CN103123356B - A kind of time-resolved fluorescence method comprehensive detection cancer of the uterus kit and application thereof - Google Patents
A kind of time-resolved fluorescence method comprehensive detection cancer of the uterus kit and application thereof Download PDFInfo
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Abstract
The present invention relates to biology and technical field of medical detection, be specifically related to the time-resolved fluoroimmunoassay detection kit of a kind of simultaneously comprehensive detection cancer of the uterus tumor markers SCC, CEA, CA125, β-HCG in same reaction system, also relate to the application of this kit in the comprehensive detection cancer of the uterus tumor markers SCC, CEA, CA125, β-HCG simultaneously.This kit comprises: the microwell plate of anti-SCC, CEA, CA125, β-HCG first strain monoclonal antibody mixed antibody bag quilt respectively; Use Eu
3+anti-SCC, Sm of mark
3+anti-CEA, Tb of mark
3+anti-CA 125, the Dy of mark
3+anti-β-HCG second strain monoclonal antibody the mixed antibody of mark; Analysis buffer; Share fluorescence-enhancing agent; Cleansing solution; Quality-control product.Kit of the present invention can be used for the clinical assistant diagnosis of the cancer of the uterus, observation of curative effect and Index for diagnosis, to the treatment of uterus tumor with prevent significant, detect four tumor markerses simultaneously, simplify detecting step, improve the specificity and sensitivity that detect aggregation of data analysis.
Description
Technical field
The present invention relates to biology and technical field of medical detection, be specifically related to the time-resolved fluoroimmunoassay detection kit of a kind of simultaneously comprehensive detection cancer of the uterus tumor markers index S CC, CA125, CEA, β-HCG in same reaction system, also relate to the application of this kit in the comprehensive detection cancer of the uterus tumor markers SCC, CA125, CEA, β-HCG simultaneously.
Background technology
The cancer of the uterus is the general name of a series of malignant tumour occurring in position, uterus, and the cancer occurred in uterus the most common is carcinoma of endometrium and cervix cancer.The annual newfound cancer of the uterus case of China is about 13.15 ten thousand, and early detection cure rate is higher.Therefore, the key that the cancer of the uterus is still this disease for the treatment of is made a definite diagnosis in early days, more accurately.
At present, the conventional Testing index of the cancer of the uterus is diagnosed to have SCC, CA125, CEA, β-HCG etc. several.When the reality diagnosis cancer of the uterus, be generally make a definite diagnosis the cancer of the uterus with SCC, CA125, CEA, β-HCG four kinds of index comprehensives because four kinds of comprehensive analyses in specificity and susceptibility apparently higher than single index.Routine Test Lab or medical institutions adopt single agents box usually for detecting this four indices, and namely kit is for a kind of detection of tumor marker, and the shortcoming that this kind of mode exists is: 1, detecting step is various, wastes time and energy; 2, current four kinds of labels adopt four kinds of single index kits to detect respectively, and then carry out comprehensive descision, because the kit of different manufacturer there are differences, and the different kits of same producer due to detect label difference may exist controlled condition, parameter difference and cause testing result to there is error, this kind of error can produce larger impact in the specificity and susceptibility of comprehensive analysis four kinds of labels, and judged result may be caused to differ greatly.Therefore, develop a kind of kit that simultaneously can detect these four kinds of uterus tumor labels under relatively same testing conditions, just can reduce the metrical error because artificial origin causes, greatly improve comprehensive specificity and the susceptibility of analyzing testing result, make conclusion accurately for next step research or judgement and foundation is more fully provided.
Summary of the invention
The object of the invention is to solve problems of the prior art, the time-resolved fluoroimmunoassay detection kit of a kind of simultaneously comprehensive detection cancer of the uterus tumor markers index S CC, CA125, CEA, β-HCG in same reaction system is provided.
The present invention also aims to the application that this kit a kind of is provided.
