CN101865916B - Tetraiodothyroxide test kit and use method thereof - Google Patents
Tetraiodothyroxide test kit and use method thereof Download PDFInfo
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- CN101865916B CN101865916B CN200910049307.XA CN200910049307A CN101865916B CN 101865916 B CN101865916 B CN 101865916B CN 200910049307 A CN200910049307 A CN 200910049307A CN 101865916 B CN101865916 B CN 101865916B
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Abstract
The invention relates to an auxiliary diagnostic kit of thyroid diseases, and discloses a tetraiodothyroxide test kit which comprises tetraiodothyroxide testing particles, tetraiodothyroxide resisting antibody marked by biotin and disintegrating agent; wherein triiodothyronine testing particles are luminous particles coated by diiodothyronine, and the disintegrating agent is 8-anilino-1- naphthalenesulfonic acid ammonium salt hydrate. The invention also discloses a use method of a triiodothyronine kit. The kit can be combined with other serum and clinical information for the auxiliary diagnosis of thyroid diseases and the monitoring of the treatment effect of relevant patients.
Description
Technical field
The present invention relates to the detection kit of TA, be specifically related to detection kit and the using method thereof of the TA based on light-induced chemiluminescent principle.
Background technology
Thyroid disease is one group of more common endocrine system disease, comprises goitre that goiter due to iodine deficiency, other reasons cause, hyperthyroidism, thyroid adenoma, thyroiditis, hypothyroidism etc.The incidence of thyropathy is higher.
Hyperthyroidism is hyperthyroidism namely, and it is a kind of very common endocrine system disease clinically.Refer to by a variety of causes and cause thyroid function to strengthen, thyroid hormone secretion too much or increase a series of hypermetabolism syndromes and high Agitation and the eye symptom of the multisystems such as caused organism nervous system, the circulation system, digestive system cardiovascular system because of thyronine (T4, T4) level in blood.
Hypothyroidism is the synthetic and hyposecretion of thyroid hormone TSH, or thyroid hormone physiological effect is bad, biological effect is not enough and the systemic disease that causes.Can be divided into clinically cretinism, childhood first low, adult's first is low.If hypofunction starts from fetus or neonatal period, be called cretinism; Before starting from sexual development, children claim juvenile form first to subtract; Starting from into people claims adult first to subtract.
Thyroid adenoma is common clinical, and wherein the overwhelming majority is benign lesion, and minority is cancer, sarcoma, malignant lymphoma etc.This disease women incidence of disease is apparently higher than the male sex, men and women's ratio approximately 1: 2～3 of falling ill.
Thyroiditis is the thyroid disease taking inflammation as main manifestations, comprises infectious and non-infectious.Not rare.Upper said acute, the subacute and chronic thyroiditis of clinical authority, only show the length of young lysis, between them, without inner link, also do not change mutually, every kind has and delivers the different causes of disease and have clinical characters, pathologic process and intrinsic final result.
TA (3, 5, 3 ', 5 '-tetraiodo-L-thyronine, abbreviation T4), it is the hormone producing from thyroid follicle epithelial secretion, be released into after blood, the T4 overwhelming majority all with TBG (thyroid binding globulin) combination, be combined by special receptor protein in nucleus, induce this albumen to produce activity conformation, make the effective combination of privileged site of its DNA binding site and DNA, thereby activate the transcription process of DNA, reach the biological function that regulatory gene is expressed, wherein approximately 0.03% total serum T4 is free state, with in conjunction with hormone mobile equilibrium, maintain normal physiological function.Large number of biological is learned and physiological the results show, the body heat regulation of the normal development of thyroid hormone to cell and differentiation, animal and various sugar, fat and protein metabolism balance maintained important regulation and control effect.Therefore, detect diagnosis or the antidiastole of serum T 4 for hyperthyroidism, Hypothyroidism and thyroid tumors, in evolution, curative effect and prognosis evaluation to thyroid function relevant disease, tool is of great significance.
In serum, T4 concentration is higher than normal value, Ke Nengshi: (1) hyperthyroidism; (2) drug influence: as take thyroxine, estrogens medicine and contraceptive, perphenazine etc.; (3) Other diseases: as congenital hereditary thyroid binding globulin increases disease, acute infectious hepatitis, intermittent hematoporphyria etc.; (4) physiologic factor: as gestation.
In serum, T4 concentration is lower than normal value, Ke Nengshi: (1) thyroid disease: disease, chronic lymphocytic thyroiditis under primary thyroid function; (2) drug influence: antithyroid drug; (3) Other diseases: serious liver and kidney failure, nephrotic syndrome, activity acromegalia, heredity thyroxine-binding globulin reduce disease etc.
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); Photobiology mark taking 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.
summary of the invention
The object of this invention is to provide a kind of detection kit and using method thereof that can be used for quantitatively detecting TA in serum.
