CN103116025A - Kit for comprehensive detection of gastric cancer by means of time-resolved fluorescence method and application thereof - Google Patents
Kit for comprehensive detection of gastric cancer by means of time-resolved fluorescence method and application thereof Download PDFInfo
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- CN103116025A CN103116025A CN201210510566XA CN201210510566A CN103116025A CN 103116025 A CN103116025 A CN 103116025A CN 201210510566X A CN201210510566X A CN 201210510566XA CN 201210510566 A CN201210510566 A CN 201210510566A CN 103116025 A CN103116025 A CN 103116025A
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Abstract
The invention relates to the technical field of biological and medical detection, and in particular relates to an immunodetection kit for comprehensively detecting indexes of gastric cancer tumor landmarks by means of a time-resolved fluorescence method, i.e. CA724, CEA and CA199 in a same reaction system at the same time, and further relates to an application of the kit in the comprehensive detection of the gastric cancer tumor landmarks, i.e. the CA724, the CEA and the CA199. The kit comprises microwell plate coated by an anti-CA724, CEA and CA199 first strain monoclonal antibody hybrid antibody, an Eu3+ marked anti-CA724, Tb3+ marked anti-CA199 and Sm+ marked anti-CEA type second strain monoclonal antibody hybrid antibody, an analysis buffer solution, a common fluorescence reinforcing agent, a scrubbing solution and a quality control product. The kit provided by the invention can be used for the clinical auxiliary diagnosis, the curative effect observation and the prognosis judgment on gastric cancers, and has an important meaning on the treatment and the prevention of the gastric cancer tumors, three tumor landmarks can be detected at the same time, detection steps can be simplified, and the specificity and the sensitivity of the comprehensive analysis of test data can be improved.
Description
Technical field
The present invention relates to biology and technical field of medical detection, be specifically related to a kind of in same reaction system the time-resolved fluoroimmunoassay detection kit of comprehensive detection stomach neoplasms tumor markers index CA724, CEA, CA199 simultaneously, also relate to the application of this kit in comprehensive detection stomach neoplasms tumor markers CA724, CEA, CA199 simultaneously.
Background technology
The annual new cancer of the stomach more than 100 ten thousand in the whole world, China accounts for 42%, dead approximately 800,000, and China accounts for 35%, is one of country that incidence gastric cancer rate and mortality ratio are the highest, and M & M is all that the world average level twice is many.The data demonstration, 2007, in front 10 cancers of China, the incidence gastric cancer rate was second, mortality ratio is the 3rd; The men and women's of incidence gastric cancer rate and mortality ratio ratio is 2 to 1; The incidence of disease in rural area is that 1.6 times of city, the mortality ratio in rural area are 1.9 times of city; Death/incidence rate (be equivalent to ill after the risk of dying of illness) is the 8th.The incidence of disease of cancer of the stomach significantly raises along with the increase at age, and the peak age of morbidity is at 50 years old～80 years old, but presents year by year rejuvenation trend, and in cancer of the stomach, 19～35 years old patient's ratio rises to current 3.3% from 1.7% over 40 years.Compatriots' cause of the death 1/4 is cancer at present, and 1/4 of the cancer cause of the death is cancer of the stomach, and in the alimentary system malignant tumour death, about half is died from cancer of the stomach.Cancer of the stomach curative effect and stadium morning and evening and diagnoses and treatment and means are closely related, early carcinoma of stomach 90% above patient after enough treatments can more than 5-year Survival or cure, and the late gastric cancer patient treats latter 5 years survival rate less thaies 5%, therefore making a definite diagnosis in early days is still the key of this disease for the treatment of.
