CN103149359A - Time resolution fluorescence method comprehensive detection pancreatic cancer kit and application thereof - Google Patents
Time resolution fluorescence method comprehensive detection pancreatic cancer kit and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biology and medical science detection, in particular to a detection kit of comprehensive detection pancreatic cancer tumor markers crux armature (CA) 242, carcinoma embryonic antigen (CEA), CA 50, CA 199 in the same reaction system and further relates to the application for detecting pancreatic cancer tumor markers CA 242, CEA, CA 50, CA 199 in the kit. The diction kit comprises micro-well plates and mixed antibodies and the like, wherein the micro-well plates coats respectively a first monoclonal antibody mixed antibodies for preventing CA 242, CEA, CA 50, CA 199, the mixed antibodies of a second monoclonal antibody for preventing CA 242, CEA, CA 50, CA 199are marked by Eu3+, Tb3+, Sm3+ and Dy3+. The detection kit can be used for clinical auxiliary diagnosis, curative effect observation and prognosis of the pancreatic cancer and capable of detecting for tumor markers at the same time and simplifying detecting steps and improving specificity and sensitivity of test data comprehensive analysis.
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 pancreatic tumour mark index CA242, CA50, CEA, CA199 simultaneously, also relate to the application of this kit in comprehensive detection pancreatic tumour mark CA242, CA50, CEA, CA199 simultaneously.
Background technology
Cancer of pancreas is one of common malignant tumour of alimentary canal, due to the typical clinical manifestation of early stage shortage, has belonged to middle and advanced stage when the patient more than 90% makes a definite diagnosis clinically, loses best occasion for the treatment, and its 5 years survival rates are only 5%.In the common cancer in the world, the male sex suffers from cancer of pancreas and occupies the 4th, the world, and the women occupies the 5th, and the prognosis weak effect.Study hotspot is to seek specific early diagnosis tumor markers at present, realizes the early diagnosis to cancer of pancreas, improves the survival rate of Pancreas cancer patients, and making a definite diagnosis in early days, more accurately cancer of pancreas is still the key of this disease for the treatment of.
At present, the detection index commonly used of diagnosis of pancreatic cancer has CA242, CA50, CEA, CA199 etc. several.When actual diagnosis of pancreatic cancer, be generally to make a definite diagnosis cancer of pancreas with CA242, CA50, CEA, tetra-kinds of index comprehensives of CA199 because four kinds of comprehensive analyses on specificity and susceptibility apparently higher than single index.Routine Test Lab or medical institutions adopt the single agents box usually for detection of this four indices, and 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, current four kinds of labels adopt respectively four kinds of single index kits to be detected, and then comprehensively judge, 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 four kinds of labels of analysis-by-synthesis and susceptibility, may cause judged result to differ greatly.Therefore, develop and a kind ofly can under relatively same testing conditions, detect the kit of these four kinds of pancreatic tumour labels 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 pancreatic tumour mark index CA242, CA50, 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 pancreas kit, four kinds of pancreatic tumour mark CA242, CA50, CEA, CA199 comprehensively in the same reaction system of same micropore, the employing time-resolved fluoroimmunoassay detects, and this kit comprises: anti-CA242, CA50, CEA, the coated microwell plate of CA199 the first strain monoclonal antibody mixed antibody respectively; Use Eu
3+anti-CA242, the Tb of mark
3+anti-CA50, the Sm of mark
3+anti-CA199, the Dy 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.
Wherein, the preparation method of the microwell plate that described the first strain monoclonal antibody mixed antibody is coated is: anti-CA242 respectively, CA50, 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 be placed in 4 ℃ of environment in room temperature and spend the night, then use twice of normal saline flushing microwell plate, freeze drying, make the coated microwell plate of the first strain monoclonal antibody mixed antibody.
In described antibody coating buffer, the concentration of the first strain monoclonal antibody of anti-CA242, CA50, CEA, CA199 is 0.009～0.017g/L respectively.