Know-why of the present invention:
TA detection reagent of the present invention 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 the level of the TA in serum, first use the agent of dissociating in sample, to dissociate with the TA (T4) of thyroxine-binding globulin (thyroxine-binding globulin, TBG) combination.Then under homogeneous phase condition, using inside with the photosensitive particulate (nanoscale) of dyestuff and be coated with triiodo thryonine (T3) and the potpourri of the inner luminous particle (nanoscale) with luminophor as reagent with detect sample mix.
T3 has another name called triiodo thryonine, is a kind of hormone of secreting out from thyroid gland, is one group of tyrosine containing iodine, taking iodine and tyrosine as raw material synthetic in thyroid gland gland cell.T3 secretory volume is less, but it is active large, is 5 times of T4.T3, outside thyroid gland, can be formed by thyroxinic de-iodate, and both are structurally very similar.The T3 overwhelming majority in blood is combined with TBG, and approximately 0.4% free T3 acts on target cell, maintains normal physiological function.In approximately 20% circulation, T3 is produced by thyroid gland, and all the other are produced by de-iodine (5 ' D-I) conversion of outer shroud of T4 80% mainly from liver.
T3 molecular formula is C15H12I3NO4, and structure is as follows:
T4 molecular formula is C15H11I4NO4, and structure is as follows:
Due to T4 and T3 molecular structure quite similar, can and there is faint combination for the antibody of T4 in T3 therefore.In the time there is T4 in serum, this fragile combination is destroyed, is replaced by the combination of T4 and T4 antibody.
Now nanometer photosensitive particulate can catch TA (T4) quickly and effectively with the nano luminescent particulate that is coated with surface 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 TA (T4), 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.
It is documented, 8-Anilino-1-naphthalenesulfonic acid ammonium salt (8-Anilino-1-naphthalenesulfonic acid ammoniumsalt, be called for short ANS) can by and albumen hydrophobic region between strong combination, thereby protein conformation is changed, the interaction of dissociating between albumen.(Yoshimura，T.，Monitoring protein conformational changes duringmembrane fusion.Method Enzymol.，221，72-82)
Based on above-mentioned principle, first aspect present invention provides a kind of detection kit for detection of TA (T4), comprising: TA detection of particles, biotin labeled anti-TA antibody and the agent of dissociating; Wherein TA detection of particles is the coated luminous particle of triiodo thryonine, and the agent of dissociating is 8-Anilino-1-naphthalenesulfonic acid ammonium salt, Sodium Mercurothiolate, sodium salicylate or sodium trichloroacetate.In kit, also can comprise the photosensitive particulate that Avidin is coated.
In kit, luminous particle, biotin labeled anti-TA antibody (T4Ab), dissociate agent and the coated photosensitive particulate independent packaging respectively of Avidin that above-mentioned triiodo thryonine (T3) is coated, and coated luminous particle, biotin labeled anti-TA antibody (T4Ab) and the coated photosensitive particulate of Avidin of triiodo thryonine (T3) is suspension.The solvent of the above-mentioned solution that contains the coated photosensitive particulate of the coated luminous particle of triiodo thryonine (T3), biotin labeled anti-TA antibody (T4Ab) 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 triiodo thryonine (T3) is the HEPES damping fluid of HEPES, NaCl, EDTA and BSA, and the composition of the preferred pH8.0 of solvent of biotin labeled anti-TA antibody (T4Ab) is Tris, NaCl and EDTA-Na-2H
2the Tris damping fluid of O, the preferred HEPES of solvent, NaCl and the EDTA-Na-2H of the coated photosensitive particulate of Avidin
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.
When for quantitative detection TA (T4), in mentioned reagent box, also can comprise the solution that contains multiple known TA concentration.The independent packaging respectively of the solution of above-mentioned different TA concentration.
The above-mentioned agent of dissociating is selected from 8-Anilino-1-naphthalenesulfonic acid ammonium salt, Sodium Mercurothiolate, sodium salicylate or sodium trichloroacetate, is preferably 8-Anilino-1-naphthalenesulfonic acid ammonium salt (ANS).The damping fluid of agent of dissociating can for Tris damping fluid, PBS damping fluid or distilled water, be preferably distilled water.
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 (< 100nm) 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 100-400nm, preferably between 150-300nm, 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 the joint efficiency of better triiodo thryonine (T3), reagent stability and is connected repeatedly, through testing the particulate of preferred aldehydes primary surface functional group, and has applied NaBH
3the reductive amination process of CN is as the method for attachment of antibody.
The luminous quantity of luminous particle is the final illumination effect detecting of impact directly.The general meeting of luminous particle luminous quantity that market provides is 150,000---within the scope of 350,000 photon number/100 μ g luminous particle, and preferably medium tenacity level (>=250,000 photon number/100ug) luminous particle on the upper side.
In the coated luminous particle of above-mentioned triiodo thryonine (T3), the mass ratio of luminous particle and triiodo thryonine (T3) is preferably 10: (0.01-0.05), and preferably 10: 0.02.
In above-mentioned biotin labeled antibody (T4Ab), the molecule ratio of biotin and antibody is preferably (10-50): 1, and preferably 30: 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 triiodo thryonine (T3) adopts NaBH
3the reductive amination process legal system of CN is standby, and reactions steps is as follows:
1) mix: by luminous particle and triiodo thryonine (T3) in mass ratio example be 10: (0.1-0.5) 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 solution;
4) wash products, obtains the coated luminous particle of triiodo thryonine (T3).
