CN101769927B - Detection particle of carcino-embryonic antigen as well as preparation and application thereof - Google Patents
Detection particle of carcino-embryonic antigen as well as preparation and application thereof Download PDFInfo
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- CN101769927B CN101769927B CN200810204999.6A CN200810204999A CN101769927B CN 101769927 B CN101769927 B CN 101769927B CN 200810204999 A CN200810204999 A CN 200810204999A CN 101769927 B CN101769927 B CN 101769927B
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Abstract
The invention relates to a diagnostic reagent of carcino-embryonic antigen and discloses a detection particle of the carcino-embryonic antigen, which is a luminous particle encapsulated by a carcino-embryonic antigen (CEA) antibody. The invention also discloses preparation and application of the detection particle of the carcino-embryonic antigen and further discloses an in-vitro detection kit for detecting the carcino-embryonic antigen contained in a sample and a using method thereof. The in-vitro detection kit can quantificationally detect the carcino-embryonic antigen contained in a human serum sample and can be used for the auxiliary diagnosis of cancers and the monitoring of the therapeutic effect of cancer patients by combining with other serum and clinical information.
Description
Technical field
The present invention relates to the diagnostic reagent of cancer, be specifically related to carcinomebryonic antigen (CEA) detection of particles, its preparation and application based on light-induced chemiluminescent principle.
Background technology
Immunology detection is a kind of means that the specific reaction based on antigen and antibody detects, because it can utilize isotope, enzyme, chemiluminescent substance etc., detected signal is amplified and shown, therefore be often used to detect protein, the micro-bioactivator such as hormone.
China's immunology detection has experienced radio-immunity detection (rise in 20 century 70s, be now still generally used in hospital above county level) substantially; Enzyme linked immunosorbent detection (risen the eighties in 20th century, each Clinical Institutions generally uses); Biological marker take chemiluminescence as representative and immunoassay technology (start to promote the use of the nineties in 20th century, and product steps into the growth stage) three phases.This development process mainly the improving constantly of demand of the susceptibility based on to detection method, accuracy and property simple to operation determines.
Chemiluminescence immune assay (Chemiluminescence Immunoassay) is worldwide to develop nearly ten years very fast on-radiation immuno analytical method.Detecting principle is using luminescent substance as signal amplifying system and nationality helps its luminous intensity directly to measure immune combination.Due to its high sensitivity, the advantage such as sensing range is wide, has become the substituent of radioimmunoassay and normal enzyme immunoassay, is the important developing direction of immunology detection.
But the at present domestic chemiluminescence detection reagent of development voluntarily, is mostly heterogeneous reaction, adopts the direct mark of chemical substrate, excites by chemical reaction.Its analytic process and traditional enzyme mark detection type seemingly, need clean and separate repeatedly, detect length consuming time, and automaticity is not high.Reagent detects with luminous host and test format and different in external producer.With Abbott, the chemi-excitation that the companies such as Bayer and Chiron are representative, carries out immunoassays with the direct labelled antigen of chemical luminous substrate or antibody.The most frequently used is acridinium ester, can be by hydrogen peroxide oxidation in alkaline environment and luminous.BD is with AKP, and golden steel gastral cavity is that matrix adopts enzyme-catalyzed chemical luminescence.Roche is for adopting electrochemiluminescence (electrochemiluminescence, ECL), and the tris (bipyridine) ruthenium of luminous substrate divalence and reaction partner tripropyl amine (TPA) lose electronics and oxidized at electrode surface.The tripropyl amine (TPA) of oxidation loses a H+ and becomes strong reductant, the trivalent ruthenium of oxidized form is reduced to the divalent ruthenium of excited state, discharges photon immediately and reverts to the luminous substrate of ground state.This process is carried out again and again at electrode surface, constantly sends photon and often keeps the constant of concentration of substrate.Because involving separation cleaning in course of reaction, the Automation Design complexity, instrument is quite expensive.In addition, the stability of luminous host is also a large problem.
Light-induced chemiluminescent method, by introducing laser technology and Nano microsphere technology, has successfully solved above-mentioned deficiency.Carry out in homogeneous phase because of reaction, both accelerated reaction velocity, avoided again repeatedly separating and cleaning step, can effectively reduce detection background value, reduce the reaction time, and can realize automation mechanized operation.At present, PE company has released the light-induced chemiluminescent reagent A lpha-Screen using for biological study.But in clinical detection field, the light-induced chemiluminescent also not emerging detects reagent, in particular for tumor markers and hepatitis test item.
Summary of the invention
The object of this invention is to provide the detection of particles that one can be used for quantitatively detecting carcinomebryonic antigen (CEA), its preparation, testing conditions and application.
Know-why of the present invention:
Carcinomebryonic antigen of the present invention (CEA) detection reagent and kit are relevant to light-induced chemiluminescent detection technique, and photo-induced chemiluminescence immunoassay technology is a kind of method of utilizing the light wave of chemiluminescent substance transmitting to carry out immunoassays.This technology has mainly been integrated high molecular particle technology, organic synthesis, the research of protein chemistry and clinical detection association area.
Why the present invention can detect carcinomebryonic antigen (CEA), due under homogeneous phase condition, using inside with the photosensitive particulate (nanoscale) of dyestuff and be coated with anti-CEA antibody and the potpourri of the inner luminous particle (nanoscale) with luminophor as reagent with detect sample mix.Now nanometer photosensitive particulate can catch CEA quickly and effectively with the nano luminescent particulate that is coated with anti-CEA antibody, and within closely, three forms immune sandwich complex.After exciting light irradiates, the dyestuff in nanometer photosensitive particulate is induced to activate, and discharges the active oxygen ion (singlet oxygen) of high-energy state.The active oxygen ion of this high-energy state is captured by in-plant nano luminescent particulate, thereby transferring energy is to activate the luminophor in described luminous particle.After number microsecond, the luminophor in luminous particle will discharge high level ruddiness.Measure these high level photons with photon counter, and by computer, photon number is scaled to concentration of target molecules, the number of photon number has accurately reflected the concentration of target molecule.And in the time that sample does not contain CEA, cannot closely form immune sandwich complex, active oxygen ion also cannot be passed to luminous particle surface.Active oxygen ion is decay rapidly in liquid phase, when detection, produces without high level ruddiness.Concrete principle is referring to Fig. 1 and Fig. 2.