The described Eu that uses
3+anti-CA242, the Tb of mark
3+anti-CA50, the Sm of mark
3+anti-CA199, the Dy of mark
3+in the anti-CEA second strain monoclonal antibody mixed antibody of mark, use Eu
3+the anti-CA242 antibody of mark, Tb
3+the anti-CA50 antibody of mark, Sm
3+the anti-CA199 antibody of mark, Dy
3+the quality proportioning of the anti-CEA antibody of mark is: Eu
3+the anti-CA242 antibody of mark: Tb
3+the anti-CA50 antibody of mark: Sm
3+the anti-CA199 antibody of mark: Dy
3+anti-CEA antibody=(2～6) of mark: (5～9): (6～11): (7～10).
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 CA242, CA50, CEA, CA199 standard items.
The application of a kind of time-resolved fluorescence method comprehensive detection cancer of pancreas kit in comprehensive detection pancreatic tumour mark CA242, CA50, CEA, CA199.
The application of time-resolved fluorescence method comprehensive detection cancer of pancreas kit in comprehensive detection pancreatic tumour mark CA242, CA50, 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 CA242, CA50, CEA, tetra-kinds of standard items of CA199;
(2) four kinds of standard items mixed solutions that prepared from step (1) by testing sample add respectively in the different holes of the coated microwell plate of anti-CA242, CA50, 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 CA242, CA50, 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 CA242, CA50, CEA, tetra-kinds of standard items of CA199 with series concentration gradient of measuring;
(4) luminous value with CA242, CA50, CEA, CA199 typical curve separately in CA242, CA50, CEA, CA199 luminous value and standard items in the testing sample of measuring compares, and draws CA242 in testing sample, CA50, CEA, CA199 content separately.
Wherein, described testing sample is human serum.
Further, the preparation method of described coated damping fluid is: getting concentration is the carbonate buffer solution that 0.03mol/L, pH value are 9.0, 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 0.01mol/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 pancreatic tumour mark CA242, CA50, 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.
Pancreatic tumour mark CA242 provided by the invention, CA50, CEA, CA199 time-resolved fluoroimmunoassay detection kit, have the extraordinary range of linearity, and the range of linearity that detects CA242 is 10U/ml～400U/ml, detects and be limited to 3U/ml; The range of linearity that detects CA50 is 15U/ml～300U/ml, detects and is limited to 7U/ml; The range of linearity that detects CEA is 1.0ng/ml～100ng/ml, detects and is limited to 0.5ng/ml; The range of linearity that detects CA199 is 15U/ml～350U/ml, detects and is limited to 5U/ml.Each normal reference value that detects index is respectively: CA242 is 0～22U/ml; CA50 is 0～25U/ml; CEA is 0～5ng/ml; CA199 is 0～37U/ml.Pancreatic tumour mark CA242 provided by the invention, CA50, 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 pancreas, to the treatment of pancreatic tumour with prevent significant.
Adopt kit provided by the invention can realize in same reaction system comprehensive detection pancreatic tumour mark four indices CA242, CA50, CEA, CA199 simultaneously, detect simple to operately, step is few, and required time is short, convenient and swift; Testing result is accurate, avoided four kinds of individual event kits of existing employing to detect respectively that four indices CA242, CA50, CEA, CA199 comprehensively judge again and the error in judgement that causes, 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;
The kit that Fig. 2 provides for the embodiment of the present invention 1 and the single index kit of CanAg company test CA242 correlation regression analysis chart;
Fig. 3 tests CA50 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. 4 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. 5 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
The time-resolved fluorescence method comprehensive detection cancer of pancreas kit that the present embodiment provides comprises: the coated microwell plate of mixed antibody that tetra-kinds of monoclonal antibodies of the antibody MAM02-008 of the antibody ab3982 of the antibody A M09238PU-N of the antibody C3918 of anti-CA242, anti-CA50, anti-CA199, anti-CEA form; Use Eu
3+the C3624 antibody of mark (antibody of anti-CA242), use Tb
3+the AM09239PU-N antibody of mark (antibody of anti-CA50), use Sm
3+the ab116024 antibody of mark (antibody of anti-CA199), use Dy
3+the mixed antibody that four kinds of monoclonal antibodies of the MAM02-881 antibody of mark (antibody of anti-CEA) form; Analysis buffer; Share fluorescence-enhancing agent; Cleansing solution; CA242, CA50, CEA, tetra-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 the kit that the present embodiment provides, anti-CA242 antibody C3918, C3624 are self-control; Anti-CA199 antibody ab3982, ab116024 are purchased from Abcam company; The antibody A M09238PU-N of anti-CA50, AM09239PU-N are purchased from Acris company; Anti-CEA antibody MAM02-008, MAM02-881 antibody are purchased from Meridian company.