At present, the detection index commonly used of diagnosis of gastric cancer has CA724, CEA, CA199 etc. several.When actual diagnosis of gastric cancer, be generally to make a definite diagnosis cancer of the stomach with CA724, CEA, tri-kinds of index comprehensives of CA199 because three index comprehensive analyses on specificity and susceptibility apparently higher than single index.At present Routine Test Lab or medical institutions adopt respectively the single agents box usually for detection of these three indexs, and a kit is detected for a kind of tumor marker, and the shortcoming that this kind of mode exists is: 1, detecting step is various, wastes time and energy; 2, several labels adopt respectively several single index kits to detect, because the kit of different manufacturers there are differences, and the different kits of same producer are because the label difference detected may exist the difference of controlled condition, parameter to cause testing result to have error, this kind of error can produce larger impact on the specificity of three kinds of labels of analysis-by-synthesis and susceptibility, may cause judged result to differ greatly.Therefore, develop a kind of kit that can be under relatively same testing conditions can detect these three kinds of tumor markers simultaneously, just can reduce the detection error caused due to the artificial origin, greatly improve specificity and the susceptibility of analysis-by-synthesis testing result, for next step research or judgement are made conclusion accurately foundation more fully is provided.
Summary of the invention
The objective of the invention is in order to solve problems of the prior art, provide a kind of in same reaction system the time-resolved fluoroimmunoassay detection kit of comprehensive detection stomach neoplasms tumor markers index CA724, CEA, CA199 simultaneously.
The present invention also aims to provide a kind of application of this kit.
In order to realize above purpose, the technical solution adopted in the present invention is: a kind of time-resolved fluorescence method comprehensive detection cancer of the stomach kit, three kinds of stomach neoplasms tumor markers CA724, CEA, CA199 comprehensively in the same reaction system of same micropore, the employing time-resolved fluoroimmunoassay detects, and this kit comprises: anti-CA724, CEA, the coated microwell plate of CA199 the first strain monoclonal antibody mixed antibody respectively; Use Eu
3+anti-CA724, the Tb of mark
3+anti-CA199, the Sm of mark
3+the anti-CEA second strain monoclonal antibody mixed antibody of mark; Analysis buffer; Share fluorescence-enhancing agent; Cleansing solution; Quality-control product.
The preparation method of the microwell plate that described the first strain monoclonal antibody mixed antibody is coated is: anti-CA724 respectively, CEA, coated damping fluid dilution for CA199 the first strain monoclonal antibody mixed antibody, make the antibody coating buffer, then to the described antibody coating buffer that adds respectively 100 μ l in each hole of microwell plate, be coated with 2 hours in 37 ℃, then use the normal saline flushing microwell plate three times, and then to the shrouding liquid that adds respectively 200 μ l in each hole of microwell plate, seal 2 hours or spend the night in 4 ℃ of sealings in room temperature, then use twice of normal saline flushing microwell plate, freeze drying, make the coated microwell plate of the first strain monoclonal antibody mixed antibody.
Wherein, in described antibody coating buffer, the concentration of the first strain monoclonal antibody of anti-CA724, CEA, CA199 is 0.010～0.019g/L respectively.
The described Eu that uses
3+anti-CA724, the Tb of mark
3+anti-CA199, the Sm of mark
3+in the anti-CEA second strain monoclonal antibody mixed antibody of mark, use Eu
3+the anti-CA724 antibody of mark, Tb
3+the anti-CA199 antibody of mark, Sm
3+the quality proportioning of the anti-CEA antibody of mark is: Eu
3+the anti-CA724 antibody of mark: Tb
3+the anti-CA199 antibody of mark: Sm
3+anti-CEA antibody=(3～6) of mark: (3～7): (5～9).
The preparation method of analysis buffer is: to every liter of concentration, be to add 9g NaCl, 0.5g NaN in 0.05mol/L, the pH value Tris-Hcl damping fluid that is 8.0
3, 10g BSA, 0.1ml Tween-40,5g polyglycol, mix, make analysis buffer.
The preparation method of described shared fluorescence-enhancing agent is: the part of dissociating: the ethanol of the Triton X-100 of the yttria of the PTA of 70～200 μ mol, 2～7 μ mol, 0.6g and 300ml is mixed, water is settled to 1 liter, with acetic acid adjust pH to 3.5, make the part of dissociating; Strengthen part: the Tris of the Phen of 0.1～0.4mmol and 0.2mol is mixed, and water is settled to 1L, makes the enhancing part.
The preparation method of described cleansing solution is: be to add Tween-20 in 0.01mol/L, the pH value PBS damping fluid that is 7.4 in concentration, mix, the PBS solution that to make the Tween-20 mass percent concentration be 0.05%, the NaN that is then 0.2% with mass percent concentration
3the solution preservative treatment, make cleansing solution.