Wherein, in blend step, the mass ratio of luminous particle and triiodo thryonine (T3) is 10: (0.1-0.4), and preferably 10: 0.2.Reaction buffer can be MES damping fluid, phosphate buffer, preferably MES damping fluid, and the preferred 0.05M of concentration, 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 triiodo thryonine (T3) can adopt following method to make:
1) mix: luminous particle and triiodo thryonine (T3) are mixed in damping fluid.Preferably, damping fluid is MES damping fluid, and the mass ratio of luminous particle and triiodo thryonine (T3) is 10: (0.01-0.05), and preferably 10: 0.02.
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 preferably 25: 1 with the mass ratio of EDAC and mixes.Reaction conditions is 37 DEG C 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 5: 8.Reaction conditions is 37 DEG C of revolving reactions.General approximately 16 hours sufficient reactings.
4) wash products, obtains the coated luminous particle of triiodo thryonine (T3).
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 10 hour, and the cross-linking efficiency of luminous particle and diiodothyronine obtained raising, has saved diiodothyronine 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.
Second aspect present invention, discloses the using method of the detection kit of above-mentioned TA, and the detection kit of TA is utilized light-induced chemiluminescent principle, adopts double antibody sandwich method, can quantitatively detect the TA in serum.
The using method of the detection kit of TA (T4) comprises the following steps:
First use the agent of dissociating to carry out pre-service to sample; then by the detection of particles for detection of TA, biotin labeled free anti-TA antibody (T4Ab) and the coated photosensitive particulate hybrid reaction of Avidin in sample and kit; then irradiate reacting hole with exciting light, measure the luminous photon amount of each reacting hole and obtain optical signal value.
Specifically comprise the following steps:
1) in reacting hole, add sample, then add successively the coated luminous particle of T3, dissociate agent and biotin labeled anti-T4 antibody (T4Ab), obtain 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.
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.
The detection kit of above-mentioned TA (T4), in the time being applied in quantitative detection TA (T4), need be calculated T4 content in testing sample according to typical curve.
Above-mentioned steps 1) reaction conditions be 37 DEG C of incubation 10-30 minute, preferably 20 minutes, step 2) reaction conditions be also 37 DEG C of incubation 10-30 minute, preferably 15 minutes.
In above-mentioned initial reaction solution, the concentration of the coated luminous particle of triiodo thryonine (T3), without particular restriction, can be 50-200 μ g/ml, preferably 150 μ g/ml.
The working concentration of the above-mentioned agent of dissociating can be 0.5mg/ml-1.5mg/ml, is preferably 1mg/ml.
In initial reaction solution, the concentration of biotin labeled anti-TA antibody, without particular restriction, can be 0.5-1.5 μ g/ml, is preferably 1 μ g/ml.
In end reaction solution, the concentration general control of the coated photosensitive particulate of Avidin, at 10-100 μ g/ml, can be 30-60 μ g/ml, preferably 40 μ g/ml.
In said method, the volume of initial reaction solution, without particular restriction, can be 60-100 μ l, preferably 100 μ l; The volume of end reaction solution, also without particular restriction, can be 120-275 μ l, preferably 275 μ l.
Above-mentioned sample comprises serum, blood plasma, whole blood or standard items.
In the time that detection kit of the present invention is used for quantitatively detecting TA (T4), also need to calculate according to typical curve the content of TA in testing sample
The present invention adopts light-induced chemiluminescent detection technique, coordinate light-induced chemiluminescent analytic system to measure the quantitative external diagnosis reagent case of TA (T4) in human serum sample, can combine with other serum and clinical information for the auxiliary diagnosis of thyroid disease and the monitoring of related diseases human therapy effect.TA of the present invention (T4) light-induced chemiluminescent detection kit is highly sensitive, accuracy good, sensing range is wide and easy and simple to handle, quick, cost is low, good stability, non-environmental-pollution and radiologic hazard, be with a wide range of applications clinically.
Brief description of the drawings
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: comparison test test result schematic diagram.
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
T3 Biochemika
Gly Sigma ≥95％
HEPES is through HTC of section
Tris traditional Chinese medicines
NaBH
3CN Acros
ANS Sigma
Anti-T4 monoclonal antibody Biospecfic
PentaTek company of the photosensitive particulate U.S.
PentaTek company of the luminous particle U.S.
8-Anilino-1-naphthalenesulfonic acid ammonium salt (ANS) Sigma
Instrument model producer
Light-induced chemiluminescent analyser HT Shanghai Bo Yang Medical Instruments company
Ultraviolet spectrophotometer 752P Shanghai spectrum company
High speed freezing centrifuge CR21G HITACHI
Analytical balance ALC-2100.2 ACCULAB
Analytical balance AG285 METTLER
PH meter DELTA320 METTLER
Particle instrument Model 370 Nicomp
Microplate reader MultiSKAN MK3 labsystem
Ultrasonic cleaner KQ5200E Kunshan ultrasonic instrument company
Its woods Bel of eddy mixer QL-901
China of magnetic stirring apparatus (79-1) 2003-03 state
The preparation of the coated luminous particle of embodiment 1 T3
Antibody and luminous particle preparation method:
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: T3 dialyses 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 2mg/ml.