In order to realize above object, the technical solution adopted in the present invention is: a kind of time-resolved fluorescence method comprehensive detection cancer of the uterus kit, four kinds of uterus tumor mark SCC, CA125, CEA, β-HCG comprehensively in the same reaction system of same micropore, employing time-resolved fluoroimmunoassay detects, and this kit comprises: the microwell plate of anti-SCC, CA125, CEA, β-HCG first strain monoclonal antibody mixed antibody bag quilt respectively; Use Eu
3+anti-SCC, Tb of mark
3+anti-CA 125, the Sm of mark
3+anti-CEA, Dy of mark
3+anti-β-HCG second strain monoclonal antibody the mixed antibody of mark; Analysis buffer; Share fluorescence-enhancing agent; Cleansing solution; Quality-control product.
Wherein, the preparation method of the microwell plate of the first described strain monoclonal antibody mixed antibody bag quilt is: anti-SCC respectively, CA125, CEA, β-HCG first strain monoclonal antibody mixed antibody bag is buffered liquid dilution, obtained antibody coating buffer, then in each hole of microwell plate, add the described antibody coating buffer of 100 μ l respectively, in 37 DEG C of bags by 2 hours, then normal saline flushing microwell plate is used three times, and then in each hole of microwell plate, add the shrouding liquid of 200 μ l respectively, close 2 hours in room temperature or be placed in 4 DEG C of environment and spend the night, then normal saline flushing microwell plate twice is used, freeze drying, the microwell plate of obtained first strain monoclonal antibody mixed antibody bag quilt.
In described antibody coating buffer, the concentration of the first strain monoclonal antibody of anti-SCC, CA125, CEA, β-HCG is 0.009 ~ 0.018g/L respectively.
Described uses Eu
3+anti-SCC, Tb of mark
3+anti-CA 125, the Sm of mark
3+anti-CEA, Dy of mark
3+in the anti-β-HCG second strain monoclonal antibody mixed antibody of mark, use Eu
3+anti-SCC antibody, the Tb of mark
3+anti-CA 125 antibody, the Sm of mark
3+anti-CEA antibody, the Dy of mark
3+the quality proportioning of the anti-β-HCG antibody of mark is: Eu
3+the anti-SCC antibody of mark: Tb
3+the anti-CA 125 antibody of mark: Sm
3+the anti-CEA antibody of mark: Dy
3+anti-β-HCG antibody=(5 ~ 10) of mark: (6 ~ 9): (6 ~ 11): (11 ~ 15).
The preparation method of analysis buffer is: to often liter of concentration be 0.05mol/L, pH value is add 9g NaCl, 0.5g NaN in the Tris-Hcl damping fluid of 8.0
3, 10g BSA, 0.1ml Tween-40,5g polyglycol, mix, i.e. obtained analysis buffer.
The preparation method of described shared fluorescence-enhancing agent is: part of dissociating: mixed by the ethanol of the yttria of the PTA of 70 ~ 200 μm of ol, 2 ~ 7 μm of ol, Triton X-100 and 300ml of 0.6g, 1 liter is settled to water, with acetic acid adjust pH to 3.5, obtained part of dissociating; Strengthen part: mixed by the Tris of the Phen of 0.1 ~ 0.4mmol and 0.2mol, be settled to 1L with water, obtained enhancing part.
The preparation method of described cleansing solution is: concentration be 0.01mol/L, pH value is add Tween-20 in the PBS damping fluid of 7.4, mixing, obtained Tween-20 mass percent concentration is the PBS solution of 0.05%, is then the NaN of 0.2% with mass percent concentration
3solution preservation process, obtained cleansing solution.
Described quality-control product is the potpourri of SCC, CA125, CEA, β-HCG standard items.
The application of a kind of time-resolved fluorescence method comprehensive detection cancer of the uterus kit in the comprehensive detection cancer of the uterus tumor markers SCC, CA125, CEA, β-HCG.