The potpourri that described quality-control product is CA724, CEA, CA199 standard items.
The application of a kind of time-resolved fluorescence method comprehensive detection cancer of the stomach kit in comprehensive detection stomach neoplasms tumor markers CA724, CEA, CA199.
The application of described kit in comprehensive detection stomach neoplasms tumor markers CA724, CEA, CA199 comprises the following steps:
(1) dilute quality-control product by analysis buffer, make and there is the mixed solution that the series concentration gradient includes CA724, CEA, CA199 standard items;
(2) the standard items mixed solution prepared from step (1) by testing sample adds respectively in the different holes of the coated microwell plate of anti-CA724, CEA, CA199 the first strain monoclonal antibody mixed antibody, and then Xiang Kongzhong adds the second strain monoclonal antibody mixed antibody that is marked with the rare earth ion chelate that can be combined with CA724, CEA, CA199 respectively, add afterwards shared fluorescence-enhancing agent, carry out the time-resolved fluoroimmunoassay detection, obtain luminous value;
(3) do typical curve according to the luminous value of the mixed solution that comprises CA724, CEA, CA199 standard items with series concentration gradient of measuring;
(4) luminous value with CA724, CEA, CA199 typical curve separately in CA724, CEA, CA199 luminous value and standard items in the testing sample of measuring compares, and draws CA724 in testing sample, CEA, CA199 content separately.
Described testing sample is human serum.
Further, the preparation method of described coated damping fluid is: getting concentration is the carbonate buffer solution that 0.07mol/L, pH value are 9.3, the NaN that is then 0.2% with mass percent concentration
3the solution preservative treatment, make coated damping fluid.
The preparation method of described shrouding liquid is: to concentration, be to add BSA in 0.05mol/L, the pH value PBS damping fluid that is 7.4, and the PBS damping fluid of the BSA that to be mixed with the BSA mass percent concentration be 1%, the NaN that is then 0.2% with mass percent concentration
3the solution preservative treatment, make shrouding liquid.
Kit provided by the invention, adopt the time-resolved fluoroimmunoassay detection method, realizes the purpose of comprehensive detection three stomach neoplasms tumor markers CA724, CEA, CA199 simultaneously.Time resolved fluoro-immunoassay (TRFIA) is a kind ofly to take rare earth ion as label, according to the luminous characteristics of rare earth ion chelate, measures the technical method of specificity fluorescent by the time resolution techniques.TRFIA utilizes the exciting light of rare earth ion different with radiative wavelength, utilizing emitted light has long die-away time simultaneously, can effectively 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, can utilize the different wavelength of transmitted light of different rare earth ions and different die-away times, realize in same reaction system detecting a plurality of marks simultaneously, such and single index separate detection is compared and can be saved time, personnel, reagent etc., just in time the applicable while of the present invention is detected many indexs to these characteristics in same reaction system.
Stomach neoplasms tumor markers CA724 provided by the invention, CEA, CA199 time-resolved fluoroimmunoassay detection kit, have the extraordinary range of linearity, and the range of linearity that detects CA724 is 2U/ml～300U/ml, detects and be limited to 1U/ml; The range of linearity that detects CEA is 2.0ng/ml～120ng/ml, detects and is limited to 0.5ng/ml; The range of linearity that detects CA199 is 12U/ml～400U/ml, detects and is limited to 6U/ml.Each positive reference value that detects index is respectively: CA724>7U/ml; CEA>5ng/ml; CA199>30U/ml.Stomach neoplasms tumor markers CA724 provided by the invention, CEA, CA199 time-resolved fluoroimmunoassay detection kit can be used for clinical assistant diagnosis, observation of curative effect and the prognosis judgement of cancer of the stomach, to the treatment of cancer of the stomach tumour with prevent significant.
Adopt kit provided by the invention can realize in same reaction system comprehensive detection stomach neoplasms tumor markers index CA724, CEA, CA199 simultaneously, detect simple to operately, step is few, and required time is short, convenient and swift; Testing result is accurate, avoided three kinds of individual event kits of existing employing to detect respectively index CA724, CEA, CA199, the error in judgement that comprehensively judges again and cause, specificity and the susceptibility of testing result have greatly been improved, for next step research or judgement are made conclusion accurately reliable basis is provided.
