CN103575902B - A kind of time-resolved fluorescence method four comprehensive detection oophoroma kits and application thereof - Google Patents
A kind of time-resolved fluorescence method four comprehensive detection oophoroma kits and application thereof Download PDFInfo
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- CN103575902B CN103575902B CN201210276519.3A CN201210276519A CN103575902B CN 103575902 B CN103575902 B CN 103575902B CN 201210276519 A CN201210276519 A CN 201210276519A CN 103575902 B CN103575902 B CN 103575902B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57449—Specifically defined cancers of ovaries
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/36—Gynecology or obstetrics
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7023—(Hyper)proliferation
- G01N2800/7028—Cancer
Abstract
The present invention relates to biology and technical field of medical detection, be specifically related to the time-resolved fluoroimmunoassay detection kit of a kind of simultaneously comprehensive detection oophoroma tumor marker four indices HE4, CA125, CEA, TPS in same reaction system, also relate to the application of this kit in comprehensive detection four oophoroma tumor markers HE4, CA125, CEA, TPS simultaneously.This kit comprises: anti-HE4, CA125, CEA, TPS first microwell plate of strain monoclonal antibody mixed antibody bag quilt respectively; Use Sm
3+anti-HE4, Eu of mark
3+anti-CA 125, the Tb of mark
3+anti-CEA, Dy of mark
3+the anti-TPS second strain monoclonal antibody mixed antibody of mark; Analysis buffer; Share fluorescence-enhancing agent; Cleansing solution; Quality-control product.Kit provided by the invention can be used for the clinical assistant diagnosis of oophoroma, observation of curative effect and Index for diagnosis, to the treatment of oophoroma tumor with prevent significant, detect four tumor markerses simultaneously, simplify detecting step, improve the specificity and sensitivity that detect aggregation of data analysis.
Description
Technical field
The present invention relates to biology and technical field of medical detection, be specifically related to the time-resolved fluoroimmunoassay detection kit of a kind of simultaneously comprehensive detection oophoroma tumor marker four indices HE4, CA125, CEA, TPS in same reaction system, also relate to the application of this kit in comprehensive detection four oophoroma tumor markers HE4, CA125, CEA, TPS simultaneously.
Background technology
Ovarian tumors is hidden, and poor prognosis, is described as one of cancer the most fatal.The early symptom of oophoroma is very not obvious, and initial symptoms is that abdominal pain or expansion, gastrointestinal disturbances are bad, and not easily cause enough attention, all reach an advanced stage when general ovarian cancer patients finds, therefore cure rate is extremely low.If can find that oophoroma just can improve the cure rate of oophoroma greatly in early days, in order to reach this purpose, people have carried out a large amount of research work.
CA125 is detected a kind of glycoprotein that can be combined by monoclonal antibody OC125 nineteen eighty-three from ovarian epithelial carcinoma antigen by people such as Bast, be present in the serum of epithelial ovarian cancer tissue and patient, be mainly used in the pernicious serous ovarian cancer of auxiliary diagnosis, ovarian epithelial carcinoma is also the index of observation of curative effect after ovarian cancer post operation, chemotherapy simultaneously.CA125 is a kind of diagnosis of ovarian cancer and monitors one of the most responsive index of its recurrence, but CA125 often can't detect in early days morbidity, and its clinical practice has certain limitation.Given this, experts and scholars both domestic and external are devoted to find more efficiently mark, can make a definite diagnosis oophoroma in early days, more accurately.
At present, expert, the scholar of lot of domestic and international make a definite diagnosis oophoroma with HE4, CA125, CEA, TPS tetra-kinds of index comprehensives one after another, four kinds of comprehensive analyses in specificity and susceptibility apparently higher than single index.Routine Test Lab or medical institutions adopt single agents box usually for detecting this four indices, and namely kit is for a kind of detection of tumor marker, and the shortcoming that this kind of mode exists is: 1, detecting step is various, wastes time and energy; 2, four kinds of labels adopt 4 kinds of kits to detect respectively, because the kit of different manufacturer there are differences, and the different kits of same producer due to detect label difference may exist controlled condition, parameter difference and cause testing result to there is error, this kind of error can produce larger impact in the specificity and susceptibility of comprehensive analysis four kinds of labels, and judged result may be caused to differ greatly.Therefore, develop a kind of kit that simultaneously can detect these four kinds of tumor markers under relatively same testing conditions, just can reduce the metrical error because artificial origin causes, greatly improve comprehensive specificity and the susceptibility of analyzing testing result, conclusion is accurately made to next step research or judgement foundation is more fully provided.