The application of time-resolved fluorescence method comprehensive detection cancer of the uterus kit in the comprehensive detection cancer of the uterus tumor markers SCC, CA125, CEA, β-HCG, comprises the following steps:
(1) dilute quality-control product by analysis buffer, make the mixed solution that there is series concentration gradient and include SCC, CA125, CEA, β-HCG four kinds of standard items;
(2) four kinds of standard items mixed solutions prepared by testing sample and step (1) are added respectively in the different holes of microwell plate of anti-SCC, CA125, CEA, β-HCG first strain monoclonal antibody mixed antibody bag quilt, and then Xiang Kongzhong adds the second strain monoclonal antibody mixed antibody being marked with rare earth ion chelate that can be combined with SCC, CA125, CEA, β-HCG respectively, add shared fluorescence-enhancing agent afterwards, carry out time-resolved fluoroimmunoassay detection, obtain luminous value;
(3) typical curve is done according to the luminous value comprising the mixed solution of SCC, CA125, CEA, β-HCG four kinds of standard items with series concentration gradient measured;
(4) compare with the luminous value of the respective typical curve of SCC, CA125, CEA, β-HCG in SCC, CA125, CEA, β-HCG luminous value in the testing sample measured and standard items, draw the content that in testing sample, SCC, CA125, CEA, β-HCG is respective.
Wherein, described testing sample is human serum.
Further, the preparation method that described bag is buffered liquid is: get that concentration is 0.05mol/L, pH value is the carbonate buffer solution of 8.6, is then the NaN of 0.2% with mass percent concentration
3solution preservation process, obtained bag is buffered liquid.
The preparation method of described shrouding liquid is: to concentration be 0.01mol/L, pH value is add BSA in the PBS damping fluid of 7.4, is mixed with the PBS damping fluid that BSA mass percent concentration is the BSA of 1%, is then the NaN of 0.2% with mass percent concentration
3solution preservation process, obtained shrouding liquid.
Kit provided by the invention, adopts time-resolved fluoroimmunoassay detection method, realizes the object of the comprehensive detection cancer of the uterus tumor markers SCC, CA125, CEA, β-HCG simultaneously.Time resolved fluoro-immunoassay (TRFIA) is a kind of is label with rare earth ion, according to the luminous characteristics of rare earth ion chelate, measures the technical method of specificity fluorescent by TIME RESOLVED TECHNIQUE.TRFIA utilizes the exciting light of rare earth ion different with radiative wavelength, utilizing emitted light has long die-away time simultaneously, effectively can get rid of the interference of non-specific fluorescence like this, there is the range of linearity wide, the advantages such as highly sensitive and good stability, in addition, the different wavelength of transmitted light of different rare earth ions and different die-away times can be utilized, realize detecting multiple mark in same reaction system simultaneously, such and single index separate detection is compared and can be saved time, personnel, reagent etc., these features detect multi objective while of being just in time applicable to of the present invention in same reaction system.
The cancer of the uterus tumor markers SCC, CA125, CEA, β-HCG time-resolved fluoroimmunoassay detection kit provided by the invention, has the extraordinary range of linearity, and the range of linearity detecting SCC is 1ng/ml ~ 90ng/ml, detects and is limited to 0.5ng/ml; The range of linearity detecting CA125 is 25U/ml ~ 500U/ml, detects and is limited to 9U/ml; The range of linearity detecting CEA is 2ng/ml ~ 400ng/ml, detects and is limited to 1ng/ml; The range of linearity detecting β-HCG is 1mIU/ml ~ 9000mIU/ml, detects and is limited to 0.5mIU/ml.The normal reference value of each Testing index is respectively: SCC is 0 ~ 2.5ng/ml; CA125 is 0 ~ 35U/ml; CEA is 0 ~ 6.5ng/ml; β-HCG is 0 ~ 5mIU/ml.The cancer of the uterus tumor markers SCC, CA125, CEA, β-HCG time-resolved fluoroimmunoassay detection kit provided by the invention can be used for the clinical assistant diagnosis of the cancer of the uterus, observation of curative effect and Index for diagnosis, to the treatment of uterus tumor with prevent significant.
The comprehensive detection cancer of the uterus tumor markers four indices SCC, CA125, CEA, β-HCG while of adopting kit provided by the invention can realize in same reaction system, detect simple to operate, step is few, and required time is short, convenient and swift; Testing result is accurate, avoid existing employing four kinds of individual event kits to detect four indices SCC, CA125, CEA, β-HCG respectively and carry out comprehensive descision again and the error in judgement caused, substantially increase specificity and the susceptibility of testing result, make conclusion accurately for next step research or judgement and provide reliable basis.