3) T3 (MES damping fluid) of the luminous particle suspension of MES damping fluid, 100mg/ml (MES damping fluid) and 2mg/ml mixes with the volume ratio of 1: 10: 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 DEG C 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 DEG C 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 processing: draw a certain amount of luminous particle centrifugal in high speed freezing centrifuge, supernatant discarded, adds a certain amount of pH6.00.05M MES damping fluid (hereinafter to be referred as MES damping fluid), ultrasonic to particulate Eddy diffusion.
2) mix: T3 is mixed with the solution of 1mg/ml with 0.2N NaOH.According to 10mg luminous particle: 0.2mg T3 adds T3 solution, and adding MES damping fluid to luminous particle solid content is 25mg/ml, vibration mixes and obtains reactant liquor.
3) reaction: with the NaBH3CN solution of MES damping fluid preparation 25mg/mL, according to adding with the reactant liquor volume ratio of 3: 250, mix.37 DEG C of revolving reaction 68-70 hour.
4) sealing: with 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 DEG C of revolving reactions 2 hours.
5) clean: in completely reacted luminous antibody, add 0.1M Na
2cO
3solution, high speed freezing centrifuge is centrifugal, abandons supernatant, adds fresh 0.1M Na
2cO
3, Eddy diffusion, again centrifugal, so clean 4 times, finally suspend with a small amount of luminescence reagent damping fluid.
6) solid content, the particle diameter of the cleaned luminous antibody of sampling and measuring, regulates luminous antibody concentration 10mg/mL to prepare to dilute with luminescence reagent damping fluid.
Both prepare the comparison of result:
1. saved preparation time:
| Original preparation technology | New preparation process | |
| The luminous particle of preparation 100mg cross-linking antibody | 74h | 64h |
2. improve antibody linked efficiency, saved antibody consumption:
| Original preparation technology | New preparation process | |
| The antibody linked particulate of 1mg | 50mg | 500mg |
3. strengthened reaction signal:
| Original preparation technology calibrates product 1 signal value | New preparation process calibration product 1 signal value | |
| The anti-T4 antibody response of biotinylation of the luminous particle that 150ug/ml T3 is coated and 1 μ g/ml | 120000 | 170000 |
4. improved sensitivity:
| Original preparation technology calibrates product 2 signal values/calibration product 1 signal value | New preparation process calibration product 2 signal values/calibration product 1 signal value | |
| The anti-T4 antibody response of biotinylation of the luminous particle that 150ug/ml T3 is coated and 1 μ g/ml | 75000/120000＝0.625 | 90000/170000＝0.53 |
5. expanded sensing range:
| Original preparation technology's sensing range | New preparation process sensing range | |
| The anti-T4 antibody response of biotinylation of the luminous particle that 150ug/ml T3 is coated and 1 μ g/ml | 0-16ug/dl | 0-32ug/dl |
Prepare the coated luminous particle of T3 with reference to above-mentioned preparation method, and test by properties the selection research of relatively having carried out concrete technology condition from following several respects:
The detection method of the parameters relating in the present embodiment is as follows:
1. light signal detection method:
In reacting hole, add respectively 25 μ l samples, then add successively 25 μ l luminescence reagents (150 μ g/ml), 25ul dissociate agent ANS (1mg/ml), 25 μ l biotinylated antibody reagent (1 μ g/ml).Then put into instrument (light-induced chemiluminescent analytic system), automatically operated according to the following steps by instrument: vibration, 37 DEG C of incubations 20 minutes, more automatically add the rear 37 DEG C of incubations of the coated photosensitive particulate reagent of 175 μ l Avidins (40 μ g/ml) 15 minutes.Instrument automatically produces Ear Mucosa Treated by He Ne Laser Irradiation micropore and calculates the luminous photon amount in every hole.
2. accuracy detection method:
Adopt respectively the T4 kit of 2 lot numbers to carry out 1 detection to quality-control product QCL, QCH, each repetition measurement 10 holes, every duplicate samples detects 20 times altogether.Obtain following result:
From the above results, in its batch of this product, accuracy is less than 5%, and between batch, accuracy is less than 10%.
3. sensitivity detection method:
Detect 10 hole calibration object 0 μ g/dl, 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, the reagent of two lot numbers after testing, its sensitivity for analysis is respectively 0.46ug/dL, 0.50ug/dL, all not higher than 1 μ g/dl.
4. process conditions are selected:
1) investigation of antiseptic
For the consideration of and standing storage anticorrosion to reagent, we select 5/10000ths Proclin 300 to investigate as antiseptic.