The 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;
Fig. 2 tests CA724 correlation regression analysis chart for the single index kit of the new industry of kit and Shenzhen company that the embodiment of the present invention 1 provides;
Fig. 3 tests CA199 correlation regression analysis chart for the single index kit of the new industry of kit and Shenzhen company that the embodiment of the present invention 1 provides;
The kit that Fig. 4 provides for the embodiment of the present invention 1 and the single index kit of Abbott company test CEA correlation regression analysis chart.
Embodiment
Below by specific embodiment, technical scheme of the present invention is elaborated.
Embodiment 1
Stomach neoplasms tumor markers CA724, the CEA that the present embodiment provides, CA199 time-resolved fluoroimmunoassay detection kit comprise: the coated microwell plate of mixed antibody that tri-kinds of monoclonal antibodies of the antibody MAM02-008 of the antibody ab3982 of the antibody C7189 of anti-CA724, anti-CA199, anti-CEA form; Use Eu
3+the C7164 antibody of mark (antibody of anti-CA724), use Tb
3+the ab116024 antibody of mark (antibody of anti-CA199), use Sm
3+the mixed antibody that three kinds of monoclonal antibodies of the MAM02-881 antibody of mark (antibody of anti-CEA) form; Analysis buffer; Share fluorescence-enhancing agent; Cleansing solution; CA724, CEA, tri-kinds of standard items of CA199 mix the quality-control product formed.This kit also comprises other related reagents such as negative control, positive control, substrate.
In described kit, C7189, C7164 antibody are self-control; Ab3982, ab116024 antibody are purchased from abcam company; MAM02-008, MAM02-881 antibody are purchased from Meridian company.
C7189, ab3982, the preparation method of the microwell plate that the MAM02-008 mixed antibody is coated is: coated damping fluid dilution for mixed antibody, make the mixed antibody coating buffer, in the mixed antibody coating buffer, anti-CA724 respectively, CEA, the concentration of the first strain monoclonal antibody of CA199 is 0.014g/L, be in the mixed antibody coating buffer, antibody C7189, ab3982, the concentration of MAM02-008 is 0.014g/L, then to the mixed antibody coating buffer that adds respectively 100 μ l in each hole of microwell plate, be coated with 2 hours in 37 ℃, then use the normal saline flushing microwell plate three times, and then to the shrouding liquid that adds respectively 200 μ l in each hole of microwell plate, in room temperature sealing 2 hours, then use twice of normal saline flushing microwell plate, freeze drying, make the coated microwell plate of mixed antibody, be sealed in aluminium foil bag, 4 ℃ save backup.
Wherein, the preparation method of coated damping fluid is: getting concentration is the carbonate buffer solution that 0.07mol/L, pH value are 9.3, the NaN that is then 0.2% with mass percent concentration
3the solution preservative treatment, make coated damping fluid.The preparation method of shrouding liquid is: to concentration, be to add BSA in 0.05mol/L, the pH value PBS damping fluid that is 7.4, and the PBS damping fluid of the BSA that to be mixed with the BSA mass percent concentration be 1%, the NaN that is then 0.2% with mass percent concentration
3the solution preservative treatment, make shrouding liquid.
Use Eu
3+the C7164 antibody of mark, use Tb
3+the ab116024 antibody of mark, use Sm
3+the preparation method of the mixed antibody that three kinds of monoclonal antibodies of the MAM02-881 antibody of mark form is: by Eu
3+the C7164 antibody of mark, Tb
3+the ab116024 antibody of mark, Sm
3+the MAM02-881 antibody of mark is with mass ratio: Eu
3+the C7164 antibody of mark: Tb
3+the ab116024 antibody of mark: Sm
3+the MAM02-881 antibody of mark=5:5:7 mixes, and makes mixed antibody.