Accompanying drawing explanation
Same reaction system reaction principle schematic diagram in the same micropore of kit that Fig. 1 provides for the embodiment of the present invention 1;
The single index kit of the kit that Fig. 2 provides for the embodiment of the present invention 1 and Abbott company tests SCC correlation regression analysis figure;
The single index kit of the kit that Fig. 3 provides for the embodiment of the present invention 1 and Roche Holding Ag tests CA125 correlation regression analysis figure;
The single index kit of the kit that Fig. 4 provides for the embodiment of the present invention 1 and Abbott company tests CEA correlation regression analysis figure;
The single index kit of the kit that Fig. 5 provides for the embodiment of the present invention 1 and Siemens company tests β-HCG correlation regression analysis figure.
Embodiment
Below by specific embodiment, technical scheme of the present invention is described in detail.
Embodiment 1
The time-resolved fluorescence method comprehensive detection cancer of the uterus kit that the present embodiment provides, comprising: the microwell plate of the mixed antibody bag quilt of the antibody MAM02-008 of the antibody H00006317-M01 of anti-SCC, the antibody M32112M of anti-CA 125, anti-CEA, the antibody 10R-7515 of anti-β-HCG tetra-kinds of monoclonal antibody compositions; Use Eu
3+mark H00025816-A01 antibody (antibody of anti-SCC), use Tb
3+mark M86306M antibody (antibody of anti-CA 125), use Sm
3+mark MAM02-881 antibody (antibody of anti-CEA), use Dy
3+the mixed antibody of 10R-7751 antibody (antibody of anti-β-HCG) four kinds of monoclonal antibody compositions of mark; Analysis buffer; Share fluorescence-enhancing agent; Cleansing solution; The quality-control product of SCC, CA125, CEA, β-HCG four kinds of standard items mixing compositions.This kit also comprises other related reagents such as negative control, positive control, substrate.
In the kit that the present embodiment provides, anti-SCC antibody H00006317-M01, H00025816-A01 are purchased from Abnova company; Anti-β-HCG antibody 10R-7515,10R-7751 are purchased from Fitzgerald company; Antibody M32112M, M86306M antibody of anti-CA 125 and the monoclonal antibody MAM02-008 of anti-CEA, MAM02-881 antibody are all purchased from Meridian company.
H00006317-M01, M32112M, MAM02-008, the preparation method of the microwell plate of the mixed antibody bag quilt of 10R-7515 tetra-kinds of monoclonal antibody compositions is: mixed antibody bag is buffered liquid dilution, obtained mixed antibody coating buffer, in mixed antibody coating buffer, anti-SCC respectively, CA125, CEA, the concentration of the first strain monoclonal antibody of β-HCG is 0.012g/L, namely in mixed antibody coating buffer, antibody H00006317-M01, M32112M, MAM02-008, the concentration of 10R-7515 is 0.012g/L, then in each hole of microwell plate, add the mixed antibody coating buffer of 100 μ l respectively, in 37 DEG C of bags by 2 hours, then normal saline flushing microwell plate is used three times, and then in each hole of microwell plate, add the shrouding liquid of 200 μ l respectively, 2 hours are closed in room temperature, then normal saline flushing microwell plate twice is used, freeze drying, the microwell plate of obtained mixed antibody bag quilt, be sealed in aluminium foil bag, 4 DEG C save backup.
Wherein, the preparation method that bag is buffered liquid is: get that concentration is 0.05mol/L, pH value is the carbonate buffer solution of 8.6, is then the NaN of 0.2% with mass percent concentration
3solution preservation process, obtained bag is buffered liquid.The preparation method of shrouding liquid is: to concentration be 0.01mol/L, pH value is add BSA in the PBS damping fluid of 7.4, is mixed with the PBS damping fluid that BSA mass percent concentration is the BSA of 1%, is then the NaN of 0.2% with mass percent concentration
3solution preservation process, obtained shrouding liquid.