C3918, AM09238PU-N, ab3982, the preparation method of the microwell plate that the mixed antibody that tetra-kinds of monoclonal antibodies of MAM02-008 form is coated is: coated damping fluid dilution for mixed antibody, make the mixed antibody coating buffer, in the mixed antibody coating buffer, anti-CA242 respectively, CA50, CEA, the concentration of the first strain monoclonal antibody of CA199 is 0.012g/L, be in the mixed antibody coating buffer, antibody C3918, AM09238PU-N, ab3982, the concentration of MAM02-008 is 0.012g/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 0.03mol/L, pH value are 9.0, 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 0.01mol/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 C3624 antibody of mark, use Tb
3+the AM09239PU-N antibody of mark, use Sm
3+the ab116024 antibody of mark, use Dy
3+the preparation method of the mixed antibody that four kinds of monoclonal antibodies of the MAM02-881 antibody of mark form is: by Eu
3+the C3624 antibody of mark, Tb
3+the AM09239PU-N antibody of mark, Sm
3+ab 116024 antibody of mark, Dy
3+the MAM02-881 antibody of mark is with mass ratio: Eu
3+the C3624 antibody of mark: Tb
3+the AM09239PU-N antibody of mark: Sm
3+the ab116024 antibody of mark: Dy
3+the MAM02-881 antibody of mark=4:8:10:9 mixes, and makes mixed antibody.
Wherein use Eu
3+the C3624 antibody of mark, use Tb
3+the AM09239PU-N antibody of mark, use Sm
3+the ab116024 antibody of mark, use Dy
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-CA242, CA50, CA199, CEA respectively corresponding rare earth ion chelate label be N1-to isothiocyanic acid benzyl-DTTA-Eu, N1-to isothiocyanic acid benzyl-DTTA-Tb, N1-to isothiocyanic acid benzyl-DTTA-Sm, N1-to isothiocyanic acid benzyl-DTTA-Dy.The N1-of below take is example to the isothiocyanic acid benzyl-anti-CA242 antibody of DTTA-Eu mark C3624, and its concrete preparation method and step are:
(1) centrifugal column that the application molecular cut off is 50kDa before mark carries out purification process before mark to C3624 antibody, and concrete steps are as follows: the unsettled 1mg C3624 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 antibody C3624 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 C3624 to be marked, mix; Be placed on shaking table 4 ℃ and spend the night, make the antibody C3624 that mark is good, use Eu
3+the C3624 antibody of mark;
, draw and use Eu with pipettor as column equilibration liquid balance Superdex 2001 * 30cm post with the PBS damping fluid of three times of column volumes
3+the slow loading of C3624 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, 7 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 CA242, CA50, CEA, CA199 standard items.The CA242 standard items are purchased from Meridian company, and the CA50 standard items are purchased from Fitzgerald company, and the CEA standard items are purchased from Abnova company, and the CA199 standard items are purchased from Acris company.