Detect and do not add antiseptic and add serum, normal human serum, the serum of T4 measured value >=25 μ g/dL, the 1%BSA 0.01M PBS of 5/10000ths Proclin 300 as the T4 measured value≤2 μ g/dL of antiseptic.Every kind of condition detects two holes, gets RLU and detects average.The results are shown in following table:
| RLU average | Utmost point low value serum | Normal human serum | High value serum | 1％BSA 0.01M PBS |
| Do not add antiseptic | 168597 | 23810 | 1202 | 164102 |
| Add antiseptic | 167926 | 24339 | 1227 | 160455 |
Can find test findings not to be had a significant effect as antiseptic using 5/10000ths Proclin 300 according to above result, therefore select 5/10000ths Proclin 300 as antiseptic.
2) selection of calibration object dilution
For the calibration object dilution that obtains long-term stability and more easily obtain replaces the not serum containing T4, with the clinical serum of 20 parts of T4 measured value≤2 μ g/dL as with reference to investigating alternative calibration object dilution, alternative calibration object dilution have respectively physiological saline, distilled water, interpolation 1%BSA 0.01M pH 7.4PBS, remove hormone cow's serum, every part of survey single hole of 20 parts of negative serums, the RLU that calculates this 20 hole detects average; Every kind of detection two holes of alternative calibration object dilution, get RLU and detect average.Testing result is as follows
| Physiological saline | Distilled water | Remove hormone cow's serum | 1％BSA PBS | With reference to serum | |
| RLU (average) | 112461 | 126750 | 193919 | 168175 | 168523 |
The optical signal level that can find 1%BSA 0.01M PBS according to above result is suitable with reference to serum with clinical T4, so we select 1%BSA 0.01M PBS as calibration object dilution.
3) reaction ratio of luminous particle and T3
Luminous particle: T3 is coated in the ratio of 10mg/0.01mg, 10mg/0.02mg, 10mg/0.05mg respectively, relatively chooses best coated reaction ratio.
Detect the coated luminous particle of T3 that three kinds of differential responses ratios prepare, its sensitivity, accuracy, linearity.Testing result is as following table:
The comparison of luminous particle and T3 reaction ratio
Known according to above experimental result, the coated substantially proportional relation of antigen amount in antigen addition and luminous particle, but the antigen of T3 antigen on coated while continuing to increase reaches capacity.Consider QC result and Cost Problems, select the reaction ratio of 10mg luminous particle and 0.02mg T3 can obtain good linearity and sensitivity.
4) luminescence reagent damping fluid
HEPES damping fluid, PBS damping fluid for luminous particle coated T3 are made into respectively to the concentration of 150 μ g/ml, relatively choose optimized buffer liquid.
HEPES damping fluid: pH8.0,0.05M HEPES+0.3M NaCl+25mM EDTA+1.6%BSA+ surfactant+antiseptic;
PBS damping fluid: pH7.4,0.02M PBS+1%BSA+ surfactant+antiseptic;
With the coated luminous particle of T3 of two kinds of damping fluids preparation, detect its sensitivity, 37 DEG C and destroy 7 days stability, linearity.Testing result is as following table:
The luminous particle damping fluid comparison that T3 is coated
Known according to above experimental result, 37 DEG C of luminous particle coated T3 with two kinds of damping fluid preparations are destroyed after 7d, entirety RLU has decline to a certain degree, linear in significant change, but the degree that HEPES damping fluid declines than the sensitivity of PBS damping fluid is smaller, therefore selects the damping fluid of HEPES as reagent 1.
5) selection of antiseptic
For the consideration of and standing storage anticorrosion to reagent, we select 5/10000ths Proclin 300 to investigate as antiseptic.
Detect and do not add antiseptic and add serum, normal human serum, the serum of T4 measured value >=25 μ g/dL, the 1%BSA 0.01M PBS of 5/10000ths Proclin 300 as the T4 measured value≤2 μ g/dL of antiseptic.Every kind of condition detects two holes, gets RLU and detects average.The results are shown in following table:
| RLU average | Utmost point low value serum | Normal human serum | High value serum | 1％BSA 0.01M PBS |
| Do not add antiseptic | 168597 | 23810 | 1202 | 164102 |
| Add antiseptic | 167926 | 24339 | 1227 | 160455 |
Can find test findings not to be had a significant effect as antiseptic using 5/10000ths Proclin 300 according to above result, therefore select 5/10000ths Proclin 300 as antiseptic.
6) selection of cleaning way
Dialysis cleaning step: 100 times of volume dialysis, each 4-5 hour, exchange buffering liquid 2 times.The dialysis cleaning operation time is longer, and cleans not thorough.
Centrifugal 30 minutes of centrifuge method cleaning step: 12000rpm, cleans 3 times, precipitates resuspended with ultrasonic method.It is shorter that this method is cleaned the time used, effective.Consider and select centrifuge method to clean.
The final particle diameter of selecting 250nm, luminous quantity is >=250, the luminous particle of 000 photon number/100ug, the 0.01M pH 7.4PBS of 1%BSA is as dilution, 5/10000ths Proclin 300 are as antiseptic, the luminous particle reaction density of 20mg/ml, the FG-bead of 10: 0.02 and the ratio of T3, centrifuge method is cleaned as optimum preparation condition.