Use Eu
3+mark H00025816-A01 antibody, use Tb
3+mark M86306M antibody, use Sm
3+mark MAM02-881 antibody, use Dy
3+the preparation method of the mixed antibody of 10R-7751 antibody four kinds of monoclonal antibody compositions of mark is: by Eu
3+h00025816-A01 antibody, the Tb of mark
3+m86306M antibody, the Sm of mark
3+mAM02-881 antibody, the Dy of mark
3+the 10R-7751 antibody of mark is with mass ratio: Eu
3+the H00025816-A01 antibody of mark: Tb
3+the M86306M antibody of mark: Sm
3+the MAM02-881 antibody of mark: Dy
3+10R-7751 antibody=7:6:11:13 the mixing of mark, obtained mixed antibody.
Wherein use Eu
3+mark H00025816-A01 antibody, use Tb
3+mark M86306M antibody, use Sm
3+mark MAM02-881 antibody, use Dy
3+the preparation method of 10R-7751 antibody of mark is: the mark process step of often kind of antibody is identical, the rare earth ion chelate label that just antibody of anti-SCC, CA125, CEA, β-HCG is corresponding respectively be respectively N1-to isothiocycmatobenzyl-DTTA-Eu, N1-to isothiocycmatobenzyl-DTTA-Tb, N1-to isothiocycmatobenzyl-DTTA-Sm, N1-to isothiocycmatobenzyl-DTTA-Dy.Mark anti-SCC antibody H00025816-A01 for N1-to isothiocycmatobenzyl-DTTA-Eu below, its concrete preparation method and step are:
(1) before mark, application molecular cut off is that the centrifugal column of 50kDa marks front purification process to H00025816-A01 antibody, concrete steps are as follows: the unsettled 1mg H00025816-A01 antibody that adds is in the centrifugal column of 50kDa, centrifugal 8 minutes of 9000rpm, discards filtrate; In centrifugal column, add 200 μ l mark damping fluid, centrifugal 8 minutes of 9000rpm, discard filtrate, this step repeats four times, is taken out by centrifuge tube for the last time, discards filtrate; In centrifugal column, add 50 μ l mark damping fluid, allow it leave standstill 1 minute, then the reversion of the filter membrane of centrifugal column is loaded in centrifuge tube, centrifugal 6 minutes of 8000rpm, collect filtrate, namely obtain antibody H00025816-A01 to be marked; Wherein, mark that damping fluid is 50mmol/L, pH value is the carbonate buffer solution of 9.0;
(2) take rare earth ion chelate label N 1-to isothiocycmatobenzyl-DTTA-Eu 0.2mg, add 80 μ l ultrapure waters and dissolve, make chelating reagent; Chelating reagent is joined in the EP pipe filling antibody H00025816-A01 to be marked, mix; To be placed on shaking table 4 DEG C to spend the night, the obtained antibody H00025816-A01 marked, namely uses Eu
3+the H00025816-A01 antibody of mark;
Superdex 2001 × 30cm post is balanced, with pipettor absorption Eu as column equilibration liquid with the PBS damping fluid of three times of column volumes
3+the slow loading of H00025816-A01 antibody of mark, carries out wash-out with eluent, and flow control is at 1ml/min, and Protein Detection instrument detects that protein peak collects sample, by degerming through 0.22 μm of filter membrane for the object product collected; With specific activity assay method, labelled antibody is carried out to calculating and the analysis of mark rate.Highly purified BSA can join in labelled antibody solution by standing storage labelled antibody, and the final concentration of BSA is 0.1%, 4 DEG C can be placed in or-20 DEG C preserve.
The preparation method of analysis buffer is: to often liter of concentration be 0.05mol/L, pH value is add 9g NaCl, 0.5g NaN in the Tris-Hcl damping fluid of 8.0
3, 10g BSA, 0.1ml Tween-40,5g polyglycol, mix, i.e. obtained analysis buffer.
The preparation method sharing fluorescence-enhancing agent is: part of dissociating: mixed by the ethanol of the yttria of the PTA of 100 μm of ol, 5 μm of ol, Triton X-100 and 300ml of 0.6g, be settled to 1 liter with water, with acetic acid adjust pH to 3.5, and obtained part of dissociating; Strengthen part: mixed by the Tris of the Phen of 0.3mmol and 0.2mol, be settled to 1 liter with water, obtained enhancing part.