Summary of the invention
The object of the invention is to solve problems of the prior art, the time-resolved fluoroimmunoassay detection kit of a kind of simultaneously comprehensive detection oophoroma tumor marker four indices HE4, CA125, CEA, TPS in same reaction system is provided.
The present invention also aims to the application that this kit a kind of is provided.
In order to realize above object, the technical solution adopted in the present invention is: a kind of time-resolved fluorescence method four comprehensive detection oophoroma kits, four kinds of oophoroma tumor markers HE4, CA125, CEA, TPS comprehensively in the same reaction system of same micropore, employing time-resolved fluoroimmunoassay detects, and this kit comprises: anti-HE4, CA125, CEA, TPS first microwell plate of strain monoclonal antibody mixed antibody bag quilt respectively; Use Sm
3+anti-HE4, Eu of mark
3+anti-CA 125, the Tb of mark
3+anti-CEA, Dy of mark
3+the anti-TPS second strain monoclonal antibody mixed antibody of mark; Analysis buffer; Share fluorescence-enhancing agent; Cleansing solution; Quality-control product.
Wherein, the preparation method of the microwell plate of the first described strain monoclonal antibody mixed antibody bag quilt is: anti-HE4 respectively, CA125, CEA, the mixed antibody bag of the first strain monoclonal antibody composition of TPS is buffered liquid dilution, obtained antibody coating buffer, then in each hole of microwell plate, add the antibody coating buffer of 50 μ l respectively, in 37 DEG C of bags by 2 hours, then normal saline flushing microwell plate is used three times, and then in each hole of microwell plate, add the shrouding liquid of 100 μ l respectively, 2 hours are closed in room temperature, then normal saline flushing microwell plate twice is used, freeze drying, the microwell plate of obtained mixed antibody bag quilt.This microwell plate is sealed in aluminium foil bag, and 4 DEG C save backup.
In described antibody coating buffer, the concentration of the first strain monoclonal antibody of anti-HE4, CA125, CEA, TPS is 0.01 ~ 0.016g/L respectively.
Further, the preparation method that described bag is buffered liquid is: get that concentration is 0.05mol/L, pH value is the carbonate buffer solution of 9.6, is then the NaN of 0.2% with mass percent concentration
3solution preservation process, obtained bag is buffered liquid.
The preparation method of described shrouding liquid is: to concentration be 0.01mol/L, pH value is add BSA in the PBS damping fluid of 7.4, is mixed with the PBS damping fluid that BSA mass percent concentration is the BSA of 1%, is then the NaN of 0.2% with mass percent concentration
3solution preservation process, obtained shrouding liquid.
