combine just more firm.

The tenth, anti-hTK1 monoclonal antibody 2A ₆And the concentration of rabbit anti-mouse igg polyclonal antibody is wherein more commonly used with 1.0g/L usually between 0.8-1.6g/L.Be lower than 1.0g/L, may influence the antigen-antibody combination, cause the antigen-antibody combination rate to reduce; May cause unnecessary waste and be higher than 1.0g/L, perhaps because be not to be the optium concentration of antigen-antibody combination, the combination of influence or interference antigen-antibody on the contrary.Particularly different antigen and antibody, its concentration do not have consistent standard, some in addition can be in a word with 0.5g/L., determining of optium concentration still must be according to experiment situation final decision.

The 11, be starting point with well 3 during assembling chip, it is followed successively by in proper order: filter sample paper (separation of whole blood film), water adsorption glass fibrous paper, polyester film (collaurum-anti-hTK1 monoclonal antibody 3E ₈Bond), the cellulose nitrate miillpore filter (is coated with anti-hTK1 monoclonal antibody 2A ₆With the rabbit anti-mouse igg polyclonal antibody) and absorbent filter, and directly be pasted on the PVC plastic sheet of white, and on this diaphragm, cover layer protecting film, and after the vacuum drying, shear, add the drying agent sealing, place cool place, black out, drying, ventilation to preserve.It should be noted especially, be layered on need through damping fluids processing the same with the NC film of filter sample paper (separation of whole blood film) below the well 3 with the water adsorption glass fibrous paper, excessive and influence result's repetition and accuracy with the PH variation that prevents test sample.

The 12, as it is too fast at this breast cancer gene albumen early warning chip chromatography to detect sample (whole blood or serum or blood plasma), hTK1 be not able to do in time as yet with the NC film on hTK1 antibody specificity association reaction completely takes place, just crossed detection line 2, cause chromogenic reaction not obvious, and false negative result occurs.And it is too fast at this breast cancer gene albumen early warning chip chromatography to detect sample, also can cause the very fast colour developing of nature controlling line 1 that is used to contrast, and the also not colour developing of weak positive sample, and the tester thinks that judgement time has arrived as a result by mistake, and and false negative result appears.Therefore, detecting the sample translational speed too fast is not good thing, should attract great attention, and be controlled.

The 13, detect very few, the detection sample deficiency by breast cancer gene albumen early warning chip in other words of sample (whole blood or serum or blood plasma), cause detecting the nature controlling line 1 that sample can't reach detection line 2 and be used to contrast, promptly can't make the composition to be measured (hTK1) that detects in the sample can't with hTK1 monoclonal antibody on the NC film or IgG polyclonal antibody generation association reaction, can't sentence read result or take for early warning chip and lost efficacy to such an extent as to cause.Therefore, when actual detected, should guarantee has enough detection samples.

The 14, it is too much to detect sample (whole blood or serum or blood plasma), perhaps detecting sample (whole blood or serum or blood plasma) causes excessive, make breast cancer gene albumen early warning chip can't stop haemocyte effectively, thereby cause haemocyte and serum or blood plasma to move to detection line 2 together, cause detection line 2 fuzzy, disturb and influenced judgement as a result, make the failure of an experiment.

The 15, prevent that the temperature when use winter from crossing low phenomenon, because temperature is low excessively, detection sample (whole blood or serum or blood plasma) is slowed down at this breast cancer gene albumen early warning chip chromatography, but also the reaction velocity of antigen and antibody specific combination is reduced, sometimes, the color reaction of some weak positive samples even can't show fully at 30min.Therefore, when temperature is low, should prolong reaction time and judgement time as a result as far as possible, in order to avoid false positive results occurs, cause occurring mistaken diagnosis or fail to pinpoint a disease in diagnosis.

The 16, use this breast cancer gene albumen early warning chip, should in the laboratory of cleaning, carry out as far as possible, and prevent that dust from dropping on the early warning chip.So there is dust to fall on the breast cancer gene albumen early warning chip, then can influences and detect sample moving or rate of propagation on this breast cancer gene albumen early warning chip, cause the result can not normally observe or occur error in judgement as a result.

The 17, use this breast cancer gene albumen early warning chip, should as far as possible it be placed on the table top of level.Table top is uneven or crooked, also may influence to detect sample (whole blood or serum or blood plasma) moving or rate of propagation on this breast cancer gene albumen early warning chip, causes the result can not normally observe or occur error in judgement as a result.

The 18, breast cancer gene albumen early warning chip should place cool place, drying, ventilation at ordinary times, and don't fail to place drying agent in the packing, and notes changing at any time, to prevent the early warning chip humidity.Because, the not only contaminated easily and growth bacterium of breast cancer gene albumen early warning chip, but also can cause its complete failure.

The 19, need sometimes in the actual detected sample to be diluted according to the situation that detects sample (whole blood or serum or blood plasma).Therefore, after adding the detection sample, to examine the speed that it moves or spreads.As if finding that detection sample rate of propagation is very slow or stopping, should adding dilution as early as possible, in order to avoid add to such an extent that can't promote too late.In addition, dilution is can not liquid feeding too many, otherwise haemocyte can be moved thereupon or be diffused into detection line 2, causes the judged result difficulty.