Based on above-mentioned principle, first aspect present invention provides a kind of detection of particles for detection of carcinomebryonic antigen (CEA), is the coated luminous particle of anti-CEA antibody.
Second aspect present invention provides the detection kit of a kind of carcinomebryonic antigen (CEA), comprises the coated luminous particle of above-mentioned anti-CEA antibody, also can comprise biotin labeled anti-CEA antibody and/or the coated photosensitive particulate of Avidin in kit.
In kit, coated luminous particle, biotin labeled anti-CEA antibody and the coated photosensitive particulate of Avidin of above-mentioned anti-CEA antibody is placed in separately independently container with the form of solution respectively.The solvent of the above-mentioned solution that contains the coated photosensitive particulate of the coated luminous particle of anti-CEA antibody, biotin labeled anti-CEA antibody or Avidin can be the dicyandiamide solution of the applicable antigen-antibody reaction of routine, as HEPES buffer system, Tris buffer system.The composition of the preferred pH8.0 of solvent of the coated luminous particle of anti-CEA antibody is HEPES, NaCl and EDTA-Na-2H
2the HEPES damping fluid of O, the composition of the preferred pH8.0 of solvent of biotin labeled anti-CEA antibody is Tris, NaCl and EDTA-Na-2H
2the Tris damping fluid of O, the solvent preferred component of the coated photosensitive particulate of Avidin is HEPES, NaCl and EDTA-Na-2H
2the HEPES damping fluid of O; sealing and the material of protein protection effect in above-mentioned various solvent, also can be added if BSA and the stable reagent that prevents particles agglomerate are as Tween20; consideration for and standing storage anticorrosion to reagent also can be added antiseptic in solvent, and the Proclin 300 that the preferred 100U/ml gentamicin of antiseptic and mass percent are 5/10000ths is as antiseptic.
In kit, also can comprise positive control and negative control.
When for quantitative detection carcinomebryonic antigen (CEA), in mentioned reagent box, also can comprise the solution that contains multiple known carcinomebryonic antigen (CEA) concentration.The independent packaging respectively of the solution of above-mentioned different carcinoma embryonal antigen (CEA) concentration.
Above-mentioned luminous particle refers to the high molecular particle that is filled with luminophor and lanthanide compound.Luminophor can be the derivant of Dioxene (dioxine) or thioxene (thioxene) etc., and lanthanide compound can be Eu (TTA)
3/ TOPO or Eu (TTA)
3/ Phen etc., this particulate can be by buying on market.
Research is found, because final detection reaction is homogeneous reaction, the particle diameter (mean diameter of particulate) of luminous particle is large (> 400nm) meeting natural subsidence too, impact detects effect, the particle diameter too little (< 50nm) of particulate, can make the cleaning in preparation process more difficult, antagonist connection work is unfavorable, therefore the particle size range of luminous particle should be between 50-400nm, preferably between 100-300nm, preferably 250nm.
The surface functional group of luminous particle can be the group of any energy connexin matter, but the most frequently used particulate that mainly contains carboxyl and aldehyde radical surface functional group.Use different microparticle surfaces functional groups, the reactive mode and the condition that connect antibody are not identical yet.
In view of obtaining better anti-CEA linkage rate of antibody, reagent stability and is connected repeatability, the particulate of preferred carboxyl surface functional group when method of attachment as antibody of improving one's methods of record after employing.
The luminous quantity of luminous particle is the final illumination effect detecting of impact directly.Luminous particle luminous quantity that market provides generally can be 50,000---and 10,000, within the scope of 000 photon number/100ug luminous particle, preferably 150,000-350,000 photon number/100ug) luminous particle.
In above-mentioned biotin labeled anti-CEA antibody, the molecule ratio of biotin and antibody is preferably (30-50): 1, and preferably 40: 1.
Above-mentioned photosensitive particulate is the high molecular particle that is filled with Photoactive compounds.Under 670-690nm optical excitation, can produce singlet oxygen ion, when itself and luminous particle distance is enough near situation, single line oxonium ion is delivered to luminous particle, react with the luminophor in luminous particle, produce ultraviolet light, ultraviolet light further excites lanthanide compound again, produces 520-620nm wavelength photon.Photoactive compounds can be phthalocyanine dye etc., and this particulate also can be by buying on market.
In the coated photosensitive particulate of above-mentioned Avidin, the mass ratio of Avidin and photosensitive particulate, without particular restriction, is preferably 1: (3-10), and preferably 1: 5.
Commercially available photosensitive particulate is all applicable to the present invention, and diameter of particle is preferably 180-260nm, preferably 220nm.
The coated luminous particle of anti-CEA antibody can adopt NaBH
3the reductive amination process method of CN makes, NaBH
3the reductive amination process step of CN is as follows:
1) mix: by luminous particle and anti-CEA antibody in mass ratio example be 10: (1-4) be mixed in damping fluid;
2) reaction: the NaBH that adds damping fluid preparation
3cN solution mixes and reacts;
3) sealing: the Gly and the NaBH that add damping fluid preparation
3cN solution, mixes after reaction, then adds the sealing of BSA (bovineserum albumin) solution;
4) wash products, obtains the coated luminous particle of anti-CEA antibody.
Wherein, in blend step, the mass ratio of luminous particle and anti-CEA antibody is 10: (1-4) preferably 10: 2.Reaction buffer can be MES damping fluid, phosphate buffer, and preferably MES rushes liquid, the preferred 0.05M of concentration, and in reactions steps, in reaction solution, the concentration of luminous particle can be 10～40mg/ml, preferably 20mg/ml.