Use Sm
3+anti-HE4, Eu of mark
3+anti-CA 125, the Tb of mark
3+anti-CEA, Dy of mark
3+the preparation method of the anti-TPS second strain monoclonal antibody mixed antibody of mark, wherein the mark process step of often kind of antibody is identical, the rare earth ion chelate label that just antibody of anti-HE4, CA125, CEA, TPS is corresponding respectively be respectively N1-to isothiocycmatobenzyl-DTTA-Sm, N1-to isothiocycmatobenzyl-DTTA-Eu, N1-to isothiocycmatobenzyl-DTTA-Tb, N1-to isothiocycmatobenzyl-DTTA-Dy, respective antibody is referred to as labelled antibody below, each self-corresponding label is referred to as rare earth ion chelate, and concrete preparation method is:
(1) antibody labeling pre-treatment
Carry out marking front purification process with the centrifugal column antagonist that molecular cut off is 50kDa, concrete steps are as follows: the unsettled 1mg antibody that adds is in the centrifugal column of 50kDa, and centrifugal 8 minutes of 9000rpm, discards filtrate; In centrifugal column, add 200 μ l mark damping fluid, centrifugal 8 minutes of 9000rpm, discard filtrate, this step repeats four times, is taken out by centrifuge tube for the last time, discards filtrate; In centrifugal column, add 50 μ l mark damping fluid, allow it leave standstill 1 minute, then the reversion of the filter membrane of centrifugal column is loaded in centrifuge tube, centrifugal 6 minutes of 8000rpm, collect filtrate, namely obtain antibody to be marked;
(2) antibody labeling process
Take rare earth ion chelate 0.1 ~ 0.4mg, add 50 ~ 100 μ l ultrapure waters and dissolve; Chelating reagent is joined and fills in the EP pipe of antibody to be marked, mix; To be placed on shaking table 4 DEG C to spend the night, the obtained antibody marked;
Superdex 200 1X 30cm post is balanced as column equilibration liquid with the PBS damping fluid of three times of column volumes, the slow loading of antibody marked is drawn with pipettor, wash-out is carried out with eluent, flow control is at 1ml/min, Protein Detection instrument detects that protein peak collects sample, by degerming through 0.22 micron membrane filter for the object product collected; With specific activity assay method, labelled antibody is carried out to calculating and the analysis of mark rate.Highly purified BSA can join in labelled antibody solution by standing storage labelled antibody, and the final concentration of BSA is 0.1%, 4 DEG C can be placed in or-20 DEG C preserve.
The preparation method of described shared fluorescence-enhancing agent is: part of dissociating: mixed by the ethanol of the yttria of the PTA of 70 ~ 200 μm of ol, 2 ~ 7 μm of ol, Triton X-100 and 300ml of 0.6g, 1 liter is settled to water, with acetic acid adjust pH to 3.5, obtained part of dissociating; Strengthen part: mixed by the Tris of the Phen of 0.1 ~ 0.4mmol and 0.2mol, be settled to 1 liter with water, obtained enhancing part.
The preparation method of described cleansing solution is: concentration be 0.01mol/L, pH value is add Tween-20 in the PBS damping fluid of 7.4, mixing, obtained Tween-20 mass percent concentration is the PBS solution of 0.05%, then be the NaN3 solution preservation process of 0.2% with mass percent concentration, obtained cleansing solution.
Described quality-control product is the potpourri of HE4, CA125, CEA, TPS standard items.
A kind of described application of kit in comprehensive detection four oophoroma tumor markers HE4, CA125, CEA, TPS.
The application of kit in comprehensive detection four oophoroma tumor markers HE4, CA125, CEA, TPS, specifically comprises the following steps:
(1) dilute quality-control product by analysis buffer, make the mixed solution that there is series concentration gradient and include HE4, CA125, CEA, TPS tetra-kinds of standard items;
(2) four kinds of standard items mixed solutions prepared by testing sample and step (1) are added respectively anti-HE4, CA125, CEA, TPS first strain monoclonal antibody mixed antibody bag quilt microwell plate different holes in, and then Xiang Kongzhong adds the second strain monoclonal antibody mixed antibody being marked with rare earth ion chelate that can be combined with HE4, CA125, CEA, TPS respectively, add shared fluorescence-enhancing agent afterwards, carry out time-resolved fluoroimmunoassay detection, obtain luminous value;
(3) typical curve is done according to the luminous value comprising the mixed solution of HE4, CA125, CEA, TPS tetra-kinds of standard items with series concentration gradient measured;
(4) compare with the luminous value of HE4, CA125, CEA, TPS typical curve separately in HE4, CA125, CEA, TPS luminous value in the testing sample measured and standard items, draw HE4, CA125, CEA, TPS content separately in testing sample.
Wherein, described testing sample is human serum.