The preparation of embodiment 2 biotin labeling antibody
Preparation method:
1) antibody treatment: anti-T4 monoclonal antibody (mark) is dialysed in 0.1M NaHCO3 solution, measure antibody concentration and be adjusted to 1mg/mL;
2) with the Biotin solution of DMSO preparation 16.17mg/mL;
3) mark: get the anti-T4 monoclonal antibody of the 1mg/mL handling well (mark) and the Biotin solution preparing, the two mixes according to the volume ratio of 10000: 54, mixes rapidly; 2～8 DEG C leave standstill reaction 12～16 hours;
4) dialysis: completely reacted biotin labeling antibody is dialysed in biotin labeling dialysis buffer liquid;
5) the biotinylated antibody sucking-off of having dialysed is transferred in clean centrifuge tube, sampling and measuring antibody concentration, uses biotin labeling dialysis buffer liquid to regulate biotin labeling antibody concentration to 0.5mg/mL.
The detection method of the parameters relating in the present embodiment is as follows:
1, antibody reacted according to different proportion with Biotin and detect:
By Biotin-X-X-NHS: antibody carries out mark according to the different proportion of 20: 1,30: 1,40: 1, relatively chooses best mark ratio.
Detect the three kinds of biotin that isolabeling ratio does not prepare-anti-T4, its sensitivity, accuracy and linearity.Testing result is as following table:
The mark ratio of antibody and biotin
Known according to above experimental result, biotin: anti-T4 mark ratio is 30: 1 o'clock, and its sensitivity is better, and cost is also lower.Therefore select the ratio of 30: 1 as biotin: the mark ratio of anti-T4.
2, choosing of biotin labeling antibody damping fluid:
By anti-biotin labeling for T4 antibody Tris damping fluid, PBS damping fluid be made into respectively the concentration of 1 μ g/ml, relatively choose optimized buffer liquid.
Tris damping fluid: pH 8.0,0.1M Tris+0.3M NaCl+25mM EDTA+0.1%BSA+ surfactant+antiseptic;
PBS damping fluid: pH7.4,0.02M PBS+1%BSA+ surfactant+antiseptic;
With the anti-T4 of biotin labeling of two kinds of damping fluids preparation, detect its sensitivity, 37 DEG C and destroy 7 days stability, linearity.Testing result is as following table:
The comparison of the anti-T4 damping fluid of biotin labeling
Known according to above experimental result, the anti-T437 of biotin labeling DEG C for preparing with two kinds of damping fluids was destroyed after 7 days, entirety RLU has decline to a certain degree, linear in significant change, but it is smaller that Tris damping fluid declines than the sensitivity of PBS damping fluid, therefore selects the damping fluid of Tris as reagent 2.
3, choosing of biotin labeling antibody-solutions working concentration:
3.1. the anti-T4 working concentration of biotin labeling is chosen
Anti-biotin labeling in reagent 2 T4 is made into respectively to the concentration of 0.5 μ g/ml, 1 μ g/ml, 1.5 μ g/ml with Tris damping fluid, relatively chooses best effort concentration.
With the reagent 2 of the anti-T4 of biotin labeling of three kinds of variable concentrations, detect its sensitivity, linearity.Testing result is as following table:
Choosing of the anti-T4 working concentration of biotin labeling
Known according to above experimental result, when in reagent 2, the working concentration of the anti-T4 of biotin labeling is 1 μ g/ml, its sensitivity, linearity are better.
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 DEG C 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 DEG C 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 DEG C 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 preparation that embodiment 4 dissociates agent ANS
The agent of dissociating is used 8-Anilino-1-naphthalenesulfonic acid ammonium salt (ANS) to be dissolved in 95% ethanol and is mixed with 10mg/mL concentrate, then adds 5/10000ths the anticorrosion storage of Proclin-300.
The detection method of the parameters relating in the present embodiment is as follows:
1, dissociate the choosing of agent damping fluid:
Tris damping fluid, PBS damping fluid, distilled water for 8-Anilino-1-naphthalenesulfonic acid ammonium salt (ANS) are made into respectively to the concentration of 1mg/ml, relatively choose optimized buffer liquid.
Tris damping fluid: pH 8.0,0.1M Tris+0.3M NaCl+25mM EDTA+0.1%BSA+ surfactant+antiseptic;
PBS damping fluid: pH7.4,0.02M PBS+1%BSA+ surfactant+antiseptic;
With the agent of dissociating of three kinds of damping fluids preparation, detect its sensitivity, 37 DEG C and destroy 7 days stability, linearity.Testing result is as following table:
The agent damping fluid comparison of dissociating
Known according to above experimental result, dissociating after 37 DEG C of destruction 7d of agent with three kinds of damping fluid preparations, entirety RLU has decline to a certain degree, linear in significant change, but use distilled water more smaller than Tris damping fluid and PBS damping fluid sensitivity decline, therefore select the damping fluid of distilled water as the agent of dissociating.