Improved, the coated luminous particle of anti-CEA antibody can adopt following method to make:
1) mix: luminous particle and anti-CEA antibody are mixed in damping fluid.Preferably, damping fluid is MES damping fluid, and the mass ratio of luminous particle and anti-CEA antibody is (8-15): 1, and preferably 12.5: 1.
2) reaction: add EDAC (1-ethyl-(3-dimethylaminopropyl) the phosphinylidyne diimmonium salt hydrochlorate) solution of damping fluid preparation mix and react.Preferably, damping fluid is MES damping fluid, and in mixed solution, luminous particle is (15-40) with the mass ratio of EDAC: 1 mixes, and preferred mass ratio is 25: 1.Reaction conditions is 37 ℃ of revolving reactions.General approximately 48 hours sufficient reactings.
3) in the reactant liquor obtaining toward step 2, add the BSA solution of damping fluid preparation mix and react.Preferably, damping fluid is MES damping fluid.Reactant liquor volume that BSA solution and step 2 obtain is better is 1: 1-1: 2.Reaction conditions is 37 ℃ of revolving reactions.General approximately 16 hours sufficient reactings.
4) wash products, obtains the coated luminous particle of anti-CEA antibody.
The mode of cleaning can be cleaned or dialysis cleaning for centrifuge method.
Dialysis cleaning step: adopt reaction buffer dialysis, each 4-5 hour, exchange buffering liquid 4 times.The dialysis cleaning operation time is longer, and particle loss is more, causes yield to reduce.
Centrifuge method cleaning step comprises the centrifugal supernatant that goes, and adds reaction buffer washing, and precipitation is opened in ultrasonic processing, 3-5 time so repeatedly.When centrifuge method is cleaned, centrifugal force can make luminous particle temporal aggregate together, but can be easy to again disperse to open through ultrasonic processing, and this method running time is short, and yield is higher.Therefore preferably adopt this kind of cleaning way.
Above-mentioned improved preparation method and NaBH
3the main difference of the reductive amination process method of CN is key reaction reagent N aBH
3cN has replaced to EDAC, and the replacement of this reagent not only makes reaction time and NaBH
3the reductive amination process method of CN is compared and has been shortened more than 4 hour, and the cross-linking efficiency of luminous particle and anti-CEA antibody obtained raising, has saved antibody consumption, and in addition, test shows, the detection of particles that the method makes and NaBH
3the detection of particles that the reductive amination process method of CN makes is compared, and reaction signal is stronger under the same conditions, and sensitivity is higher, and sensing range is also wider.
The coated photosensitive particulate of Avidin can adopt NaBH
3the reductive amination process legal system of CN is standby.
Third aspect present invention, discloses the using method of mentioned reagent box, comprises the following steps:
By the detection of particles for detection of carcinomebryonic antigen (CEA), biotin labeled anti-CEA antibody and the coated photosensitive particulate hybrid reaction of Avidin in testing sample and kit; then irradiate reacting hole with exciting light, measure the luminous photon amount of each reacting hole and obtain optical signal value.
Above-mentioned exciting light sources wavelength coverage is 600-700nm, preferably 640-680nm; The luminous utilizing emitted light optical source wavelength scope of reacting hole is 600-680nm, preferably 610-620nm; Utilizing emitted light scope time delay is 100ms-1000ms; The power bracket of exciting light sources is 5-100mw, preferably 40-60w.
Preferably, the using method of mentioned reagent box is specially:
1) in reacting hole, add detection of particles and the biotin mark for detection of carcinomebryonic antigen (CEA) in sample, kit
The anti-CEA antibody of note, obtains initial reaction solution, hybrid reaction;
2) adding the coated photosensitive particulate of Avidin to obtain end reaction solution reacts again;
3) exciting light irradiates reacting hole, measures the luminous photon amount of each reacting hole and obtains optical signal value.
The reaction conditions of above-mentioned steps 1 is 37 ℃ of incubation 10-30 minute, and preferably 20 minutes, the reaction conditions of step 2 was also 37 ℃ of incubation 10-30 minute, preferably 15 minutes.
In step 1, the coated luminous particle of anti-CEA antibody is suspension, and the concentration of the coated luminous particle of anti-CEA antibody in suspension, without particular restriction, can be 200-400ug/ml, preferably 300ug/ml.
In step 1, biotin labeled anti-CEA antibody is also suspension, and the concentration of biotin labeled anti-CEA antibody in suspension, without particular restriction, can be 30-50ug/ml, preferably 50ug/ml.
In step 2, the coated photosensitive particulate of Avidin is suspension, and the concentration general control of the coated photosensitive particulate of Avidin in suspension, at 20-100ug/ml, can be 20-60ug/ml, preferably 40ug/ml.
In said method, the volume of initial reaction solution, without particular restriction, can be 45-75ul, preferably 75ul; The volume of end reaction solution, also without particular restriction, can be 150-250ul, preferably 250ul.
Above-mentioned sample comprises serum, blood plasma and whole blood.
When detection of particles of the present invention and detection kit are when quantitatively detecting carcinomebryonic antigen (CEA), also need to calculate testing sample CEA content according to typical curve.
The present invention adopts light-induced chemiluminescent detection technique, the external diagnosis reagent case of carcinomebryonic antigen in quantitative measurement human serum sample (CEA), can combine with other serum and clinical information for the auxiliary diagnosis of cancer and the monitoring of cancer patient result for the treatment of.
Carcinomebryonic antigen (CEA) enzyme that the present invention and detected object are close exempt from method kit in methodology relatively, enzyme is exempted from method with OD value embodiment pattern detection value, OD value is can sensing range narrow, only can do qualitative detection, and sensitivity is low.Kit of the present invention adopts light-induced chemiluminescent method to measure the CEA in sample, has the features such as highly sensitive, sensing range is wide, exempts from method can reach higher sensitivity and more excellent sensing range in methodology compared with enzyme.
