Background technology
The more special biomacromolecule of one class is arranged: cytoskeletal protein in various tumor markers.This cell component once once had been considered to a kind of supporting structure of cell, and what biological function is stable in properties do not have.But; the research of more than ten years has recently made us have to re-recognize this important cell component: this huge family not only supports the normal morphology of keeping cell and tissue; but also the differentiation and the growth of participation cell tissue; participate in the interaction (comprising the conduction of signal) of cell and cell, cell and extracellular matrix and stimulating the generation reaction to external world; the protection cell is avoided machinery, plays a role in the infringement of medicine etc.Tumour cell in conversion process, differentiation state, many-sides such as cellular morphology change, and also show the expression pattern of cytoskeleton composition, the change of aspects such as distributing position.Thereby this composition has been widely used in diagnosing tumor, have in addition may be used for tumor treatment.Clinically, and cytokeratin in this cell component (cytokeartins, cKs) most widely used general: as to be used for the judgement of tumor cell tissue's type, source, differentiation degree on the SABC; Be used for the early diagnosis of tumours such as lung cancer, colorectal cancer, cancer of the stomach, breast cancer on the serology, good pernicious differentiation, clinical stages, the result of treatment monitoring, the early detection of small transfer and patient follow up a case by regular visits to.
Cytokeratin is a most complicated histone in the cytoskeleton composition one median fiber silk family, approximately comprise 20 members, molecular weight is between 40 1 6OKDa, isoelectric point difference according to them can be divided into amphitypy: I type (alkaline keratin, CK1 one CK8, PI:6.1 1), 11 types (acid keratin, CK9 one CK20, PI:4.9 1).CYFRA21-1, TPAcyk, TPS use preferably at the immune analysis method of cytokeratin composition at present clinically, and they are respectively at cytokeratin 19 fragments; Thin chest keratin 8,18,19: cytokeratin 18M3 epi-position.CYFRA21-1 is applied to lung cancer, especially squama cancer, it is 61% to the total recall rate of lung cancer, average recall rate to small-cell carcinoma of the lung, squama cancer, gland cancer is respectively 52%, 79%, 54%, can be used as the foundation of small-cell carcinoma of the lung and non-little thin lung cancer antidiastole with the NSE use in conjunction, can be used for the state of illness monitoring behind the lung cancer chemicotherapy, the early detection of recurrence and time-to-live prediction are the most effective tumor markerses of present non-small cell lung cancer.
TPS is the soluble fragments of the tissue antigen discerned by cytokeratin 18 antibody, the malignant tumour of epithelial origin and metastatic tumor have than high expressed, especially the TPS high expressed is also gone into blood in a large number between tumour cell increment active period, can embody the biological behaviour of tumour better.Reports such as Zheng Youwei, the TPS positive rate is 55.1% in the patients with lung cancer serum, and positive rate is 100% in bronchoalveolar lavage fluid, points out as the mark TPS of lung cancer more responsive than CEA simultaneously.
The research of labelled immune analytical technology and application development are rapid over past ten years, have been widely used in each field of biomedical fundamental research and clinical disease diagnosis.The method that is used to detect serological index mainly comprises radioactive isotope immunoassay, enzyme linked immunological adsorption method and chemiluminescence immune assay.These methods both can be used as the primary dcreening operation test and also can be used as validation test, and this method is widely used at present.
What the cytokeratin 18 that we develop was measured kits (chemoluminescence method) employing is double-antibody sandwich single stage method reaction pattern, adopt cytokeratin 18 Monoclonal Antibody solid phases, specific monoclonal antibody purity height, no infectiousness, non-specific binding reaction is little, does not need special experimental laboratory just can produce in enormous quantities.
Kit of the present invention can be applicable to open chemiluminescence measuring instrument, and use cost is low, easier applying.
Summary of the invention
The purpose of this invention is to provide a kind of cytokeratin 18 (CyK-18) chemiluminescence immune analysis quantitative determination reagent kit.
Another object of the present invention provides a kind of method for preparing the mentioned reagent box.
Kit according to the present invention comprises: the 1) monoclonal antibody of the cytokeratin 18 of enzyme labeling; 2) be coated with the carrier of the monoclonal antibody of cytokeratin 18; 3) cytokeratin 18 calibration objects; And 4) chemical luminous substrate that above-mentioned enzyme acted on.