Wherein use Eu
3+the C7164 antibody of mark, use Tb
3+the ab116024 antibody of mark, use Sm
3+the preparation method of the MAM02-881 antibody of mark is: the mark process step of every kind of antibody is identical, just respectively the antibody of anti-CA724, CEA, CA199 respectively corresponding rare earth ion chelate label be N1-to isothiocyanic acid benzyl-DTTA-Eu, N1-to isothiocyanic acid benzyl-DTTA-Sm, N1-to isothiocyanic acid benzyl-DTTA-Tb.The N1-of below take is example to the C7164 antibody of isothiocyanic acid benzyl-DTTA-Eu mark, and concrete preparation method and the step of its mark are:
(1) centrifugal column that the application molecular cut off is 50kDa before mark carries out purification process before mark to C7164 antibody, and concrete steps are as follows: the unsettled 1mg C7164 antibody that adds is in the centrifugal column of 50kDa, and centrifugal 8 minutes of 9000rpm, discard filtrate; Add 200 μ l mark damping fluids in centrifugal column, centrifugal 8 minutes of 9000rpm, discard filtrate, and this step repeats four times, for the last time centrifuge tube taken out, and discards filtrate; Add 50 μ l mark damping fluids in centrifugal column, allow its standing 1 minute, in the centrifuge tube of then reversion of the filter membrane of centrifugal column being packed into, centrifugal 6 minutes of 8000rpm, collect filtrate, obtains C7164 antibody to be marked; Wherein, the mark damping fluid is the carbonate buffer solution that 50mmol/L, pH value are 9.0;
(2) take rare earth ion chelate label N1-to isothiocyanic acid benzyl-DTTA-Eu 0.2mg, add 80 μ l ultrapure waters to be dissolved, make chelating reagent; Chelating reagent is joined in the EP pipe that fills antibody C7164 to be marked, mix; Be placed on shaking table 4 ℃ and spend the night, make the antibody C7164 that mark is good, use Eu
3+the C7164 antibody of mark;
, draw and use Eu with pipettor as column equilibration liquid balance Superdex 200 1 * 30cm post with the PBS damping fluid of three times of column volumes
3+the slow loading of C7164 antibody of mark, carry out wash-out with eluent, and flow control is at 1ml/min, and the Protein Detection instrument detects protein peak and collects sample, by the purpose product collected through 0.22 μ m filter membrane degerming; With the specific activity assay method, labelled antibody is carried out calculating and the analysis of mark rate.The standing storage labelled antibody can join highly purified BSA in labelled antibody solution, and the final concentration of BSA is 0.1%, can be placed in 4 ℃ or-20 ℃ and be preserved.
The preparation method of analysis buffer is: to every liter of concentration, be to add 9g NaCl, 0.5g NaN in 0.05mol/L, the pH value Tris-Hcl damping fluid that is 8.0
3, 10g BSA, 0.1ml Tween-40,5g polyglycol, mix, make analysis buffer.
The preparation method who shares fluorescence-enhancing agent is: the part of dissociating: the ethanol of the Triton X-100 of the yttria of the PTA of 100 μ mol, 4 μ mol, 0.6g and 300ml is mixed, and water is settled to 1 liter, with acetic acid adjust pH to 3.5, makes the part of dissociating; Strengthen part: the Tris of the Phen of 0.3mmol and 0.2mol is mixed, and water is settled to 1 liter, makes the enhancing part.
The preparation method of cleansing solution is: be to add Tween-20 in 0.01mol/L, the pH value PBS damping fluid that is 7.4 in concentration, mix, the PBS solution that to make the Tween-20 mass percent concentration be 0.05%, the NaN that is then 0.2% with mass percent concentration
3the solution preservative treatment, make cleansing solution.
The potpourri that quality-control product is CA724, CEA, CA199 standard items.The CA724 self-control, CEA is purchased from Abnova company, and CA199 is purchased from Acris company.
