CN103105384A - Time-resolved fluorescence comprehensive detection breast cancer kit and application thereof - Google Patents
Time-resolved fluorescence comprehensive detection breast cancer kit and application thereof Download PDFInfo
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- CN103105384A CN103105384A CN2012105127300A CN201210512730A CN103105384A CN 103105384 A CN103105384 A CN 103105384A CN 2012105127300 A CN2012105127300 A CN 2012105127300A CN 201210512730 A CN201210512730 A CN 201210512730A CN 103105384 A CN103105384 A CN 103105384A
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Abstract
The invention relates to the technical field of biological and medical detection and in particular relates to a time-resolved fluorescence comprehensive detection breast cancer kit for simultaneously and comprehensively detecting breast cancer tumor marker indexes such as CA125, CEA and CA153 in the same reaction system, and also relates to application of the kit in comprehensive detection of breast cancer tumor markers CA125, CEA and CA153. The kit comprises a porous plate coated by a first monoclonal antibody mixed antibody which resists CA125, CEA and CA153, a second monoclonal antibody mixed antibody for Eu3+ marked anti-CA125, Tb3+ marked anti-CEA and Sm3+ marked anti-CA153, an analytical buffer solution, a common fluorescent enhancement agent, a scrubbing solution and a quality control. The provided kit can be used for clinical auxiliary diagnosis, curative effect observation and prognosis of breast cancer and has significance for treating and preventing the breast cancer tumor; and meanwhile, three tumor markers are detected, the detection steps are simplified, and the specificity and sensitivity of comprehensive analysis of detected data are 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 breast cancer tumour mark index CA125, CEA, CA153 simultaneously, also relate to the application of this kit in comprehensive detection breast cancer tumour mark CA125, CEA, CA153 simultaneously.
Background technology
The pathogenesis of breast carcinoma age distribution is different in country between east and west, in district occurred frequently as countries such as Northern Europe, North Americas, breast cancer was since appearance in about 20 years old, kept the fast rise impetus before being 45-50 year climacteric, approximately the incidence of disease rising of every growths 10-20 of age year is 1 times, after climacteric, rise relatively slow, 75-85 year reaches the highest.And in Asia etc. the low area of sending out, the incidence of disease of breast cancer can slightly descend after menopause, the onset peak of general breast cancer is between 45-55 year, the Asian still keeps this Characteristics of age distribution after migrating western countries.Early detection, making a definite diagnosis breast cancer, implement excision, is still the most effective methods for the treatment of of this disease at present.Therefore, make a definite diagnosis in early days, more accurately the key that breast cancer is still this disease for the treatment of.
At present, the detection index commonly used of diagnosing mammary cancer has CA125, CEA, CA153 etc. several.When actual diagnosing mammary cancer, be generally to make a definite diagnosis breast cancer with CA125, CEA, tri-kinds of index comprehensives of CA153 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 breast cancer tumour mark index CA125, CEA, CA153 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 breast cancer kit, three kinds of breast cancer tumour mark CA125, CEA, CA153 comprehensively in the same reaction system of same micropore, the employing time-resolved fluoroimmunoassay detects, and this kit comprises: difference anti-CA 125, CEA, the coated microwell plate of CA153 the first strain monoclonal antibody mixed antibody; Use Eu
3+the anti-CA 125 of mark, Tb
3+anti-CEA, the Sm of mark
3+the anti-CA153 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: the difference anti-CA 125, CEA, coated damping fluid dilution for CA153 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, in room temperature sealing 2 hours, 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-CA 125, CEA, CA153 is 0.009～0.014g/L respectively.
The described Eu that uses
3+the anti-CA 125 of mark, Tb
3+anti-CEA, the Sm of mark
3+in the anti-CA153 second strain monoclonal antibody mixed antibody of mark, use Eu
3+the anti-CA 125 antibody of mark, Tb
3+the anti-CEA antibody of mark, Sm
3+the quality proportioning of the anti-CA153 antibody of mark is: Eu
3+the anti-CA 125 antibody of mark: Tb
3+the anti-CEA antibody of mark: Sm
3+anti-CA153 antibody=(7～13) of mark: (6～11): (6.5～13).
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 CA125, CEA, CA153 standard items.
The application of a kind of time-resolved fluorescence method comprehensive detection breast cancer kit in comprehensive detection breast cancer tumour mark CA125, CEA, CA153.
The application of described kit in comprehensive detection breast cancer tumour mark CA125, CEA, CA153 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 CA125, CEA, CA153 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-CA 125, CEA, CA153 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 CA125, CEA, CA153 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 CA125, CEA, CA153 standard items with series concentration gradient of measuring;
(4) luminous value with CA125, CEA, CA153 typical curve separately in CA125, CEA, CA153 luminous value and standard items in the testing sample of measuring compares, and draws CA125 in testing sample, CEA, CA153 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 0.05mol/L, pH value are 8.5, 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 0.02mol/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 three breast cancer tumour mark CA125 of comprehensive detection, CEA, CA153 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.