Further, the method for preparing the mentioned reagent box according to the present invention may further comprise the steps:
1) monoclonal antibody of usefulness enzyme labeling cytokeratin 18; 2) with the monoclonal antibody bag suppressed by vector of cytokeratin 18; 3) with cytokeratin 18 pure product preparation cytokeratin 18 calibration objects; 4) monoclonal antibody of the cytokeratin 18 of above-mentioned cytokeratin 18 calibration objects of packing, enzyme labeling and the chemical luminous substrate that this enzyme is acted on; And 5) be assembled into finished product.
Above-mentioned according to kit of the present invention and preparation method thereof in, described carrier can be solid phase carrier, preferred microporous plate, plastic bead, plastic tube or magnetic-particle.Described enzyme can be horseradish peroxidase or alkaline phosphatase.Described chemical luminous substrate can for, luminol or different luminol, 1,2-two oxidative ethane analog derivatives, comprising (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes (AMPPD), CSPD or CDP-Star.
In the method according to the invention, the step 2 of wherein said bag suppressed by vector) comprise following process:
Comprise the coating buffer of the citric acid of the trisodium citrate of 7.30g and 4.45g with 1000mL, the pH value is that 4.5~4.8 coating buffer mixes back loading on solid phase carrier with the monoclonal antibody of CyK-18, the physiological saline washing; Seal with phosphate buffer.Described confining liquid comprises 0.2gNaH based on the described confining liquid of 1000mL
2PO
42H
2O, 2.9gNaH
2PO
412H
2O, 10gBSA and 1mL Proclin 300, the pH value is 7.0~7.5.
Concrete mentioned reagent box can comprise CyK-18 calibration object, antibody sandwich plate, enzyme labeling thing and chemical luminous substrate liquid, 20 times of concentrated cleaning solutions etc.Wherein, described CyK-18 calibration object is a standard level, and micropore lath, the enzyme labeling CyK-18 monoclonal antibody that purity is not less than 90%, the antibody sandwich plate is 48 or 96 holes is that coupling alkaline phosphatase or horseradish peroxidase, chemical luminous substrate liquid are that AMPPD or luminol, concentrated cleaning solution are Tris-HCl.
The present invention's " cytokeratin 18 chemiluminescence immune analysis quantitative determination reagent kits " detection by quantitative exactly goes out the content of patient CyK-18, can be according to CyK-18 content, to the state of an illness of the tumour of epithelial origins such as alimentary canal, mammary gland, urogenital tract, respiratory tract monitor, the judgement of the early detection of therapeutic evaluation, recurrence and prognosis is significant.It has high specific, high sensitivity, high precision, high accuracy, easy advantage such as fast.Every index that this CyK-18 measures kit (chemoluminescence method) all reaches enzyme-linked immunosorbent assay.And, according to detection system of the present invention is open-sky technique, easy to be quick, does not need the expensive luminous measuring instrument of full-automatic chemical, be particularly suitable for vast middle and small hospital and promote the use of, for clinical diagnosis and research work provide a kind of very valuable detection means.According to kit of the present invention, the CyK-18 antigen of the CyK-18 monoclonal antibody of bag quilt and sample forms " double-antibody sandwich " structure on the CyK-18 monoclonal antibody of enzyme labeling and the carrier, therefore " double-antibody sandwich single stage method " reaction pattern of adopting of the present invention had not only effectively utilized the chemiluminescence principle, but also had guaranteed the sensitivity that detects.
What kit of the present invention was used is the enzymatic luminous substrate, by the chromogenic substrate in the light signal replacement enzyme-linked immuno assay that detects the luminous substrate generation, thereby have a specificity equal with enzyme-linked immuno assay, and sensitivity improves greatly, improve about 10 times than now Enzyme Linked Immunoadsorbent Assay sensitivity, the state of an illness that can be the tumour of epithelial origins such as alimentary canal, mammary gland, urogenital tract, respiratory tract is monitored, therapeutic evaluation, the early detection of recurrence and the judgement of prognosis provide more special, quick, reliable foundation.
In research process of the present invention, the present inventor has at first carried out screening experiment and Quality Identification to used starting material, comprises the luminous intensity of activity, chemical luminous substrate of the absorption property of activity, carrier (as lighttight white microwell plate) of labelled antibody and coated antibody and variation size, enzyme and luminous duration etc.Then method for coating is studied, be cushioned liquid and protect liquid to experimentize, select optimal bag and be cushioned liquid and protection liquid, tested by concentration by the different bags of antibody and find best concentration conditions with different bags.Mark for ALP can have diverse ways, by explore repeatedly and the contrast experiment finally found easy, productive rate is high, cost is low, the reliable quality labeling method, and different enzyme dilutions has been carried out long-term investigation tested, made and can make the activity stabilized dilution of the long-term maintenance of enzyme labeling thing.