Embodiment 2
The application of the kit that the embodiment of the present invention 1 provides in comprehensive detection stomach neoplasms tumor markers CA724, CEA, CA199, the kit that adopts the embodiment of the present invention 1 to provide, while comprehensive detection stomach neoplasms tumor markers CA724, CEA, CA199 time-resolved fluoroimmunoassay detection method, its reaction principle as shown in Figure 1, adopt double antibody sandwich method, comprise the following steps:
(1) dilute CA724, CEA, CA199 protein standard substance potpourri by analysis buffer, quality-control product, make and have the mixed solution that the series concentration gradient includes CA724, CEA, CA199 protein standard substance;
(2) get respectively protein standard substance mixed solution prepared by 100 μ l testing sample human serums and step (1), add antibody C7189, ab3982, in the different holes of the microwell plate that the mixed antibody that tri-kinds of monoclonal antibodies of MAM02-008 form is coated, 37 ℃ of incubations 1 hour, (during washing, the compound method of cleansing solution used is: in every liter of PBS damping fluid, add 9g sodium chloride in three washings, 0.5ml Tween-20, shake up, get final product), add afterwards 100 μ l mixed mark antibody working fluids (by analysis buffer, with volume ratio 1:3000, to dilute mixed mark antibody storage liquid, make mixed mark antibody working fluid, contain Eu in this mixed mark antibody working fluid of every 100 μ l
3+c7164 antibody 50ng, the Tb of mark
3+ab116024 antibody 50ng, the Sm of mark
3+the MAM02-881 antibody 70ng of mark, all the other are analysis buffer), concussion evenly, 37 ℃ of incubations 1 hour, with cleansing solution, wash 5 times, on thieving paper, control is done, and adds 200 μ l to share the part of dissociating of fluorescence-enhancing agent, shakes 5 minutes, add 20 μ l to share the enhancing part of fluorescence-enhancing agent, shake 8 minutes, on the time-resolved fluorescence instrument, measure fluorescence intensity, excitation wavelength is 315nm, 647nm, 612nm, 544nm are respectively Sm
3+, Eu
3+, Tb
3+the strongest emission photopeak, 500 microseconds, 200 microseconds, 50 microseconds are respectively Eu
3+, Tb
3+, Sm
3+time delay, measure respectively luminous value separately.Each index in the CA724 of variable concentrations, CEA, CA199 albumen hybrid standard product respectively and luminous value separately do typical curve, obtain the typical curve separately of different indexs, the concentration of testing sample human serum calculates by typical curve.Obtain the precision of kit: CA724 analyze in and analyze between the coefficient of variation be respectively 2.9%～4.8%, 4.1%～6.5%; The coefficient of variation that CA199 analyzes between interior and analysis is respectively 3.9%～6.7%, 4.8%～7.6%; The coefficient of variation that CEA analyzes between interior and analysis is respectively 4.7%～7.5%, 5.9%～8.9%.
Embodiment 3
Detect 40 parts of CA724 that serum sample obtains with kit of the present invention, CEA, the numerical value of tri-indexs of CA199 and with 40 parts of same serum samples respectively with the single index kit of Shenzhen new industry company test CA724, the single index kit test CA199 of Shenzhen new industry company, the resulting data of single index kit test CEA of Abbott company are done respectively the recurrence correlation analysis, difference respective figure 2, 3, 4, from accompanying drawing, can find out, each achievement data that kit test of the present invention obtains and the data that obtain with the test of single index kit respectively have good correlativity.
Claims (10)
1. a time-resolved fluorescence method comprehensive detection cancer of the stomach kit, three kinds of stomach neoplasms tumor markers CA724, CEA, CA199 comprehensively in the same reaction system of same micropore, the employing time-resolved fluoroimmunoassay detects, and it is characterized in that: this kit comprises: anti-CA724, CEA, the coated microwell plate of CA199 the first strain monoclonal antibody mixed antibody respectively; Use Eu
3+anti-CA724, the Tb of mark
3+anti-CA199, the Sm of mark
3+the anti-CEA second strain monoclonal antibody mixed antibody of mark; Analysis buffer; Share fluorescence-enhancing agent; Cleansing solution; Quality-control product.
2. time-resolved fluorescence method comprehensive detection cancer of the stomach kit according to claim 1, it is characterized in that: the preparation method of the microwell plate that described the first strain monoclonal antibody mixed antibody is coated is: anti-CA724 respectively, CEA, coated damping fluid dilution for CA199 the first strain monoclonal antibody mixed antibody, make the antibody coating buffer, then to the described antibody coating buffer that adds respectively 100 μ l in each hole of microwell plate, be coated with 2 hours in 37 ℃, then use the normal saline flushing microwell plate three times, and then to the shrouding liquid that adds respectively 200 μ l in each hole of microwell plate, seal 2 hours or spend the night in 4 ℃ of sealings in room temperature, then use twice of normal saline flushing microwell plate, freeze drying, make the coated microwell plate of mixed antibody.