Kit provided by the invention, adopts time-resolved fluoroimmunoassay detection method, realizes the object of comprehensive detection four oophoroma tumor markers HE4, CA125, CEA, TPS simultaneously.Time resolved fluoro-immunoassay (TRFIA) is a kind of is label with rare earth ion, according to the luminous characteristics of rare earth ion chelate, measures the technical method of specificity fluorescent by TIME RESOLVED TECHNIQUE.TRFIA utilizes the exciting light of rare earth ion different with radiative wavelength, utilizing emitted light has long die-away time simultaneously, effectively can get rid of the interference of non-specific fluorescence like this, there is the range of linearity wide, the advantages such as highly sensitive and good stability, in addition, the different wavelength of transmitted light of different rare earth ions and different die-away times can be utilized, realize detecting multiple mark in same reaction system simultaneously, such and single index separate detection is compared and can be saved time, personnel, reagent etc., these features detect multi objective while of being just in time applicable to of the present invention in same reaction system.
Oophoroma tumor marker HE4, CA125, CEA, TPS time-resolved fluoroimmunoassay detection kit provided by the invention, has the extraordinary range of linearity, and the range of linearity detecting HE4 is 35pmol/L ~ 1000pmol/L, detects and is limited to 25pmol/L; The range of linearity detecting CA125 is 20U/ml ~ 560U/ml, detects and is limited to 6U/ml; The range of linearity detecting CEA is 2ng/ml ~ 500ng/ml, detects and is limited to 0.5ng/ml; The range of linearity detecting TPS is 45U/L ~ 520U/L, detects and is limited to 15U/L.The normal reference value of each Testing index is respectively: HE4 is 0 ~ 120pmol/L; CA125 is 0 ~ 35U/ml; CEA is 0 ~ 6.5ng/ml; TPS is 0 ~ 80U/L.Oophoroma tumor marker HE4, CA125, CEA, TPS time-resolved fluoroimmunoassay detection kit provided by the invention can be used for the clinical assistant diagnosis of oophoroma, observation of curative effect and Index for diagnosis, to the treatment of oophoroma tumor with prevent significant.
Comprehensive detection oophoroma tumor marker four indices HE4, CA125, CEA, TPS while of adopting kit provided by the invention can realize in same reaction system, detect simple to operate, step is few, and required time is short, convenient and swift; Testing result is accurate, avoid existing employing four kinds of individual event kits to detect four indices HE4, CA125, CEA, TPS respectively and carry out comprehensive descision again and the error in judgement caused, substantially increase specificity and the susceptibility of testing result, conclusion is accurately made to next step research or judgement and provides reliable basis.
Accompanying drawing explanation
Same reaction system reaction principle schematic diagram in the same micropore of kit that Fig. 1 provides for the embodiment of the present invention 1;
The kit of the kit that Fig. 2 provides for the embodiment of the present invention 1 and CanAg company tests HE4 correlation regression analysis figure (r=0.978, p < 0.05);
The kit of the kit that Fig. 3 provides for the embodiment of the present invention 1 and Roche Holding Ag tests CA125 correlation regression analysis figure (r=0.985, p<0.05);
The kit of the kit that Fig. 4 provides for the embodiment of the present invention 1 and Abbott company tests CEA correlation regression analysis figure (r=0.948, p < 0.05);
The kit of the kit that Fig. 5 provides for the embodiment of the present invention 1 and ID BiotechAB company tests TPS correlation regression analysis figure (r=0.969, p<0.05).
Embodiment
Below in conjunction with the drawings and specific embodiments, technical scheme of the present invention is described in detail.
Embodiment 1
The kit that the present embodiment provides, comprising: the microwell plate of the mixed antibody bag quilt of antibody 3A6 tetra-kinds of monoclonal antibody compositions of the antibody 9F3 of anti-HE4, the antibody M32112M of anti-CA 125, the antibody MAM02-008 of anti-CEA, anti-TPS; Use Sm
3+mark 10E1 antibody (antibody of anti-HE4), use Eu
3+mark M86306M antibody (antibody of anti-CA 125), use Tb
3+mark MAM02-881 antibody (antibody of anti-CEA), use Dy
3+the mixed antibody of 4B9 antibody (antibody of anti-TPS) four kinds of monoclonal antibody compositions of mark; Analysis buffer; Share fluorescence-enhancing agent; Cleansing solution; The quality-control product of HE4, CA125, CEA, TPS tetra-kinds of standard items mixing compositions.