Accompanying drawing explanation
Fig. 1: photo-induced chemiluminescence immunoassay technical schematic diagram: particulate, in conjunction with forming dimer, produces photon signal
Fig. 2: photo-induced chemiluminescence immunoassay technical schematic diagram: not in conjunction with particulate, produce without photon signal
Fig. 3: singlet oxygen is with particulate spacing decay, in the distance of general 600nm, does not have single line oxygen substantially, therefore not luminous yet
FG BEAD: luminous particle, includes light emitting molecule;
GG BEAD: photosensitive particulate, includes light sensitive molecule;
Singlet Oxygen: singlet oxygen, active oxygen ion;
A/B: both are the bioactive molecule of specific bond directly or indirectly.As in double antibodies sandwich detects, A, B is the monoclonal antibody of the epi-position different for target molecule C
Fig. 4: kit of the present invention and import reagent box detect comparison test result
Embodiment
Further set forth the present invention below in conjunction with embodiment.Should be understood that these embodiment are only for the present invention is described, but not limit the scope of the invention.In the following example, the experimental technique of unreceipted actual conditions and the reagent of undeclared formula are according to normal condition as people such as Sambrook, molecular cloning: the condition of the condition described in test handbook (New York:Cold Spring Harbor LaboratoryPress, 1989) or manufacturer's suggestion is carried out or configures.
Experiment Instrumental raw material sources and agent prescription:
Raw material and reagent producer
Biotin-X-X-NHS Sigma TLC≥95％
BSA Equitech /protease free
Gly Sigma≥95％
HEPES is magnificent
Tris Sigma
NaBH
3CN Acros
Avidin Pierce
EDAC Sigma
Anti-CEA antibody biospacific
PentaTek company of the photosensitive particulate U.S.
PentaTek company of the luminous particle U.S.
Instrument model producer
Particle instrument Model 370 Nicomp
Microplate reader MultiSKAN MK3 labsystem
Ultraviolet-visible pectrophotometer 752P Shanghai spectrum company limited
Fluorospectrophotometer F95 Prism Optical Technology Co
The rich positive biology/Shanghai of light-induced chemiluminescent analytic system Bei Bin photon
The preparation of the coated luminous particle of embodiment 1 carcinomebryonic antigen antibody
The coated improved preparation method of luminous particle of carcinomebryonic antigen antibody is as follows:
1) luminous particle suspension processing: draw a certain amount of carboxyl luminous particle centrifugal in high speed freezing centrifuge; supernatant discarded; add a certain amount of MES damping fluid, ultrasonication, to particulate Eddy diffusion, adds MES damping fluid to regulate luminous particle concentration to 100mg/ml.
2) antibody treatment: CEA antibody is dialysed in the MES of 0.05M pH6.0 damping fluid (hereinafter to be referred as MES damping fluid), the rear mensuration concentration of having dialysed, and regulate concentration to 8mg/ml.
3) an anti-CEA antibody-solutions (MES damping fluid) of the luminous particle suspension of MES damping fluid, 100mg/ml (MES damping fluid) and 8mg/ml, mixes with the volume ratio of 1: 1: 1, mixes rapidly, obtains reactant liquor.
4) with the EDAC solution of MES damping fluid preparation 40mg/ml, according to adding with the ratio of luminous particle 100mg/100uL EDAC, mix rapidly 37 ℃ of revolving reactions 48 hours.
5) add the BSA solution (MES damping fluid) of 200mg/ml, it is 5: 8 with reactant liquor volume ratio, mixes rapidly 37 ℃ of revolving reactions 16 hours.
6) use MES damping fluid eccentric cleaning four times, finally suspend with luminescence reagent damping fluid, measure particle diameter and solid content, regulate concentration to 10mg/ml.
By said method and employing NaBH
3the detection of particles that the reductive amination process method of CN makes compares.
NaBH
3the preparation method of CN reductive amination process method:
1) luminous particle suspension processing: draw a certain amount of luminous particle centrifugal in high speed freezing centrifuge; supernatant discarded; add a certain amount of Mes damping fluid, ultrasonication, to particulate Eddy diffusion, adds phosphate buffer to regulate luminous particle concentration to 100mg/ml.
2) antibody treatment: anti-CEA antibody is dialysed in the Mes of 0.02M pH7.0 damping fluid, the rear mensuration concentration of having dialysed, and regulate concentration to 8mg/ml.
3) the anti-CEA (Mes damping fluid) of the luminous particle suspension of Mes damping fluid, 100mg/ml (Mes damping fluid) and 8mg/ml, mixes with the volume ratio of 1: 2: 5, mixes rapidly, obtains reactant liquor.
4) use Mes damping fluid to prepare the NaBH of 25mg/ml
3cN solution, according to adding with the reactant liquor volume ratio of 1: 25, mixes rapidly, 37 ℃ of revolving reactions 48 hours.
5) the Gly solution of Mes damping fluid preparation 75mg/ml and the NaBH of 25mg/ml
3cN solution, according to adding in above-mentioned solution with the reactant liquor volume ratio of 2: 1: 10, mixes, 37 ℃ of revolving reactions 2 hours.The BSA solution (Mes damping fluid) that adds again 200mg/ml, it is 5: 8 with reactant liquor volume ratio, mixes rapidly 37 ℃ of revolving reactions 16 hours.
6) use Mes damping fluid eccentric cleaning four times, finally suspend with luminescence reagent damping fluid, measure particle diameter and solid content, regulate concentration to 10mg/ml.