2, dissociate the choosing of agent working concentration:
The ANS dissociating in agent is made into respectively to the concentration of 0.5mg/ml, 1mg/ml, 1.5mg/ml with distilled water, relatively chooses best effort concentration.
Use the agent of dissociating of the ANS of three kinds of variable concentrations, detect its sensitivity, linearity.Testing result is as following table:
Dissociate the choosing of agent working concentration
Known according to above experimental result, when in the agent of dissociating, ANS working concentration is 1mg/ml, its sensitivity, linearity are better.
3, dissociate the choosing of agent application of sample pattern:
Add respectively reagent 1 and reagent 2 to be mixed with the working concentration of 1mg/ml ANS, compare with the dissociate application of sample pattern of agent+reagent 2 of reagent 1+.
Pattern 1: reagent 1+ANS+ reagent 2;
Pattern 2:ANS adds in reagent 1;
Mode 3: ANS adds in reagent 2;
Destroy 7 days stability, linearity with three kinds of its sensitivity of application of sample mode detection, 37 DEG C.Testing result is as following table:
Known according to upper table, no matter ANS is to add in reagent 1, still adds in reagent 2, and 37 DEG C are destroyed reaction system after 7 days and all can greatly be affected, therefore preference pattern 1.
The preparation of embodiment 5 standard items
Standard items: the quantitative reference material of TA that uses Ministry of Public Health's visiting center to produce, taking PBS as dilution, according to concentration 0ug/dl, 1ug/dl, 2.4ug/dl, 6.5ug/dl, 15ug/dl, 32ug/dl prepares 6 standard items.
Embodiment 6 TAs quantitatively detect
Then in reacting hole, add respectively sample, then add successively luminescence reagent (luminous particle that T3 is coated), agent and the anti-T4 antibody of biotin labeling dissociate.Then put into instrument (light-induced chemiluminescent analytic system), automatically operated according to the following steps by instrument: vibration, 37 DEG C of incubations.Automatically add again after the coated photosensitive particulate of Avidin 37 DEG C 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 standard items concentration, it is linear that typical curve is.According to typical curve, calculate the T4 content of each sample by sample measured light signal value, unit is μ g/dL.Finally print test report.
The Optimum Experiment of testing conditions:
1, determining of incubative time:
Test material: adopt the coated luminous particle reagent of T3 (the luminous antibody reagent of lower abbreviation) of 150 coated μ g/ml of antibody and the biotin labeling antibody reagent of 1 μ g/ml, the agent ANS that dissociates of 1mg/ml, and the coated photosensitive particulate reagent of the Avidin of 40 μ g/ml.
Test samples: sensitivity reference material and quality-control product QcL, QcH.
Initial reaction condition: application of sample amount is sample 25 μ l, luminous particle reagent 25 μ l, the agent 25 μ l that dissociate, biotin labeling antibody reagent 25 μ l, the coated photosensitive particulate reagent of Avidin is 175 μ l, two Buwen bathe.
Determining of 1.1 first step incubative times
The first step is tested respectively incubative time and is respectively 10min, 15min, 20min, 30min, and second step incubative time is 15min, relatively chooses the suitableeest incubative time.
Detect its sensitivity, precision, linearity with four kinds of different incubative times.Testing result is as following table:
The comparison of first step incubative time
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.
Determining of 1.2 second step incubative times
First step incubative time is 20min, and second step is tested respectively incubative time and is respectively 10min, 15min, 20min, 30min, relatively chooses the suitableeest incubative time.
Detect its sensitivity, precision, linearity with four kinds of different incubative times.Testing result is as following table:
The comparison of second step incubative time
Known according to above experimental result, 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, therefore second step incubative time is defined as to 15min.
2, the investigation of application of sample pattern
According to the documents and materials about the antigen-antibody reaction time, and we have designed two kinds of application of sample patterns and have investigated in conjunction with the feature of this method.
Luminous antibody reagent+the 25ul of the pattern 1:25ul sample+25ul coated photosensitive particulate of agent+25ul biotinylated antibody reagent+175ul Avidin that dissociates;
Luminous antibody reagent+the 15ul of the pattern 2:15ul sample+15ul coated photosensitive particulate of agent+15ul biotinylated antibody reagent+60ul Avidin that dissociates.
According to above two kinds of application of sample patterns, investigate respectively sensitivity, precision, linearity.The results are shown in following table:
The comparison of application of sample pattern
Known according to above experimental result, pattern 1 sensitivity, accuracy are all better than pattern 2, therefore preference pattern 1.
3, the screening of biotinylated antibody concentration:
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 25 μ l samples, 25 μ l luminous particle reagent, 25 μ l dissociate agent ANS, 25 μ l biotin labeling antibody reagents, the coated photosensitive particulate of 175 μ l Avidin, two Buwen bathe, wherein the first warm bath time was 20min, and the second warm bath time was 15min.
Anti-biotin labeling T4 antibody is made into respectively to the concentration of 0.5 μ g/ml, 1 μ g/ml, 1.5 μ g/ml with Tris damping fluid, relatively chooses best effort concentration.