Embodiment 2
The application of the kit that the embodiment of the present invention 1 provides in comprehensive detection pancreatic tumour mark CA242, CA50, CEA, CA199, the kit that adopts the embodiment of the present invention 1 to provide, while comprehensive detection pancreatic tumour mark CA242, CA50, 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 CA242, CA50, CEA, tetra-kinds of protein standard substance potpourris of CA199 by analysis buffer, quality-control product, make and have the mixed solution that the series concentration gradient includes CA242, CA50, CEA, tetra-kinds of protein standard substances of CA199;
(2) get respectively four kinds of protein standard substance mixed solutions prepared by 100 μ l testing sample human serums and step (1), add C3918, AM09238PU-N, ab3982, in the different holes of the microwell plate that the mixed antibody that tetra-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 1:3500, to dilute mixed mark antibody storage liquid, make mixed mark antibody working fluid, contain useful Eu in this mixed mark antibody working fluid of every 100 μ l
3+the C3624 antibody 40ng of mark, use Tb
3+the AM09239PU-N antibody 80ng of mark, use Sm
3+the ab116024 antibody 100ng of mark, use Dy
3+the MAM02-881 antibody 90ng 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, 575nm are respectively Sm
3+, Eu
3+, Tb
3+, Dy
3+the strongest emission photopeak, 500 microseconds, 200 microseconds, 50 microseconds, 20 microseconds are respectively Eu
3+, Tb
3+, Sm
3+, Dy
3+time delay, measure respectively luminous value separately.Each index in the CA242 of variable concentrations, CA50, CEA, tetra-kinds of albumen hybrid standard product of CA199 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: CA242 analyze in and analyze between the coefficient of variation be respectively 3.5%～6.0%, 5.0%～7.3%; The coefficient of variation that CA50 analyzes between interior and analysis is respectively 4.4%～7.8%, 5.9%～8.6%; The coefficient of variation that CA199 analyzes between interior and analysis is respectively 3.3%～5.8%, 4.2%～6.9%; The coefficient of variation that CEA analyzes between interior and analysis is respectively 3.5%～6.0%, 5.4%～8.1%.
Embodiment 3
Detect 40 parts of CA242 that serum sample obtains with kit of the present invention, CA50, CEA, the numerical value of tetra-indexs of CA199 and with 40 parts of same serum samples respectively with the single index kit of CanAg company test CA242, the single index kit test CA50 of Shenzhen new industry company, 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 2-5, 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 pancreas kit, four kinds of pancreatic tumour mark CA242, CA50, 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-CA242, CA50, CEA, the coated microwell plate of CA199 the first strain monoclonal antibody mixed antibody respectively; Use Eu
3+anti-CA242, the Tb of mark
3+anti-CA50, the Sm of mark
3+anti-CA199, the Dy 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 pancreas 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-CA242 respectively, CA50, 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 be placed in 4 ℃ of environment in room temperature and spend the night, then use twice of normal saline flushing microwell plate, freeze drying, make the coated microwell plate of the first strain monoclonal antibody mixed antibody.
3. time-resolved fluorescence method comprehensive detection cancer of pancreas 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-CA242, CA50, CEA, CA199 is 0.009～0.017g/L respectively.
4. time-resolved fluorescence method comprehensive detection cancer of pancreas kit according to claim 1, is characterized in that: the described Eu of using
3+anti-CA242, the Tb of mark
3+anti-CA50, the Sm of mark
3+anti-CA199, the Dy of mark
3+in the anti-CEA second strain monoclonal antibody mixed antibody of mark, use Eu
3+the anti-CA242 antibody of mark, Tb
3+the anti-CA50 antibody of mark, Sm
3+the anti-CA199 antibody of mark, Dy
3+the quality proportioning of the anti-CEA antibody of mark is: Eu
3+the anti-CA242 antibody of mark: Tb
3+the anti-CA50 antibody of mark: Sm
3+the anti-CA199 antibody of mark: Dy
3+anti-CEA antibody=(2～6) of mark: (5～9): (6～11): (7～10).
5. time-resolved fluorescence method comprehensive detection cancer of pancreas 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 pancreas 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 pancreas kit according to claim 1, is characterized in that: the potpourri that described quality-control product is CA242, CA50, CEA, CA199 standard items.
8. the application of the arbitrary described time-resolved fluorescence method comprehensive detection cancer of pancreas kit of claim 1-7 in comprehensive detection pancreatic tumour mark CA242, CA50, CEA, CA199.