The present inventor has also carried out experimental study to the reaction pattern and the reaction conditions of kit, finally determined double-antibody sandwich single stage method reaction pattern, and the influence of experimental result is tested with regard to the different reaction time, determine the optimal reaction time.
Embodiment
The preparation of embodiment 1 Cyk-18 alkali phosphatase enzyme mark monoclonal antibody
The CyK-18 monoclonal antibody is fully dialysed to PBS with glutaraldehyde method and alkaline phosphatase coupling, adds equal-volume glycerine, preserves below-20 ℃.
The preparation of embodiment 2 Cyk-18 horseradish peroxidase-labeled monoclonal antibodies
With sodium periodate oxidizing process mark Cyk-18 monoclonal antibody.Labeling process is as follows: take by weighing 5mgHRP and be dissolved in the 1ml distilled water.Add 0.1M NaIO 4 solution that 0.2ml newly joins in last liquid, lucifuge stirred 20 minutes under the room temperature.Above-mentioned solution is packed in the bag filter, and to the sodium-acetate buffer dialysis of 1mM pH4.4,4 ℃ are spent the night.Add 20 μ l 0.2M pH9.5 carbonate buffer solutions, make the pH of above hydroformylation HRP be elevated to 9.0~9.5, add 10mg antibody then immediately in 1ml 0.01M carbonate buffer solution, the room temperature lucifuge stirred 2 hours gently.Add 4mg/ml NaBH 4 liquid that 0.1ml newly joins, mixing, put again 4 ℃ 2 hours.Above-mentioned liquid is packed in the bag filter, and to 0.15M pH7.4PBS dialysis, 4 ℃ are spent the night.Under agitation dropwise add the equal-volume saturated ammonium sulfate, put 4 ℃ 1 hour.3000rpm centrifugal half an hour, abandon supernatant.Sediment is dissolved among the PBS of a small amount of 0.15M pH7.4.Above-mentioned solution is packed in the bag filter, to the PB buffer saline dialysis of 0.15M pH7.4, remove ammonium ion after, 10,000rpm removed precipitation in centrifugal 30 minutes, supernatant is enzyme conjugates, adds equivalent high-quality glycerine, after the packing ,-20 ℃ of preservations.
Embodiment 3 preparations CyK-18 quantitative determination reagent kit of the present invention
One, alkaline phosphatase enzyme labelled antibody preparation
The Tris of enzyme mark monoclonal antibody diluent preparing weighing 12.12g, 5g BSA and 1mL Proclin 300 put into clean container well with the mentioned reagent weighing, add distilled water to 1L, the dissolving mixing.
With enzyme mark monoclonal antibody diluted enzyme labelled antibody different proportion, adopt the square formation method to select the working concentration of enzyme labelled antibody greater than 1:4000.
Two, the preparation of CyK-18 calibration object
With the pure product preparation of CyK-18, totally 6 bottles of packing 0,25,50,100,200,400U/L.
Three, the preparation of solid-phase coating plate
(1) in the clean container of 1L, behind the distilled water dissolving mixing of adding 1L, PH is 4.8 to bag, adds The addition of C yK-18 monoclonal antibody mixing, adds in each hole of microwell plate every hole 130 μ L, 4 ℃ of 24h then by the citric acid of the trisodium citrate of weighing 7.30g and 4.45g.
(2) washing: it is inferior to give a baby a bath on the third day after its birth with physiological saline.
(3) NaH of sealing weighing 0.2g
2PO
42H
2O, the NaH of 2.9g
2PO
412H
2O, 10gBSA and 1mL Proclin 300 put into clean container well with the mentioned reagent weighing, add distilled water to 1L, the dissolving mixing, and measuring the pH value is 7.0.
Every hole adds confining liquid 300 μ L respectively, and room temperature was placed 3 hours.Get rid of confining liquid, on thieving paper, pat dry.Room temperature removal moisture drying 24 hours.Carry out envelope immediately.2~8 ℃ of preservations of labeling postposition.
Four, chemical luminous substrate liquid
Weighing 24g Tris, 160g NaCl, 4g KCl, 15mL HCl, 200mL 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1; 2-two oxidative ethanes and 1mL Proclin300; clean container is put in the mentioned reagent weighing well; add distilled water, dissolving mixing to 1L.
Five, 20 times of cleansing solutions
Weighing 24g Tris, 160g NaCl, 4g KCl and 15ml HCl add distilled water to 1L in clean container, and the dissolving mixing is adjusted PH to 7.4
Six, semi-manufacture and finished product are formed
The packing of above-mentioned steps products obtained therefrom is semi-manufacture.Extract three parts of process specificitys, accuracy, sensitivity and stable assay approvals out and just can be assembled into cytokeratin 18 quantitative determination reagent kits (chemoluminescence method).Be assembled into also need inspect by random samples behind the kit and just can dispatch from the factory after qualified.