The comparison of the detection of particles that both make:
1. saved preparation time
| NaBH 3CN reductive amination process method | Improved preparation technology | |
| The luminous particle of preparation 100mg cross-linking antibody | 72h | 68h |
2. improve antibody linked efficiency, saved antibody consumption
| NaBH 3CN reductive amination process method | Improved preparation technology | |
| The antibody linked particulate of 1mg | 5mg | 12.5mg |
3. strengthened reaction signal
| Reaction conditions | NaBH
3 |
Improved preparation technology calibrates |
| The luminous particle of 300ug/mlCEA antibody, the biotinylation CEA antibody photosensitive particulate coated with 40ug/ml Avidin of 50ug/ml react | 220000 | 330000 |
4. improved sensitivity
| NaBH
3The legal mark product of CN |
Improved preparation technology calibrates |
|
| The luminous particle of 300ug/mlCEA antibody, the biotinylation CEA antibody photosensitive particulate coated with 40ug/ml Avidin of 50ug/ml react | 2453/1000＝2.453 | 3256/1000＝3.256 |
5. expanded sensing range
| NaBH 3The sensing range of CN reductive amination process method | Improved preparation technology's sensing range | |
| The luminous particle of 300ug/mlCEA antibody, the biotinylation CEA antibody photosensitive particulate coated with 40ug/ml Avidin of 50ug/ml react | 0-800ng/ml | 0-1000ng/ml |
Prepare the coated luminous particle of anti-CEA antibody with reference to the preparation method of aforementioned improved, and test by properties the selection research of relatively having carried out concrete technology condition from following several respects:
1. light signal detection method:
In reacting hole, add respectively 25 μ l samples, then add successively 25 μ l luminescence reagents and 25 μ l biotinylated antibody reagent.Then put into instrument (light-induced chemiluminescent analytic system), by instrument operation according to the following steps automatically: vibration, 37 ℃ of incubations 20 minutes, more automatically add after the coated photosensitive particulate reagent of 175 μ l Avidins 37 ℃ of incubations 15 minutes.Instrument automatically produces Ear Mucosa Treated by He Ne Laser Irradiation micropore and calculates the luminous photon amount in every hole.
2. sensitivity for analysis detection method:
Detect 10 hole calibration product 1 (0ng/ml), calculate its RLU average (AVE) and two standard deviations (SD), enter typical curve with AVE ± 2SD inverse iteration, the concentration value obtaining is the sensitivity for analysis of this kit.
3. method for detecting specificity:
Detect AFP and PSA specificity reference material, must not detect false positive
AFP (alpha-fetoprotein) specificity reference material concentration: 1000ng/mL
PSA (prostate specific antigen) specificity reference material concentration: 100ng/mL
4.Hook detection method:
Detect the sample light signal that CEA concentration is respectively 10ug/ml, 50ug/ml, 100ug/ml, result should be greater than calibration product 6 (1000ng/ml).
5. accuracy detection method:
Detect the quality-control product light signal that CEA concentration is respectively 7ng/ml and 100ng/ml, each concentration is done 10 hole replications, and substitution formula calculates CV value.The accuracy of low concentration is measured and is expressed as QC L, and the accuracy of high concentration is measured and is expressed as QC H.
6. linearity test method:
Other 5 calibration product (prepared by embodiment 6) except 0 value are done to linear analysis, calculate linearly dependent coefficient r (r should be greater than 0.99)
Process conditions are selected:
A. damping fluid and pH value in reaction are selected
The luminous particle reaction density of other reaction conditions: 25mg/ml, the luminous particle of 12.5: 1 and anti-CEA antibody mass ratio, centrifuge method is cleaned
Known according to above experimental result, the sensitivity of 0.05M MES pH6.0 damping fluid, CV are better than 0.02M PB pH 7.0 damping fluids.Therefore select the reaction buffer of 0.05M MES pH 6.0 damping fluids as the coated luminous particle of anti-CEA.
B. the reaction ratio of luminous particle and antibody
Other reaction conditions: the MES buffer of 0.05M pH6.0 is as reaction buffer, and centrifuge method is cleaned.
Known according to above experimental result, in the time that luminous particle and antibody response ratio are 12.5: 1, its every numerical value is all better, therefore luminous particle and antibody response ratio are chosen as 12.5: 1.
C. cleaning way:
Other reaction conditions: the MES buffer of 0.05M pH6.0 is as reaction buffer, the luminous particle of 12.5: 1 and anti-CEA antibody ratio.
Dialysis cleaning step: dialyse with upper volume for 100 times, each more than 4 hours, exchange buffering liquid 3 times.The dialysis cleaning operation time is longer, and in dialysis procedure, particle loss is more.
Centrifugal 30 minutes of centrifuge method cleaning step: 12000rpm, cleans 4 times.The centrifugal force that centrifuge method is cleaned can make luminous particle temporal aggregate, but is easy to again to disperse to open through ultrasonic processing.And the running time is short, yield is higher.Consider production cycle and production cost, determine to adopt this kind of cleaning way.
The final particle diameter of selecting 250nm, luminous quantity is >=250, the luminous particle of 000 photon number/100ug, and the MES buffer of 0.05M pH6.0 is as reaction buffer, the luminous particle of 12.5: 1 and anti-CEA antibody ratio, dialysis is cleaned as optimum preparation condition.
The preparation of embodiment 2 biotin labeling antibody
Preparation method:
1) antibody treatment: anti-CEA antibody is dialysed in 0.1M NaHCO
3solution, measures antibody concentration and is adjusted to 1mg/ml.
2) with the Biotin solution of DMSO preparation 16.17mg/ml.
3) mark: get the anti-CEA antibody labeling of the 1mg/ml handling well antibody and the Biotin solution preparing, the two mixes in proportion, mixes rapidly.4 ℃ leave standstill reaction 12～16 hours.
4) dialysis: completely reacted biotin labeling antibody is dialysed in biotin labeling dialysis buffer liquid (pH8.0).
5) the biotinylated antibody sucking-off of having dialysed is transferred in clean centrifuge tube to sampling and measuring antibody concentration.Biotin labeling antibody concentration qualified quality inspection is adjusted to 0.5mg/ml.
Antibody is reacted according to different proportion with Biotin and detects:
Known according to above experimental result, in the time that biotin and antibody response ratio are 40: 1, its every numerical value is all better than 30: 1 and 50: 1, therefore biotin and antibody response ratio are chosen as 40: 1.
The preparation of the coated photosensitive particulate of embodiment 3 Avidins
Photosensitive particulate: adopt the photosensitive particulate (PentaTek company of the U.S.) that particle diameter is 220 ± 40nm
Preparation method:
A, the processing of photosensitive particulate suspension: draw a certain amount of photosensitive particulate centrifugal in high speed freezing centrifuge; supernatant discarded; add a certain amount of MES damping fluid, ultrasonic to particulate Eddy diffusion on ultrasonic cell disintegration instrument, add MES damping fluid to regulate photosensitive particulate concentration to 100mg/ml.