In described kit: the monoclonal antibody 3A6 of the monoclonal antibody 9F3 of anti-HE4,10E1 antibody and anti-TPS, 4B9 antibody are inventor's self-control; The monoclonal antibody MAM02-008 of the monoclonal antibody M32112M of anti-CA 125, M86306M antibody and anti-CEA, MAM02-881 antibody are purchased from Meridian company.
The preparation method that bag is buffered liquid is: get that concentration is 0.05mol/L, pH value is the carbonate buffer solution of 9.6, is then the NaN of 0.2% with mass percent concentration
3solution preservation process, obtained bag is buffered liquid.
The preparation method of shrouding liquid is: to concentration be 0.01mol/L, pH value is add BSA in the PBS damping fluid of 7.4, is mixed with the PBS damping fluid that BSA mass percent concentration is the BSA of 1%, is then the NaN of 0.2% with mass percent concentration
3solution preservation process, obtained shrouding liquid.
9F3, M32112M, MAM02-008, the preparation method of the microwell plate of 3A6 mixed antibody bag quilt is: mixed antibody bag is buffered liquid dilution, obtained mixed antibody coating buffer, in mixed antibody coating buffer, and anti-HE4 respectively, CA125, CEA, the concentration of the first strain monoclonal antibody of TPS is 0.01 ~ 0.016g/L, in each hole of microwell plate, then add the mixed antibody coating buffer of 50 μ l respectively, makes every Kong Zhongjun contain the 9F3 of 0.5 ~ 0.8 μ g, M32112M, MAM02-008, 3A6 antibody, namely every Kong Zhongjun contains the 9F3 antibody of 0.5 ~ 0.8 μ g, the M32112M antibody of 0.5 ~ 0.8 μ g, the MAM02-008 antibody of 0.5 ~ 0.8 μ g, the 3A6 antibody of 0.5 ~ 0.8 μ g, in 37 DEG C of bags by 2 hours, then normal saline flushing microwell plate is used three times, and then in each hole of microwell plate, add the shrouding liquid of 100 μ l respectively, close 2 hours in room temperature, then use normal saline flushing microwell plate twice, freeze drying, the microwell plate of obtained mixed antibody bag quilt, be sealed in aluminium foil bag, 4 DEG C save backup.
Sm
3+mark 10E1, Eu
3+mark M86306M, Tb
3+mark MAM02-881, Dy
3+the preparation method of mark 4B9 is: the mark process step of often kind of antibody is identical, the rare earth ion chelate label that just antibody of anti-HE4, CA125, CEA, TPS is corresponding respectively be respectively N1-to isothiocycmatobenzyl-DTTA-Sm, N1-to isothiocycmatobenzyl-DTTA-Eu, N1-to isothiocycmatobenzyl-DTTA-Tb, N1-to isothiocycmatobenzyl-DTTA-Dy.