Breast cancer tumour mark CA125 provided by the invention, CEA, CA153 time-resolved fluoroimmunoassay detection kit, have the extraordinary range of linearity, and the range of linearity that detects CA125 is 25U/ml～660U/ml, detects and be limited to 10U/ml; The range of linearity that detects CEA is 3ng/ml～580ng/ml, detects and is limited to 1ng/ml; The range of linearity that detects CA153 is 20U/ml～300U/ml, detects and is limited to 5U/ml.Each normal reference value that detects index is respectively: CA125 is 0～35U/ml; CEA is 0～6.5ng/ml; CA153 is 0～30U/ml.Breast cancer tumour mark CA125 provided by the invention, CEA, CA153 time-resolved fluoroimmunoassay detection kit can be used for clinical assistant diagnosis, observation of curative effect and the prognosis judgement of breast cancer, to the treatment of breast cancer tumour with prevent significant.
Adopt kit provided by the invention can realize in same reaction system comprehensive detection breast cancer tumour mark index CA125, CEA, CA153 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 CA125, CEA, CA153, 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;
The kit that Fig. 2 provides for the embodiment of the present invention 1 and the single index kit of Roche Holding Ag test CA125 correlation regression analysis chart;
The kit that Fig. 3 provides for the embodiment of the present invention 1 and the single index kit of Abbott company test CEA correlation regression analysis chart;
The kit that Fig. 4 provides for the embodiment of the present invention 1 and the single index kit of Beijing Re Jing biotech firm test CA153 correlation regression analysis chart.
Embodiment
Below by specific embodiment, technical scheme of the present invention is elaborated.
Embodiment 1
Breast cancer tumour mark CA125, the CEA that the present embodiment provides, CA153 time-resolved fluoroimmunoassay detection kit comprise: the coated microwell plate of mixed antibody that tri-kinds of monoclonal antibodies of the antibody ab28081 of the antibody MAM02-008 of the antibody M32112M of anti-CA 125, anti-CEA, anti-CA153 form; Use Eu
3+the M86306M antibody of mark (antibody of anti-CA 125), use Tb
3+the MAM02-881 antibody of mark (antibody of anti-CEA), use Sm
3+the mixed antibody that three kinds of monoclonal antibodies of the ab70475 antibody of mark (antibody of anti-CA153) form; Analysis buffer; Share fluorescence-enhancing agent; Cleansing solution; CA125, CEA, tri-kinds of standard items of CA153 mix the quality-control product formed.This kit also comprises other related reagents such as negative control, positive control, substrate.
In described kit, antibody ab28081, antibody ab70475 are purchased from abcam company; Antibody M32112M, antibody M86306M and antibody MAM02-008, antibody MAM02-881 are all purchased from Meridian company.
M32112M, MAM02-008, the preparation method of the microwell plate that the ab28081 mixed antibody is coated is: coated damping fluid dilution for mixed antibody, make the mixed antibody coating buffer, in the mixed antibody coating buffer, the difference anti-CA 125, CEA, the concentration of the first strain monoclonal antibody of CA153 is 0.010g/L, be in the mixed antibody coating buffer, antibody M32112M, MAM02-008, the concentration of ab28081 is 0.010g/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 0.05mol/L, pH value are 8.5, 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 0.02mol/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 M86306M antibody of mark, use Tb
3+the MAM02-881 antibody of mark, use Sm
3+the preparation method of the mixed antibody that three kinds of monoclonal antibodies of the ab70475 antibody of mark form is: by Eu
3+the M86306M antibody of mark, Tb
3+the MAM02-881 antibody of mark, Sm
3+the ab70475 antibody of mark is with mass ratio: Eu
3+the M86306M antibody of mark: Tb
3+the MAM02-881 antibody of mark: Sm
3+the ab70475 antibody of mark=10:8:11 mixes, and makes mixed antibody.
Wherein use Eu
3+the M86306M antibody of mark, use Tb
3+the MAM02-881 antibody of mark, use Sm
3+the preparation method of the ab70475 antibody of mark is: the mark process step of every kind of antibody is identical, just respectively the antibody of anti-CA 125, CEA, CA153 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.The N1-of below take is example to the M86306M 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 M86306M antibody, and concrete steps are as follows: the unsettled 1mg M86306M 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 M86306M 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 M86306M to be marked, mix; Be placed on shaking table 4 ℃ and spend the night, make the antibody M86306M that mark is good, use Eu
3+the M86306M 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 M86306M 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 0.1mmol 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 CA125, CEA, CA153 standard items.CA125, CEA are purchased from Abnova company, and CA153 is purchased from abcam company.