B, Avidin solution preparation: weigh a certain amount of Avidin, add MES damping fluid and be dissolved to 8mg/ml.
C, mixing: by the Avidin of the photosensitive particulate suspension of handling well, 8mg/ml and MES damping fluid, mix with the volume ratio of 2: 5: 1, mix rapidly, obtain reactant liquor.
D, reaction: the NaBH of MES damping fluid preparation 25mg/ml
3cN solution, according to adding with the reactant liquor volume ratio of 1: 25, mixes rapidly.37 ℃ of revolving reactions 48 hours.
E, sealing: the Gly solution of MES damping fluid preparation 75mg/ml and the NaBH of 25mg/ml
3cN solution, according to adding in above-mentioned solution with the reactant liquor volume ratio of 2: 1: 10, mixes, 37 ℃ of revolving reactions 2 hours.The BSA solution (MES damping fluid) that adds again 200mg/ml, it is 5: 8 with reactant liquor volume ratio, mixes rapidly 37 ℃ of revolving reactions 16 hours.
F, cleaning: in completely reacted solution, add MES damping fluid, high speed freezing centrifuge is centrifugal, abandon supernatant, add fresh MES damping fluid ultrasonic method Eddy diffusion, again centrifugal, so clean 3 times, finally suspend with a small amount of sensitization reagent damping fluid, measure solid content, use sensitization reagent damping fluid to regulate concentration to 10mg/ml.
The formulation of embodiment 4 normal reference values
According to the result that 414 normal non-cancer patient's blood samples are detected, 95% sample CEA level is less than 5.35ng/ml, so we can think that 0-5.35ng/ml is the clinical normal reference range of this reagent.
Embodiment 5 calibrates the preparation of product
Calibration product: the quantitatively reference material of carcinomebryonic antigen (CEA) that uses Ministry of Public Health's visiting center to produce, take NBCS as dilution, according to concentration 0ng/ml, 1ng/ml, 5ng/ml, 40ng/ml, 200ng/ml, 1000ng/ml prepares 6 calibration product.
First in reacting hole, add respectively sample, then add successively luminescence reagent (luminous particle that antibody is coated) and biotin labeling antibody.Then put into instrument (light-induced chemiluminescent analytic system), automatically operated according to the following steps by instrument: vibration, 37 ℃ of incubations.Automatically add again after the coated photosensitive particulate of Avidin 37 ℃ of incubations again.Incubation finishes rear instrument and automatically produces Ear Mucosa Treated by He Ne Laser Irradiation micropore and calculate the luminous photon amount in every hole.
Detect as stated above the luminous photon amount of each calibration product, adopt cubic spline fit mapping and get final product the optical signal value recording and corresponding calibration product concentration, it is linear that typical curve is.
In quantitative measurement, according to typical curve, calculate the CEA content of each sample by sample measured light signal value, unit is ng/ml.
The Optimum Experiment of testing conditions:
Determining of 1.1 incubative times:
Test material: adopt the luminous particle reagent (the luminous antibody reagent of lower abbreviation) of the coated 300ug/ml of antibody and the biotin labeling antibody reagent of 50ug/ml, and the coated photosensitive particulate reagent of the Avidin of 40ug/ml
Test samples: calibration product 6 and quality-control product QcL, QcH.
Initial reaction condition: application of sample amount is sample 25ul, luminous particle reagent 25ul, biotin labeling antibody reagent 25ul, the coated photosensitive particulate reagent of Avidin is 175ul, two Buwen bathe.
1.1.1 determining of first step incubative time
First step incubative time is respectively 10min, 15min, 20min, 30min, and second step incubative time is that 20min detects test samples, investigates respectively the indexs such as sensitivity for analysis, specificity, HOOK effect, precision, linearity.The results are shown in following table:
Known according to above experimental result, in the situation that all the other conditions are identical, first step incubative time is that 20min result tends towards stability, and extending first step incubative time has not had practical significance, therefore first step incubative time is defined as to 20min.
1.1.2 determining of second step incubative time
First step incubative time is 15min, and second step incubative time is respectively 10min, 15min, 20min, 30min detect test samples, investigates respectively the indexs such as sensitivity for analysis, specificity, HOOK effect, precision, linearity.The results are shown in following table:
The above results is analyzed relatively, and in the situation that all the other conditions are identical, second step incubative time is that 15min result tends towards stability, and extending second step incubative time has not had practical significance, so we are defined as second step incubative time into 15min.
The investigation of 1.2 application of sample patterns
Test material: adopt the luminous particle reagent (the luminous antibody reagent of lower abbreviation) of the coated 300ug/ml of antibody and the biotin labeling antibody reagent of 30ug/ml, and the coated photosensitive particulate reagent of the Avidin of 40ug/ml
Test samples: calibration product 6 and quality-control product QcL, QcH.
Initial reaction condition: add successively sample, luminous particle reagent, biotin labeling antibody reagent, the coated photosensitive particulate of Avidin, two Buwen bathe, and wherein the first warm bath time was 20min, and the second warm bath time was 15min.
Having designed 2 kinds of application of sample patterns investigates.
The coated photosensitive particulate reagent of the luminous antibody reagent+25ul of pattern 1:25ul sample+25ul biotinylated antibody reagent+175ul Avidin;
The coated photosensitive particulate reagent of the luminous antibody reagent+15ul of pattern 2:15ul sample+15ul biotinylated antibody reagent+105ul Avidin;
The in the situation that of identical under starting condition, according to above two kinds of application of sample patterns, internal control product are detected, investigate respectively the indexs such as sensitivity for analysis, specificity, HOOK effect, precision, linearity.The results are shown in following table:
Known according to above experimental result, pattern 1 sensitivity, accuracy and HOOK effect are all better than pattern 2, therefore preference pattern 1.