Embodiment 4 preparations CyK-18 quantitative determination reagent kit of the present invention
As enzyme labelled antibody, is luminous substrate outside with the luminol divided by horseradish peroxidase-labeled CyK-18 monoclonal antibody, and described chemical luminous substrate comprises A liquid and B liquid, wherein,
Based on the described chemical luminous substrate A of 1000mL liquid, comprise 1.7716g luminol, 0.051g4-xenol, 0.012g 4-iodobenzene boric acid, 11.4g boric acid, 4.9g borax, its pH value is 8.0~10.0;
Based on the described chemical luminous substrate B of 1000mL liquid, comprise 0.329g urea peroxide, 1mlTween20,51.58g Na
2HPO
412H
2O, 8.74g NaH
2PO
42H
2O, its pH value is 7.0~7.6.A liquid is to add Tris and dense HCl in distilled water to be made into 0.1M pH be 8.5 Tris-HCl damping fluid, comprise the Luminol of 4.0mg/mL and 0.3mg/mL to iodophenol.
B liquid is to add trisodium citrate and citric acid in distilled water, is mixed with 0.1M pH value and is 4.6 citrate buffer solution, comprises the superoxol of 200mg/mL.All the other all prepare the CyK-18 quantitative determination reagent kit with the method identical with embodiment 1.
Embodiment 5~6 preparations CyK-18 quantitative determination reagent kit of the present invention
Remove respectively with plastic bead, plastic tube is outside 7.5 as the pH value of carrier, confining liquid, and all the other all prepare the CyK-18 quantitative determination reagent kit with the method identical with embodiment 1 and 4.
Embodiment 7 preparations CyK-18 quantitative determination reagent kit of the present invention
Divided by magnetic-particle as carrier, with glutaraldehyde with the Sheet of CyK-18 anti-with the coupling of magnetic particle outside, all the other all prepare the CyK-18 quantitative measurement with the method identical with embodiment 1 and 4 and decide the agent box.
The using method of embodiment 8 kits of the present invention
The concrete operations such as the following table 1 of the CyK-18 quantitative determination reagent kit of above embodiment 1 preparation:
1) in 4 ℃ of refrigerators, takes out kit, equilibrium at room temperature 15 minutes.
2) take out coated slab, insert on the grillage.
Table 1 Cyk-18 application of sample is measured program list unit: μ L
With calibration object concentration is horizontal ordinate, and the RLU value is drawn typical curve for ordinate, finds the concentration of the CyK-18 of this serum on typical curve with each test serum RLU value.
The methodology of embodiment 9 kits of the present invention is identified
According to manufacturing conventional in this area and vertification regulation the kit of preparation among the embodiment 1 is examined and determine,
(1) kit sensitivity experiment
Carry out 10 hole replications with the S0 calibration object, its mean value adds that it is the sensitivity of kit that the twice standard deviation is brought the concentration value of curvilinear equation gained into, and its sensitivity is 10.6U/L.
(2) kit specificity experiment
Make cross reaction experiment, cross reacting rate<0.01% with its analog.
(3) kit accuracy experiment
Variation within batch
Get basic, normal, high three parts of quality controlled serum samples and carry out 10 hole parallel laboratory tests respectively, calculate the mean value (x) and the standard deviation (s) of measured value.By formula CV=s/x * 100% calculates the coefficient of variation, and variation within batch coefficient CV is respectively 6.3%, 4.9%, 4.1%.
Batch variation
Select the blood serum sample of 5 parts of variable concentrations that every part of serum is carried out replication 3 times, calculate its interassay coefficient of variation (CV%), batch variation CV is between 7.21~9.5%.
(4) kit accuracy experiment
With the calibration object raw material of high concentration, be diluted to four variable concentrations values with normal human serum, each concentration is done 5 hole parallel laboratory tests, and calculate recovery rate is in the 91-108% scope respectively.
(5) kit stability experiment
The kit storage temperature is 2-80 ℃, every index through 15 months mensuration kits all meets the demands, consider in transportation and the use that to the influence of kit, we carry out 370 ℃ of accelerated tests of 7 days, experimental result shows that every index of kit meets the requirements fully.
Sensitivity, specificity, accuracy, accuracy and the stability that " cytokeratin 18 chemiluminescence immune analysis quantitative determination reagent kits " are described is fully qualified.