The preparation method of cleansing solution is: concentration be 0.01mol/L, pH value is add Tween-20 in the PBS damping fluid of 7.4, mixing, obtained Tween-20 mass percent concentration is the PBS solution of 0.05%, is then the NaN of 0.2% with mass percent concentration
3solution preservation process, obtained cleansing solution.
Quality-control product is the potpourri of SCC, CA125, CEA, β-HCG standard items.SCC, CA125, CEA, β-HCG standard items are all purchased from Abnova company.
Embodiment 2
The application of the kit that the embodiment of the present invention 1 provides in the comprehensive detection cancer of the uterus tumor markers SCC, CA125, CEA, β-HCG, namely the kit that the embodiment of the present invention 1 provides is adopted, the comprehensive detection cancer of the uterus tumor markers SCC, CA125, CEA, β-HCG time-resolved fluoroimmunoassay detection method simultaneously, its reaction principle as shown in Figure 1, adopt double antibody sandwich method, comprise the following steps:
(1) dilute SCC, CA125, CEA, β-HCG four kinds of protein standard substance potpourris, i.e. quality-control products by analysis buffer, make the mixed solution that there is series concentration gradient and include SCC, CA125, CEA, β-HCG four kinds of protein standard substances;
(2) four kinds of protein standard substance mixed solutions prepared by 100 μ l testing sample human serums and step (1) are got respectively, add H00006317-M01, M32112M, MAM02-008, in the different holes of the microwell plate of the mixed antibody bag quilt of 10R-7515 tetra-kinds of monoclonal antibody compositions, 37 DEG C of incubations 1 hour, (during washing, the compound method of cleansing solution used is: add 9g sodium chloride in often liter of PBS damping fluid in three washings, 0.5ml Tween-20, shake up, ), add 100 μ l mixed mark antibody working fluids afterwards (by analysis buffer with volume ratio 1:3000 dilution mixture labelled antibody storage liquid, obtained mixed mark antibody working fluid, containing useful Eu in this mixed mark antibody working fluid of every 100 μ l
3+the H00025816-A01 antibody 70ng of mark, uses Tb
3+the M86306M antibody 60ng of mark, uses Sm
3+the MAM02-881 antibody 110ng of mark, uses Dy
3+the 10R-7751 antibody 130ng of mark, all the other are analysis buffer), concussion evenly, 37 DEG C of incubations 1 hour, 5 times are washed with cleansing solution, on thieving paper, control is dry, adds the part of dissociating that 200 μ l share fluorescence-enhancing agent, shakes 5 minutes, add the enhancing part that 20 μ l share fluorescence-enhancing agent, shake 8 minutes, fluorescence intensity measured by time-resolved fluorescence instrument, excitation wavelength is 315nm, 647nm, 612nm, 544nm, 575nm are respectively Sm
3+, Eu
3+, Tb
3+, Dy
3+the strongest transmitting photopeak, 500 microseconds, 200 microseconds, 50 microseconds, 20 microseconds are respectively Eu
3+, Tb
3+, Sm
3+, Dy
3+time delay, measure respective luminous value respectively.Each index in SCC, CA125, CEA, β-HCG of variable concentrations four kinds of albumen hybrid standard product is done typical curve with respective luminous value respectively, and obtain the respective typical curve of different index, the concentration of testing sample human serum calculates by typical curve.Obtain the precision of kit: the coefficient of variation in SCC analyzes and between analyzing is respectively 4.5% ~ 6.3%, 4.9% ~ 8.5%; The coefficient of variation in CA125 analyzes and between analyzing is respectively 3.3% ~ 5.4%, 4.7% ~ 7.0%; The coefficient of variation in CEA analyzes and between analyzing is respectively 4.6% ~ 6.7%, 4.9% ~ 8.5%; The coefficient of variation in β-HCG analyzes and between analyzing is respectively 3.2% ~ 5.5%, 4.2% ~ 7.7%.