The screening of the coated photosensitive particulate concentration of 1.3 luminous antibody, biotinylated antibody, Avidin
Test material: adopt the coated luminous particle reagent of antibody (the luminous antibody reagent of lower abbreviation) and biotin labeling antibody reagent, and the coated photosensitive particulate reagent of Avidin
Test samples: sensitivity reference material and quality-control product QcL, QcH.
Initial reaction condition: add successively 25ul sample, 25ul luminous particle reagent, 25ul biotin labeling antibody reagent, the coated photosensitive particulate of 175ul Avidin, two Buwen bathe, and wherein the first warm bath time was 20min, and the second warm bath time was 15min.
1.3.1 the screening of luminous antibody and biotinylated antibody concentration
The coated photosensitive particulate of Avidin adopts the concentration of 40ug/ml, compound concentration is that the luminous antibody of 200ug/ml, 300ug/ml, 400ug/ml and concentration are that the biotinylated antibody cross match of 30ug/ml, 40ug/ml, 50ug/ml detects respectively, and under starting condition, result is as follows respectively:
The luminous antibody of 200ug/ml
The luminous antibody of 300ug/ml
The luminous antibody of 400ug/ml
The above results is analyzed relatively, and the concentration of luminescence reagent is at 300 μ g/ml, and the concentration of biotinylated antibody result in the time of 50 μ g/ml is ideal, is working concentration therefore select above concentration.
1.3.2 the coated photosensitive particulate concentration screening of Avidin
Luminous antibody concentration is selected 300ug/ml, and the concentration of biotinylated antibody is selected 50ug/ml, and the coated photosensitive particulate concentration of Avidin is formulated as respectively 20ug/ml, 40ug/ml, and 60ug/ml, under initial reaction condition, result is as follows:
According to the mensuration concentration of signal to noise ratio (S/N ratio), the comparison of Hook effect and Qc L and Qc H, the coated photosensitive particulate concentration result in the time of 40ug/ml of Avidin is ideal.
Embodiment 7 evaluation tests
Reagent: adopt the coated photosensitive particulate reagent (concentration is 40ug/ml) of luminous antibody reagent (concentration 300ug/ml), biotin labeling antibody reagent (concentration is 50ug/ml) and Avidin.
Detection method: add respectively 25 μ l samples in reacting hole, then add successively 25 μ l luminescence reagents and 25 μ l biotinylated antibody reagent.Then put into instrument, by instrument operation according to the following steps automatically: vibration, 37 ℃ of incubations 20 minutes, more automatically add after the coated photosensitive particulate reagent of Avidin 175 μ l 37 ℃ of incubations 15 minutes.Instrument automatically produces Ear Mucosa Treated by He Ne Laser Irradiation micropore and calculates the luminous photon amount in every hole, can calculate sample CEA concentration according to typical curve, and unit is ng/ml, finally prints test report.
1. quantitative model performance evaluation
The detection of 1.1 sensitivity for analysis
Detect 10 hole calibration product 1 (0ng/ml), calculate its RLU average (AVE) and two standard deviations (SD), enter typical curve with AVE ± 2SD inverse iteration, the concentration value obtaining is the sensitivity for analysis of this kit
| Sensitivity for analysis (ng/ml) | 0.083 |
After testing, sensitivity for analysis is respectively 0.083ng/ml, not higher than 0.1ng/ml.
1.2 sensing range
In 3 clinical testings o'clock, 1042 normal non-cancer patients' blood sample is detected, 99% sample CEA level is less than 5.35ng/ml.And the patient samples of high concentration CEA level can cause the abnormality decline (being the hook strip effect of high titre) of RLU (photon unit relatively).In this assay method, CEA level will be greater than 1000ng/ml up to the testing result of the patient samples of 100ug/ml.
Therefore determine that sensing range is 0.1-1000ng/ml.
1.4 specific detection
Detect AFP and PSA specificity reference material, must not detect false positive
| AFP specificity reference material (ng/ml) | 2.32 |
| PSA specificity reference material (ng/ml) | 0.45 |
The detection of 1.5 linearities
Other 5 calibration product (prepared by embodiment 5) except 0 value are done to linear analysis, calculate linearly dependent coefficient r (r should be greater than 0.99), r=0.998
The detection of 1.6 accuracies
Carcinomebryonic antigen (CEA) quality-control product of the reagent that adopts respectively 2 lot numbers to high and low 2 levels detects, and repeats 10 times, by result value substitution formula, calculates CV value
1.7Hook test
| Detectable concentration | 1000ng/ml | 10ug/ml | 50ug/ml | 100ug/ml |
| Measure optical signal value | 349184 | 378922 | 423456 | 389452 |
From the above results, Hook effect is showed no at 100ug/ml
1.8 interference tests
In the clinical samples of a concentration known, add haemoglobin, triglyceride and cholerythrin and make piarhemia sample and the bilirubinic jaundice sample of 10mg/dL of the haemolysis sample of 250mg/dL haemoglobin, 500mg/dL triglyceride and detect, the deviation of measured value and original content must not exceed 10%.
| Measured value | Theoretical value | Deviation ratio (%) | |
| Haemolysis sample 1# | 266.38 | 236.21 | 12.77 |
| |
107.14 | 93.48 | 14.61 |
| Haemolysis sample 3# | 32.61 | 28.64 | 13.86 |
| Piarhemia sample 1# | 239.16 | 236.21 | 1.25 |
| |
95.22 | 93.48 | 1.86 |
| Piarhemia sample 3# | 29.64 | 28.64 | 3.49 |
| Jaundice sample 1# | 247.64 | 236.21 | 4.84 |
| |
98.24 | 93.48 | 5.09 |
| Jaundice sample 3# | 30.26 | 28.64 | 5.66 |
From the above results, 500mg/dL triglyceride and 10mg/dL cholerythrin are to this product without obvious interference, but 250mg/dL haemoglobin is on this product testing result, impact exceedes 10%, therefore avoids sample significant hemolysis as far as possible.