The 19, breast cancer gene albumen early warning chip can be used for outdoor detection, fails to pinpoint a disease in diagnosis but the mistake strong sunlight is dazzling easily; Outdoor wind-force influences result's correct judgement than also influencing the sample translational speed greatly.Therefore, the tester should conscientious especially and careful judged result in order to avoid cause erroneous judgement.

Embodiment of the present invention only is the description that preferred implementation of the present invention is carried out; be not that design of the present invention and scope are limited; do not breaking away under the preceding topic of design philosophy of the present invention; various modification and improvement that scholar in this area and engineering technical personnel make technical scheme of the present invention; all should fall into protection scope of the present invention; the technology contents that the present invention asks for protection all is documented in claims.

Claims

1, a kind of breast cancer early warning chip of easy fast measuring human I type thymidine kinase gene protein is characterized in that: described breast cancer early warning chip 4 is that (code name is 2A to an anti-hTK1 monoclonal antibody that is prepared by eukaryotic expression by an end bag ₆-McAb) and the other end then wrap by the fibre strip chromatography solid phase carrier NC film of rabbit anti-mouse igg polyclonal antibody, filter sample paper, water adsorption glass fibrous paper and be marked with collaurum and two anti-hTK1 monoclonal antibodies (code name is 3E ₈-McAb) polyester film sheet etc. of bond is pasted on white PVC plastic sheet according to this and forms.

2, the preparation method of the breast cancer early warning chip of a kind of easy fast measuring human I type thymidine kinase gene protein according to claim 1 is characterized in that may further comprise the steps:

One, preparation contains an anti-hTK1 monoclonal antibody 2A ₆The cellulose nitrate miillpore filter sheet of-McAb and rabbit anti-mouse igg polyclonal antibody:

1) with an anti-hTK1 monoclonal antibody 2A ₆-McAb and rabbit anti-mouse igg polyclonal antibody are used 10-20mmol/L respectively, and the phosphate buffer of pH7.2-7.4 (PBS) is diluted to 0.8-1.6g/L;

2) with the antibody sandwich machine they are wrapped respectively by on the cellulose nitrate miillpore filter;

3) will be coated with an anti-hTK1 monoclonal antibody 2A ₆Place 10-20mmol/L on the cellulose nitrate miillpore filter of-McAb and rabbit anti-mouse igg polyclonal antibody, in the phosphate buffer of pH7.2-7.4 (1-2%BSA), soak 60min in 37 ℃ of constant temperature;

4) use 10-20mmol/L again, the phosphate buffer of pH7.2-7.4 (PBS) washing places after room temperature or the vacuum drying standby at last.

Well 3 with the diaphragm mid-point is a starting mark, and sample paper, water adsorption glass fibrous paper, polyester film sheet (the anti-hTK1 monoclonal antibody of collaurum-two 3E will be filtered successively with hot imperial glue respectively in the left side and the right ₈-McAb bond), be coated with an anti-hTK1 monoclonal antibody 2A ₆The cellulose nitrate miillpore filter of-McAb and rabbit anti-mouse igg polyclonal antibody and absorbent filter are pasted on the PVC plastic sheet of white, and cover the effective diaphragm of one deck on this diaphragm; Can carry out vacuum drying after finishing pad pasting; At last, the batten that all posts is cut into the strip diaphragm of certain specification on cutter special, in the plastic casing of packing into, adds the drying agent sealing and preserve, breast cancer gene albumen early warning chip 4.

3, the breast cancer early warning chip of a kind of easy fast measuring human I type thymidine kinase gene protein according to claim 1 is characterized in that: this nano level collaurum comes from gold chloride (HAuCl ₄) effect of reductive agent trisodium citrate (citric acid trisodium), its electrostatic interaction makes its polymerization become a kind of stable colloidal state and is the nanogold particle of specific size, uniformity; And, because it is electronegative under the weak base environment, can pass through the static strong bonded with protein molecule positive charge group, can't influence the biological nature of gene protein antigen and monoclonal antibody protein matter.

4, the breast cancer early warning chip of a kind of easy fast measuring human I type thymidine kinase gene protein according to claim 1 is characterized in that: the strip fibrage is analysed on the solid phase carrier NC film an anti-hTK1 monoclonal antibody of the quilt that wraps (code name is 2A in this breast cancer early warning chip 4 ₆-McAb) derive from the human I type thymidine kinase (hTK1) of the expressed preparation of eucaryon; And (code name is 3E for the collaurum of mark and two anti-hTK1 monoclonal antibodies on the polyester film sheet ₈-McAb) two anti-hTK1 monoclonal antibodies in the bond (code name is 3E ₈-McAb) derive from the human I type thymidine kinase (hTK1) of the expressed preparation of eucaryon equally, but it derives from and is different from an anti-hTK1 monoclonal antibody (code name is 2A ₆-McAb) another positive clone strain.