For N1-, isothiocycmatobenzyl-DTTA-Sm is marked to the antibody 10E1 of HE4, the concrete preparation method of mark and step are:
(1) before mark, application molecular cut off is that the centrifugal column of 50kDa marks front purification process to 10E1, and concrete steps are as follows: the unsettled 1mg 10E1 antibody that adds is in the centrifugal column of 50kDa, and centrifugal 8 minutes of 9000rpm, discards filtrate; In centrifugal column, add 200 μ l mark damping fluid, centrifugal 8 minutes of 9000rpm, discard filtrate, this step repeats four times, is taken out by centrifuge tube for the last time, discards filtrate; In centrifugal column, add 50 μ l mark damping fluid, allow it leave standstill 1 minute, so and by the filter membrane of centrifugal column reversing loads in centrifuge tube, and centrifugal 6 minutes of 8000rpm, collects filtrate, namely obtain antibody 10E1 to be marked; Wherein, mark that damping fluid is 50mmol/L, pH value is the carbonate buffer solution of 9.0;
(2) take rare earth ion chelate label N1-to isothiocycmatobenzyl-DTTA-Sm 0.2mg, add 80 μ l ultrapure waters and dissolve, make chelating reagent; Chelating reagent is joined in the EP pipe filling antibody 10E1 to be marked, mix; To be placed on shaking table 4 DEG C to spend the night, the obtained antibody 10E1 marked, namely uses Sm
3+the 10E1 antibody of mark;
Superdex 200 1X 30cm post is balanced, with pipettor absorption Sm as column equilibration liquid with the PBS damping fluid of three times of column volumes
3+the slow loading of 10E1 antibody of mark, carries out wash-out with eluent, and flow control is at 1ml/min, and Protein Detection instrument detects that protein peak collects sample, by degerming through 0.22 micron membrane filter for the object product collected; With specific activity assay method, labelled antibody is carried out to calculating and the analysis of mark rate.Highly purified BSA can join in labelled antibody solution by standing storage labelled antibody, and the final concentration of BSA is 0.1%, 4 DEG C can be placed in or-20 DEG C preserve.
The preparation method of analysis buffer is: to often liter of concentration be 0.05mol/L, pH value is add 9g NaCl, 0.5g NaN in the Tris-Hcl damping fluid of 8.0
3, 10g BSA, 0.1ml Tween-40,5g polyglycol, mix, i.e. obtained analysis buffer.
The preparation method sharing fluorescence-enhancing agent is: part of dissociating: mixed by the ethanol of the yttria of the PTA of 70 ~ 200 μm of ol, 2 ~ 7 μm of ol, Triton X-100 and 300ml of 0.6g, 1 liter is settled to water, with acetic acid adjust pH to 3.5, obtained part of dissociating; Strengthen part: mixed by the Tris of the Phen of 0.1 ~ 0.4mmol and 0.2mol, be settled to 1 liter with water, obtained enhancing part.
The preparation method of cleansing solution is: concentration be 0.01mol/L, pH value is add Tween-20 in the PBS damping fluid of 7.4, mixing, obtained Tween-20 mass percent concentration is the PBS solution of 0.05%, is then the NaN of 0.2% with mass percent concentration
3solution preservation process, obtained cleansing solution.
Quality-control product is the potpourri of HE4, CA125, CEA, TPS standard items.HE4, CA125, CEA, TPS standard items are all purchased from Abnova company.
Embodiment 2
The application of the kit that the embodiment of the present invention 1 provides in comprehensive detection four oophoroma tumor markers HE4, CA125, CEA, TPS, namely the kit that the embodiment of the present invention 1 provides is adopted, comprehensive detection oophoroma tumor marker HE4, CA125, CEA, TPS time-resolved fluoroimmunoassay detection method simultaneously, its reaction principle as shown in Figure 1, adopt double antibody sandwich method, comprise the following steps:
(1) dilute HE4, CA125, CEA, TPS tetra-kinds of protein standard substance potpourris, i.e. quality-control products by analysis buffer, make the mixed solution that there is series concentration gradient and include HE4, CA125, CEA, TPS tetra-kinds of protein standard substances;