Embodiment 8 comparison tests
Reagent: adopt the coated photosensitive particulate reagent (40ug/ml) of luminous antibody reagent (300ug/ml), biotin labeling antibody reagent (50ug/ml) and Avidin.
Carried out quality level comparison take the CEA of Roche Holding Ag kit (electrochemical luminescence system) as reference, result is as follows:
| Sample | Kit of the present invention (corresponding Beyond) | Roche Holding Ag |
| Sensitivity for analysis (ng/ml) | 0.083 | 0.20 |
| Hook effect | 100ug/ml | 200ug/ml |
250 parts of the clinical samples that collection detected through Roche Holding Ag's CEA kit, then measure every part of sample with CEA light-induced chemiluminescent detection kit of the present invention, do parallel comparison, result is as Fig. 4.
Result shows the two kinds of correlativity between method: y=0.7725x+0.5144, r=0.97.Illustrate that carcinomebryonic antigen detection kit of the present invention (light-induced chemiluminescent method) compares with the similar products of external well-known manufacturer production, the technical indicator of whole product is roughly suitable.And at the bottom of this kit cost, highly sensitive, accuracy good, sensing range is wide, easy and simple to handle, save time, be suitable for to clinical expansion.
The assembly of embodiment 9 kits
By in embodiment 1-3, prepare according to the whole bag of tricks the independent packaging respectively of three kinds of reagent, after assembly, obtain basic kit.Can be used for detecting the light activation reaction light signal of sample to be tested.
After the calibration product that add the embodiment 5 of independent packaging to prepare, obtain immue quantitative detection reagent box in mentioned reagent box.Can be used for quantitatively detecting carcinomebryonic antigen (CEA).
Claims (18)
1. for detection of a detection of particles for carcinomebryonic antigen, be the coated luminous particle of anti-CEA antibody, described detection of particles makes through following technique:
A) mix: luminous particle and anti-CEA antibody are mixed in damping fluid;
B) reaction: add the EDAC solution of damping fluid preparation mix and react, reaction conditions is 37 ℃ of revolving reactions, and luminous particle is (15-40) with the mass ratio of EDAC: 1 mixes;
C) in the reactant liquor obtaining toward step b, add the BSA solution of damping fluid preparation mix and react, reaction conditions is 37 ℃ of revolving reactions;
D) wash products, obtains the coated luminous particle of anti-CEA antibody;
Described luminous particle surface functional group is selected from carboxyl; Described damping fluid is MES damping fluid.
2. as claimed in claim 1 for detection of the detection of particles of carcinomebryonic antigen, it is characterized in that, described luminous particle particle diameter is that 50nm is to 400nm.
3. as claimed in claim 1 for detection of the detection of particles of carcinomebryonic antigen, it is characterized in that, the luminous quantity of described luminous particle is 150,000-350,000 photon number/100 μ g luminous particle.
4. as claimed in claim 1 for detection of the detection of particles of carcinomebryonic antigen, it is characterized in that, in described step a, the mass ratio of luminous particle and anti-CEA antibody is (8-15): 1.
5. as claimed in claim 4 for detection of the detection of particles of carcinomebryonic antigen, it is characterized in that, described in, the mass ratio of luminous particle and anti-CEA antibody is 12.5:1.
6. as claimed in claim 1 for detection of the detection of particles of carcinomebryonic antigen, it is characterized in that, the mode of the cleaning of described steps d is that centrifuge method is cleaned.
7. a detection kit for carcinomebryonic antigen, comprises in claim 1-6 the detection of particles for detection of carcinomebryonic antigen described in arbitrary claim.
8. the detection kit of carcinomebryonic antigen as claimed in claim 7, is characterized in that, described kit also comprises one or both in biotin labeled anti-CEA antibody or the coated photosensitive particulate of Avidin.
9. the detection kit of carcinomebryonic antigen as claimed in claim 8, is characterized in that, in described biotin labeled anti-CEA antibody, the molecule ratio of biotin and antibody is (30-50): 1.
10. the detection kit of carcinomebryonic antigen as claimed in claim 8, is characterized in that, in the coated photosensitive particulate of described Avidin, the mass ratio of Avidin and photosensitive particulate is 1:(3-10).
11. detection kit of carcinomebryonic antigen as claimed in claim 8, is characterized in that, the photosensitive particulate independent packaging respectively that the described detection of particles for detection of carcinomebryonic antigen, biotin labeled anti-CEA antibody and Avidin are coated, and be suspension.
12. detection kit of carcinomebryonic antigen as claimed in claim 11, is characterized in that, the solvent of described suspension is selected from HEPES buffer system or Tris buffer system.
13. detection kit of carcinomebryonic antigen as claimed in claim 12, is characterized in that, the composition that the solvent of the described detection of particles suspension for detection of carcinomebryonic antigen is pH8.0 is the HEPES damping fluid of HEPES, NaCl and EDTA-Na-2H2O.
14. detection kit of carcinomebryonic antigen as claimed in claim 12, is characterized in that, the composition that the solvent of described biotin labeled anti-CEA antibody suspension is pH8.0 is the Tris damping fluid of Tris, NaCl and EDTA-Na-2H2O.
15. detection kit of carcinomebryonic antigen as claimed in claim 12, is characterized in that, the solvent of the coated photosensitive particulate suspension of described Avidin is that composition is the HEPES damping fluid of HEPES, NaCl and EDTA-Na-2H2O.
16. detection kit of carcinomebryonic antigen as claimed in claim 11, is characterized in that, also comprise protein protective agent, prevent one or more in stable reagent or the antiseptic of particles agglomerate in described suspension.
17. as described in arbitrary claim in claim 7-16 the detection kit of carcinomebryonic antigen, it is characterized in that, in described detection kit, also comprise positive control and negative control.
18. as described in arbitrary claim in claim 7-16 the detection kit of carcinomebryonic antigen, it is characterized in that, in described kit, also comprise the carcinomebryonic antigen solution that multiple concentration is known, the independent packaging respectively of the carcinomebryonic antigen solution of variable concentrations.
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