Embodiment 2
The application of the kit that the embodiment of the present invention 1 provides in comprehensive detection breast cancer tumour mark CA125, CEA, CA153, the kit that adopts the embodiment of the present invention 1 to provide, while comprehensive detection breast cancer tumour mark CA125, CEA, CA153 time-resolved fluoroimmunoassay detection method, its reaction principle as shown in Figure 1, adopt double antibody sandwich method, comprise the following steps:
(1) dilute CA125, CEA, CA153 protein standard substance potpourri by analysis buffer, quality-control product, make and have the mixed solution that the series concentration gradient includes CA125, CEA, CA153 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 M32112M, antibody MAM02-008, in the different holes of the microwell plate that the mixed antibody that tri-kinds of monoclonal antibodies of antibody ab28081 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+m86306M antibody 100ng, the Tb of mark
3+mAM02-881 antibody 80ng, the Sm of mark
3+the ab70475 antibody 110ng 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 CA125 of variable concentrations, CEA, CA153 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: CA125 analyze in and analyze between the coefficient of variation be respectively 3.9%～5.8%, 5.1%～7.8%; The coefficient of variation that CEA analyzes between interior and analysis is respectively 3.9%～5.9%, 4.7%～8.3%; The coefficient of variation that CA153 analyzes between interior and analysis is respectively 4.4%～6.5%, 6.0%～8.9%.
Detect 40 parts of CA125 that serum sample obtains with kit of the present invention, CEA, the numerical value of tri-indexs of CA153 and with 40 parts of same serum samples respectively with the single index kit of Roche Holding Ag test CA125, the single index kit test CEA of Abbott company, the resulting data of single index kit test CA153 of Beijing Re Jing biotech firm 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 breast cancer kit, three kinds of breast cancer tumour mark CA125, CEA, CA153 comprehensively in the same reaction system of same micropore, the employing time-resolved fluoroimmunoassay detects, and it is characterized in that: this kit comprises: difference anti-CA 125, CEA, the coated microwell plate of CA153 the first strain monoclonal antibody mixed antibody; Use Eu
3+the anti-CA 125 of mark, Tb
3+anti-CEA, the Sm of mark
3+the anti-CA153 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 breast cancer 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: the difference anti-CA 125, CEA, coated damping fluid dilution for CA153 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, 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.
3. time-resolved fluorescence method comprehensive detection breast cancer 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-CA 125, CEA, CA153 is 0.009～0.014g/L respectively.
4. time-resolved fluorescence method comprehensive detection breast cancer kit according to claim 1, is characterized in that: the described Eu of using
3+the anti-CA 125 of mark, Tb
3+anti-CEA, the Sm of mark
3+in the anti-CA153 second strain monoclonal antibody mixed antibody of mark, use Eu
3+the anti-CA 125 antibody of mark, Tb
3+the anti-CEA antibody of mark, Sm
3+the quality proportioning of the anti-CA153 antibody of mark is: Eu
3+the anti-CA 125 antibody of mark: Tb
3+the anti-CEA antibody of mark: Sm
3+anti-CA153 antibody=(7～13) of mark: (6～11): (6.5～13).
5. time-resolved fluorescence method comprehensive detection breast cancer 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 breast cancer 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 breast cancer kit according to claim 1, is characterized in that: the potpourri that described quality-control product is CA125, CEA, CA153 standard items.
8. the application of the arbitrary described time-resolved fluorescence method comprehensive detection breast cancer kit of claim 1-7 in comprehensive detection breast cancer tumour mark CA125, CEA, CA153.
9. the application of kit according to claim 8 in comprehensive detection breast cancer tumour mark CA125, CEA, CA153, 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 CA125, CEA, CA153 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-CA 125, CEA, CA153 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 CA125, CEA, CA153 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 CA125, CEA, CA153 standard items with series concentration gradient of measuring;
(4) luminous value with CA125, CEA, CA153 typical curve separately in CA125, CEA, CA153 luminous value and standard items in the testing sample of measuring compares, and draws CA125 in testing sample, CEA, CA153 content separately.
10. the application of kit according to claim 9 in comprehensive detection breast cancer tumour mark CA125, CEA, CA153, it is characterized in that: described testing sample is human serum.
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| CN104155449A (en) * | 2014-07-28 | 2014-11-19 | 广州市丰华生物工程有限公司 | Method and kit for detecting TORCH IgM antibodies and preparation method of kit |
| CN106461557A (en) * | 2014-06-27 | 2017-02-22 | 沃拉克有限公司 | A device and a method for detecting a sample contained by a sample well |
| CN111089968A (en) * | 2019-07-16 | 2020-05-01 | 南方医科大学 | Multi-index time-resolved fluorescence immunoassay detection method of single detection line |
| CN113009138A (en) * | 2020-06-17 | 2021-06-22 | 山东大学 | Kit and method for detecting breast cancer tumor marker |
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| CN113009138A (en) * | 2020-06-17 | 2021-06-22 | 山东大学 | Kit and method for detecting breast cancer tumor marker |
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