(2) four kinds of protein standard substance mixed solutions prepared by 50 μ l testing sample human serums and step (1) are got respectively, add 9F3, M32112M, MAM02-008, in the different holes of the microwell plate of 3A6 mixed antibody bag quilt, 37 DEG C of incubations 1 hour, (during washing, the compound method of cleansing solution used is: add 9g sodium chloride in often liter of PBS damping fluid in three washings, 0.5ml Tween-20, shake up, ), add 50 μ l mixed antibody working fluids afterwards (by analysis buffer with volume ratio 1:2000 dilution mixture labelled antibody storage liquid, obtained mixed antibody working fluid, consisting of of this mixed antibody working fluid: wherein containing 30ng ~ 60ng Sm
3+10E1,50ng of marking ~ 90ng Eu
3+m86306M, 40ng of marking ~ 100ng Tb
3+mAM02-881,130ng of marking ~ 200ng Dy
3+the 4B9 of mark, all the other are analysis buffer), concussion evenly, 37 DEG C of incubations 1 hour, 5 times are washed with cleansing solution, on thieving paper, control is dry, adds the part of dissociating that 200 μ l share fluorescence-enhancing agent, shakes 5 minutes, add the enhancing part that 20 μ l share fluorescence-enhancing agent, shake 8 minutes, fluorescence intensity measured by time-resolved fluorescence instrument, excitation wavelength is 315nm, 647nm, 612nm, 544nm, 575nm are respectively Sm
3+, Eu
3+, Tb
3+, Dy
3+the strongest transmitting photopeak, 500 microseconds, 200 microseconds, 50 microseconds, 20 microseconds are respectively Eu
3+, Tb
3+, Sm
3+, Dy
3+time delay, measure respective luminous value respectively.Each index in HE4, CA125, CEA, TPS of variable concentrations tetra-kinds of albumen hybrid standard product is done typical curve with respective luminous value respectively, and obtain the respective typical curve of different index, the concentration of testing sample human serum calculates by typical curve.Obtain the precision of kit: the coefficient of variation in HE4 analyzes and between analyzing is respectively 3.1% ~ 5.3%, 3.9% ~ 7.6%; The coefficient of variation in CA125 analyzes and between analyzing is respectively 2.9% ~ 4.8%, 4.1% ~ 7.0%; The coefficient of variation in CEA analyzes and between analyzing is respectively 3.9% ~ 5.9%, 4.7% ~ 8.3%; The coefficient of variation in TPS analyzes and between analyzing is respectively 3.4% ~ 5.9%, 4.0% ~ 7.9%.
Embodiment 3
The HE4 that 40 parts of serum samples obtain is detected with kit of the present invention, CA125, CEA, the numerical value of TPS tetra-indexs and with same 40 parts of serum samples respectively with the kit test HE4 of CanAg company, the kit test CA125 of Roche Holding Ag, the kit test CEA of Abbott company, the data that the kit test TPS of ID Biotech AB company obtains do regression correlations analysis respectively, respective figure 2 respectively, 3, 4, 5, as can be seen from accompanying drawing, kit of the present invention is tested each achievement data of obtaining and tested with single index kit the data obtained respectively has good correlativity.
Claims (6)
1. a time-resolved fluorescence method four comprehensive detection oophoroma kits, four kinds of oophoroma tumor markers HE4, CA125, CEA, TPS comprehensively in the same reaction system of same micropore, employing time-resolved fluoroimmunoassay detects, and it is characterized in that: this kit comprises: anti-HE4, CA125, CEA, TPS first microwell plate of strain monoclonal antibody mixed antibody bag quilt respectively; Use Sm
3+anti-HE4, Eu of mark
3+anti-CA 125, the Tb of mark
3+anti-CEA, Dy of mark
3+the anti-TPS second strain monoclonal antibody mixed antibody of mark; Analysis buffer; Share fluorescence-enhancing agent; Cleansing solution; Quality-control product.
2. time-resolved fluorescence method according to claim 1 four comprehensive detection oophoroma kits, it is characterized in that: the preparation method of the microwell plate of the first described strain monoclonal antibody mixed antibody bag quilt is: anti-HE4 respectively, CA125, CEA, TPS first strain monoclonal antibody mixed antibody bag is buffered liquid dilution, obtained antibody coating buffer, then in each hole of microwell plate, add the described antibody coating buffer of 50 μ L respectively, in 37 DEG C of bags by 2 hours, then normal saline flushing microwell plate is used three times, and then in each hole of microwell plate, add the shrouding liquid of 100 μ L respectively, 2 hours are closed in room temperature, then normal saline flushing microwell plate twice is used, freeze drying, the microwell plate of obtained mixed antibody bag quilt.