9. the application of kit according to claim 8 in comprehensive detection pancreatic tumour mark CA242, CA50, 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 CA242, CA50, CEA, tetra-kinds of standard items of CA199;
(2) four kinds of standard items mixed solutions that prepared from step (1) by testing sample add respectively in the different holes of the coated microwell plate of anti-CA242, CA50, 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 CA242, CA50, 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 CA242, CA50, CEA, tetra-kinds of standard items of CA199 with series concentration gradient of measuring;
(4) luminous value with CA242, CA50, CEA, CA199 typical curve separately in CA242, CA50, CEA, CA199 luminous value and standard items in the testing sample of measuring compares, and draws CA242 in testing sample, CA50, CEA, CA199 content separately.
10. the application of kit according to claim 9 in comprehensive detection pancreatic tumour mark CA242, CA50, CEA, CA199, it is characterized in that: described testing sample is human serum.
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| CN2012105105570A CN103149359A (en) | 2012-09-20 | 2012-12-04 | Time resolution fluorescence method comprehensive detection pancreatic cancer kit and application thereof |
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| CN201210349600 | 2012-09-20 | ||
| CN2012105105570A CN103149359A (en) | 2012-09-20 | 2012-12-04 | Time resolution fluorescence method comprehensive detection pancreatic cancer kit and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103789271A (en) * | 2014-01-26 | 2014-05-14 | 东北林业大学 | Hybridoma for resisting generation of CA242 monoclonal antibody and preparation of chemiluminescence immune assay kit |
| CN104280556A (en) * | 2014-10-31 | 2015-01-14 | 杨子学 | Detection method and kit for simultaneously determining content of lipoprotein phospholipase A2 and C reactive protein in blood plasma |
| 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 |
| CN108132347A (en) * | 2018-02-09 | 2018-06-08 | 河南省生物工程技术研究中心有限公司 | The time-resolved fluoroimmunoassay chromatograph test strip and kit of joint-detection CA19-9 and CEA |
| CN109541208A (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 CA24-2 |
| CN109541200A (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 CA50 |
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| CN101241131A (en) * | 2008-02-21 | 2008-08-13 | 江苏省原子医学研究所 | Reagent kit for simultaneously detecting ochratoxin A and aspergillus flavus toxin B1 and its detection method |
| CN102419373A (en) * | 2010-09-28 | 2012-04-18 | 广州市达瑞抗体工程技术有限公司 | Insulin and C peptide double-tagging determination kit |
| CN102507947A (en) * | 2011-11-11 | 2012-06-20 | 南方医科大学 | CEA TRFIA (time-resolved fluoroimmunoassay) kit based on IMB (immunomagnetic beads) |
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| CN101241131A (en) * | 2008-02-21 | 2008-08-13 | 江苏省原子医学研究所 | Reagent kit for simultaneously detecting ochratoxin A and aspergillus flavus toxin B1 and its detection method |
| CN102419373A (en) * | 2010-09-28 | 2012-04-18 | 广州市达瑞抗体工程技术有限公司 | Insulin and C peptide double-tagging determination kit |
| CN102507947A (en) * | 2011-11-11 | 2012-06-20 | 南方医科大学 | CEA TRFIA (time-resolved fluoroimmunoassay) kit based on IMB (immunomagnetic beads) |
Non-Patent Citations (6)
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103789271A (en) * | 2014-01-26 | 2014-05-14 | 东北林业大学 | Hybridoma for resisting generation of CA242 monoclonal antibody and preparation of chemiluminescence immune assay kit |
| CN104280556A (en) * | 2014-10-31 | 2015-01-14 | 杨子学 | Detection method and kit for simultaneously determining content of lipoprotein phospholipase A2 and C reactive protein in blood plasma |
| 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 |
| CN109541208A (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 CA24-2 |
| CN109541200A (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 CA50 |
| CN108132347A (en) * | 2018-02-09 | 2018-06-08 | 河南省生物工程技术研究中心有限公司 | The time-resolved fluoroimmunoassay chromatograph test strip and kit of joint-detection CA19-9 and CEA |
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Application publication date: 20130612 |