5, the breast cancer early warning chip of a kind of easy fast measuring human I type thymidine kinase gene protein according to claim 4, it is characterized in that: though these two kinds of hTK1 monoclonal antibodies belong to human I type thymidine kinase (hTK1) monoclonal antibody, all have same selectivity and specific immune response for its corresponding antigen, but both have different binding sites, affinity and affinity costant.

6, the breast cancer early warning chip of a kind of easy fast measuring human I type thymidine kinase gene protein according to claim 5, it is characterized in that: these two kinds of hTK1 monoclonal antibodies all can with human I type thymidine kinase (hTK1) gene protein antigen generation selectivity and specific immune response, (code name is 2A can to form an anti-hTK1 monoclonal antibody ₆-McAb) 2 and two anti-hTK1 monoclonal antibodies (code name is 3E ₈-McAb) simultaneously with the double antibodies sandwich immune complex structure (Ab1-Ag-Ab2) that combines with human I type thymidine kinase (hTK1) gene protein antigen 2A in other words ₆-McAb--hTK1 Ag--3E ₈-McAb double antibodies sandwich immune complex structure.

7, the breast cancer early warning chip of a kind of easy fast measuring human I type thymidine kinase gene protein according to claim 6, it is characterized in that:, make contained corresponding breast cancer gene albumen in the detected sample by the capillary action in the detection sample nature chromatography process and the immune response principle of double antibodies sandwich antigen--(code name is 2A to hTK1 antigen with an anti-hTK1 monoclonal antibody simultaneously ₆-McAb) (code name is 3E with two anti-hTK1 monoclonal antibodies ₈-McAb) the immune association reaction of high degree of specificity, high affinity, its double antibodies sandwich immune complex structure (Ab1-Ag-Ab2) or 2A take place ₆-McAb--hTK1 Ag--3E ₈The continuous enrichment of-McAb or hold back, and form an apparent coloured detection band (being positive findings) gradually by the color of institute's mark collaurum.

8, the breast cancer early warning chip of a kind of easy fast measuring human I type thymidine kinase gene protein according to claim 7 is characterized in that: the breast cancer gene albumen-hTK1 antigen that is contained in the detected sample can (code name be 2A by its corresponding one anti-hTK1 monoclonal antibody ₆-McAb) (code name is 3E with two anti-hTK1 monoclonal antibodies ₈-McAb) discern, also take place rapidly high specific, high-affinity immunity association reaction, confirm existing breast cancer gene albumen in this detected sample-hTK1 expression.

9, the breast cancer early warning chip of a kind of easy fast measuring human I type thymidine kinase gene protein according to claim 8, it is characterized in that: this breast cancer gene albumen-hTK1 antigen is that thymidine participates in the synthetic key enzyme of DNA as a kind of kinases, its can reversibility catalysis thymidine and the phosphoric acid deoxyribonucleoside between conversion, truly and objectively reflecting the propagation and the canceration situation of mammary glandular cell.

10, the breast cancer early warning chip of a kind of easy fast measuring human I type thymidine kinase gene protein according to claim 9, it is characterized in that: hTK1 not only accelerates to express when mammary glandular cell abnormality proliferation or canceration, and great expression also obviously increases its content or activity, the excision of particularly more notable increasing in the body fluid of the vigorous patients with mastocarcinoma of mammary glandular cell proliferative disorder or blood, and the height of hTK1 level and malignant tumour and recurrence are and reduce and trend of rising.

11, the breast cancer early warning chip of a kind of easy fast measuring human I type thymidine kinase gene protein according to claim 10, it is characterized in that: breast cancer gene albumen-hTK1 is in breast cancer cell expression showed increased, the activity of hTK1, does not go out and detect extremely low or basic detection in the body fluid of mammary gland benign tumor and normal healthy people or blood greater than 95% of total amount at breast cancer cell.

12, the breast cancer early warning chip of a kind of easy fast measuring human I type thymidine kinase gene protein according to claim 11, it is characterized in that:, then point out this detected person to have height risk or the mammary gland that breast cancer takes place to suffer from the breast cancer focus in case confirm have a large amount of breast cancer gene albumen-hTK1 to express in the detected sample.

13, the breast cancer early warning chip of a kind of easy fast measuring human I type thymidine kinase gene protein according to claim 12 is characterized in that: technique for gene engineering, colloid gold label developing technology, specific immunity detection technique and ply of paper are analysed solid phase labelling and double-antibody sandwich immuno-chromatographic assay technology that several different methods such as analytical technology organically combine.

14, the breast cancer early warning chip of a kind of easy fast measuring human I type thymidine kinase gene protein according to claim 13, it is characterized in that: do not contain corresponding breast cancer gene albumen-hTK1 antigen as detecting in the sample, then this detects sample and moves to and be coated with an anti-hTK1 monoclonal antibody (code name is 2A ₆The immune association reaction of high specific, high-affinity all can not take place in-McAb) position, promptly can not form coloured detection band (being negative findings) near the position that adds two detection lines about sampling point.