3. time-resolved fluorescence method comprehensive detection cancer of the stomach kit according to claim 2, it is characterized in that: in described antibody coating buffer, the concentration of the first strain monoclonal antibody of anti-CA724, CEA, CA199 is 0.010～0.019g/L respectively.
4. time-resolved fluorescence method comprehensive detection cancer of the stomach kit according to claim 1, is characterized in that: the described Eu of using
3+anti-CA724, the Tb of mark
3+anti-CA199, the Sm of mark
3+in the anti-CEA second strain monoclonal antibody mixed antibody of mark, use Eu
3+the anti-CA724 antibody of mark, Tb
3+the anti-CA199 antibody of mark, Sm
3+the quality proportioning of the anti-CEA antibody of mark is: Eu
3+the anti-CA724 antibody of mark: Tb
3+the anti-CA199 antibody of mark: Sm
3+anti-CEA antibody=(3～6) of mark: (3～7): (5～9).
5. time-resolved fluorescence method comprehensive detection cancer of the stomach kit according to claim 1, it is characterized in that: the preparation method of described shared fluorescence-enhancing agent is: the part of dissociating: the ethanol of the Triton X-100 of the yttria of the PTA of 70～200 μ mol, 2～7 μ mol, 0.6g and 300ml is mixed, water is settled to 1 liter, with acetic acid adjust pH to 3.5, make the part of dissociating; Strengthen part: the Tris of the Phen of 0.1～0.4mmol and 0.2mol is mixed, and water is settled to 1L, makes the enhancing part.
6. time-resolved fluorescence method comprehensive detection cancer of the stomach kit according to claim 1, it is characterized in that: the preparation method of described cleansing solution is: in concentration, be to add Tween-20 in 0.01mol/L, the pH value PBS damping fluid that is 7.4, mix, the PBS solution that to make the Tween-20 mass percent concentration be 0.05%, the NaN that is then 0.2% with mass percent concentration
3the solution preservative treatment, make cleansing solution.
7. time-resolved fluorescence method comprehensive detection cancer of the stomach kit according to claim 1, is characterized in that: the potpourri that described quality-control product is CA724, CEA, CA199 standard items.
8. the application of the arbitrary described time-resolved fluorescence method comprehensive detection cancer of the stomach kit of claim 1-7 in comprehensive detection stomach neoplasms tumor markers CA724, CEA, CA199.
9. the application of kit according to claim 8 in comprehensive detection stomach neoplasms tumor markers CA724, CEA, CA199, is characterized in that, comprises the following steps:
(1) dilute quality-control product by analysis buffer, make and there is the mixed solution that the series concentration gradient includes CA724, CEA, CA199 standard items;
(2) the standard items mixed solution prepared from step (1) by testing sample adds respectively in the different holes of the coated microwell plate of anti-CA724, CEA, CA199 the first strain monoclonal antibody mixed antibody, and then Xiang Kongzhong adds the second strain monoclonal antibody mixed antibody that is marked with the rare earth ion chelate that can be combined with CA724, CEA, CA199 respectively, add afterwards shared fluorescence-enhancing agent, carry out the time-resolved fluoroimmunoassay detection, obtain luminous value;
(3) do typical curve according to the luminous value of the mixed solution that comprises CA724, CEA, CA199 standard items with series concentration gradient of measuring;
(4) luminous value with CA724, CEA, CA199 typical curve separately in CA724, CEA, CA199 luminous value and standard items in the testing sample of measuring compares, and draws CA724 in testing sample, CEA, CA199 content separately.
10. the application of kit according to claim 9 in comprehensive detection stomach neoplasms tumor markers CA724, CEA, CA199 is characterized in that: described testing sample is human serum.
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| CN201210510566XA CN103116025A (en) | 2012-09-20 | 2012-12-04 | Kit for comprehensive detection of gastric cancer by means of time-resolved fluorescence method and application thereof |
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| 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 |
| CN109541207A (en) * | 2017-09-21 | 2019-03-29 | 苏州新波生物技术有限公司 | A kind of diagnostic kit and preparation method thereof detecting CA72-4 |
| CN110484624A (en) * | 2019-08-28 | 2019-11-22 | 南京大学 | A kind of gastric cancer biomarker and its detection method and application based on peripheral blood |
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