3. time-resolved fluorescence method according to claim 2 four comprehensive detection oophoroma kits, is characterized in that: in described antibody coating buffer, and the concentration of the first strain monoclonal antibody of anti-HE4, CA125, CEA, TPS is 0.01 ~ 0.016g/L respectively.
4. time-resolved fluorescence method according to claim 1 four comprehensive detection oophoroma kits, it is characterized in that: the preparation method of described shared fluorescence-enhancing agent is: part of dissociating: the ethanol of the yttria of the PTA of 70 ~ 200 μm of ol, 2 ~ 7 μm of ol, Triton X-100 and 300ml of 0.6g is mixed, 1 liter is settled to water, with acetic acid adjust pH to 3.5, obtained part of dissociating; Strengthen part: mixed by the Tris of the Phen of 0.1 ~ 0.4mmol and 0.2mol, be settled to 1L with water, obtained enhancing part.
5. time-resolved fluorescence method according to claim 1 four comprehensive detection oophoroma kits, it is characterized in that: the preparation method of described cleansing solution is: concentration be 0.01mol/L, pH value is add Tween-20 in the PBS damping fluid of 7.4, mixing, obtained Tween-20 mass percent concentration is the PBS solution of 0.05%, is then the NaN of 0.2% with mass percent concentration
3solution preservation process, obtained cleansing solution.
6. time-resolved fluorescence method according to claim 1 four comprehensive detection oophoroma kits, is characterized in that: described quality-control product is the potpourri of HE4, CA125, CEA, TPS standard items.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210276519.3A CN103575902B (en) | 2012-08-03 | 2012-08-03 | A kind of time-resolved fluorescence method four comprehensive detection oophoroma kits and application thereof |
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| CN104280556B (en) * | 2014-10-31 | 2016-08-17 | 杨子学 | A kind of detection method simultaneously measuring lipoprotein phospholipase A2 and c reactive protein content in blood plasma and kit |
| CN107121548A (en) * | 2016-02-25 | 2017-09-01 | 深圳市迈科龙生物技术有限公司 | Quantitatively detect test paper, preparation method and the detection method of tumor markers |
| CN109342731A (en) * | 2018-12-07 | 2019-02-15 | 江苏省原子医学研究所 | The kit of gynecological tumor marker HER-2 and HE4 is detected simultaneously |
| CN111381028A (en) * | 2018-12-29 | 2020-07-07 | 深圳市帝迈生物技术有限公司 | Kit, system, application, detection antibody composition and preparation method thereof |
| CN109444435A (en) * | 2019-01-03 | 2019-03-08 | 河南大学 | A kind of nanometer selenium kit of quick detection HE4 and CA125 |
| CN113009138B (en) * | 2020-06-17 | 2022-08-30 | 山东大学 | Kit and method for detecting breast cancer tumor marker |
| CN114280301A (en) * | 2022-03-03 | 2022-04-05 | 北京华瑞康源生物科技发展有限公司 | Method and kit for simultaneously detecting two tumor markers |
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| CN101410715A (en) * | 2006-01-27 | 2009-04-15 | 三路影像公司 | Methods for identifying patients with an increased likelihood of having ovarian cancer and compositions therefor |
| CN101460630A (en) * | 2006-01-04 | 2009-06-17 | 富士瑞必欧美国公司 | Use of he4 and other biochemical markers for assessment of endometrial and uterine cancers |
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| EP2322933A1 (en) * | 2001-08-29 | 2011-05-18 | Pacific Northwest Research Institute | Diagnosis of carcinomas |
| CN101460630A (en) * | 2006-01-04 | 2009-06-17 | 富士瑞必欧美国公司 | Use of he4 and other biochemical markers for assessment of endometrial and uterine cancers |
| CN101473041A (en) * | 2006-01-04 | 2009-07-01 | 富士瑞必欧美国公司 | Use of HE4 and other biochemical markers for assessment of endometrial and uterine cancers |
| CN101410715A (en) * | 2006-01-27 | 2009-04-15 | 三路影像公司 | Methods for identifying patients with an increased likelihood of having ovarian cancer and compositions therefor |
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