Embodiment 3
The SCC that 35 parts of serum samples obtain is detected with kit of the present invention, CA125, CEA, the numerical value of β-HCG four indexs and with same 35 parts of serum samples respectively with the single index kit test SCC of Abbott company, the single index kit test CA125 of Roche Holding Ag, the single index kit test CEA of Abbott company, the data that the single index kit test β-HCG of Siemens company obtains do regression correlations analysis respectively, respective figure 2 respectively, 3, 4, 5, as can be seen from accompanying drawing, kit of the present invention is tested each achievement data of obtaining and tested with single index kit the data obtained respectively has good correlativity.
Claims (2)
1. a time-resolved fluorescence method comprehensive detection cancer of the uterus kit, four kinds of uterus tumor mark SCC, CA125, CEA, β-HCG comprehensively in the same reaction system of same micropore, employing time-resolved fluoroimmunoassay detects, and it is characterized in that: this kit comprises: the microwell plate of anti-SCC, CA125, CEA, β-HCG first strain monoclonal antibody mixed antibody bag quilt respectively; Use Eu
3+anti-SCC, Tb of mark
3+anti-CA 125, the Sm of mark
3+anti-CEA, Dy of mark
3+anti-β-HCG second strain monoclonal antibody the mixed antibody of mark; Analysis buffer; Share fluorescence-enhancing agent; Cleansing solution; Quality-control product;
The preparation method of the microwell plate of the first described strain monoclonal antibody mixed antibody bag quilt is: anti-SCC respectively, CA125, CEA, β-HCG first strain monoclonal antibody mixed antibody bag is buffered liquid dilution, obtained antibody coating buffer, then in each hole of microwell plate, add the described antibody coating buffer of 100 μ l respectively, in 37 DEG C of bags by 2 hours, then normal saline flushing microwell plate is used three times, and then in each hole of microwell plate, add the shrouding liquid of 200 μ l respectively, close 2 hours in room temperature or be placed in 4 DEG C of environment and spend the night, then normal saline flushing microwell plate twice is used, freeze drying, the microwell plate of obtained first strain monoclonal antibody mixed antibody bag quilt,
In described antibody coating buffer, the concentration of the first strain monoclonal antibody of anti-SCC, CA125, CEA, β-HCG is 0.009 ~ 0.018g/L respectively;
Described uses Eu
3+anti-SCC, Tb of mark
3+anti-CA 125, the Sm of mark
3+anti-CEA, Dy of mark
3+in the anti-β-HCG second strain monoclonal antibody mixed antibody of mark, use Eu
3+anti-SCC antibody, the Tb of mark
3+anti-CA 125 antibody, the Sm of mark
3+anti-CEA antibody, the Dy of mark
3+the quality proportioning of the anti-β-HCG antibody of mark is: Eu
3+the anti-SCC antibody of mark: Tb
3+the anti-CA 125 antibody of mark: Sm
3+the anti-CEA antibody of mark: Dy
3+anti-β-HCG antibody=(5 ~ 10) of mark: (6 ~ 9): (6 ~ 11): (11 ~ 15);
The preparation method of described shared fluorescence-enhancing agent is: part of dissociating: mixed by the ethanol of the yttria of the PTA of 70 ~ 200 μm of ol, 2 ~ 7 μm of ol, Triton X-100 and 300ml of 0.6g, 1 liter is settled to water, with acetic acid adjust pH to 3.5, obtained part of dissociating; Strengthen part: mixed by the Tris of the Phen of 0.1 ~ 0.4mmol and 0.2mol, be settled to 1L with water, obtained enhancing part;
The preparation method of described cleansing solution is: concentration be 0.01mol/L, pH value is add Tween-20 in the PBS damping fluid of 7.4, mixing, obtained Tween-20 mass percent concentration is the PBS solution of 0.05%, is then the NaN of 0.2% with mass percent concentration
3solution preservation process, obtained cleansing solution.
2. time-resolved fluorescence method comprehensive detection cancer of the uterus kit according to claim 1, is characterized in that: described quality-control product is the potpourri of SCC, CA125, CEA, β-HCG standard items.
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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 |
| CN105929157A (en) * | 2016-05-25 | 2016-09-07 | 苏州新波生物技术有限公司 | Diagnostic kit for jointly detecting HIV antigen and HIV antibody and preparation method of diagnostic kit |
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