With the reagent 2 of the anti-T4 of biotin labeling of three kinds of variable concentrations, detect its sensitivity, linearity.Testing result is as following table:
Choosing of the anti-T4 working concentration of biotin labeling
Known according to above experimental result, when in reagent 2, the working concentration of the anti-T4 of biotin labeling is 1 μ g/ml, its sensitivity, linearity are better.
4, the screening of the concentration of the coated photosensitive particulate of Avidin:
Luminous antibody concentration is selected 150 μ g/ml, and the concentration of biotinylated antibody is selected 1 μ g/ml, and the coated photosensitive particulate concentration of Avidin is formulated as respectively 30 μ g/ml, 40 μ g/ml, and 60 μ g/ml, under initial reaction condition, result is as follows:
According to remolding sensitivity, the mensuration concentration of Qc L and Qc H, the coated photosensitive particulate concentration result in the time of 40 μ g/ml of Avidin is ideal.
Embodiment 7 evaluation tests
Reagent: adopt luminous antibody reagent (150 μ g/ml), biotin labeling antibody reagent (1 μ g/ml), coated photosensitive particulate reagent (40 μ g/ml) and the agent ANS (1mg/ml) that dissociates of Avidin.
First in reacting hole, add 25 μ l samples, then add successively 25 μ l luminescence reagents, 25 μ l dissociate agent ANS and 25 μ l biotinylated antibody reagent.Then put into instrument, by instrument operation according to the following steps automatically: vibration, 37 DEG C of incubations 20 minutes, more automatically add after the coated photosensitive particulate reagent of Avidin 175 μ l 37 DEG C 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 T4 concentration according to typical curve, and unit is μ g/dL, finally prints test report.Detection concrete steps are as follows:
1, sensing range
This kit linear detection range is 1-32 μ g/dl, and it is used to double-log model matching, and the absolute value of dose-response curve related coefficient (r) is not less than 0.9900.As T4 concentration value in working sample accurately, in sample, T4 concentration value should not exceed the concentration range of 1-32 μ g/dl calibration object curve, and the measurement result that exceeds this scope is the result of calculation drawing by calibration object curve extension.Can know the assay of a large amount of clinical samples through this kit, most of sample results is less than 32 μ g/dl, and therefore, the sensing range that this kit is set is that 1-32 μ g/dl can meet clinical practice requirement completely.
2, the detection of sensitivity
Detect 10 hole calibration object 0 μ g/dl, 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, the reagent of two lot numbers after testing, its sensitivity for analysis is respectively 0.46ug/dL, 0.50ug/dL, all not higher than 1 μ g/dl.
3, accuracy detects
Adopt respectively the T4 kit of 2 lot numbers to carry out 1 detection to quality-control product QC L, QC H, each repetition measurement 10 holes, every duplicate samples detects 20 times altogether.Obtain following result:
From the above results, in its batch of this product, accuracy is less than 5%, and between batch, accuracy is less than 10%.
4, linear detection
To other double-log or other Model fittings for 5 calibration objects except 0 value, the absolute value of dose-response curve related coefficient (r) should be not less than 0.9900.The kit of two lot numbers after testing, its linear r value is respectively 1.000,0.999.
5, interference test
In clinical samples 1#, the 2# of concentration known, 3#, add 250mg/dL haemoglobin, 500mg/dL triglyceride, 10mg/dL cholerythrin, detect with this kit, result is as following table:
Table 1 kit lot number: 20070121 (units: μ g/dl)
Table 2 kit lot number: 20070123 (units: μ g/dl)
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 (40 μ g/ml) of agent ANS (1mg/ml), luminous antibody reagent (150 μ g/ml), biotin labeling antibody reagent (1 μ g/ml) and Avidin that dissociates.
Carried out quality level comparison taking the T4 of Siemens Company kit as reference, sample used is the sensitivity reference material in embodiment 5, and result as shown in Figure 4.
Result is as shown in Figure 4 known, this kit testing result and Siemens results relevance r=0.937, 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 1-4, prepare according to the whole bag of tricks three kinds of reagent and respectively independent packaging of the agent ANS that dissociates, after assembly, obtain basic kit.Can be used for quantitatively detecting the concentration of T4 in sample.
Claims (15)
1. a detection kit for TA, comprising: TA detection of particles, biotin labeled anti-TA antibody, coated photosensitive particulate and the agent of dissociating of Avidin; Wherein TA detection of particles is the coated luminous particle of triiodo thryonine, the aqueous solution that the agent of dissociating is 8-Anilino-1-naphthalenesulfonic acid ammonium salt; In described TA detection of particles, the surface functional group of luminous particle is selected from carboxyl or aldehyde radical; In described biotin labeled anti-TA antibody, the molecule ratio of biotin and anti-TA antibody is 10～50:1; In the aqueous solution of described 8-Anilino-1-naphthalenesulfonic acid ammonium salt, the mass body volume concentrations of 8-Anilino-1-naphthalenesulfonic acid ammonium salt is 0.5-1.5mg/ml; The concentration of the coated photosensitive particulate of Avidin is 30-60 μ g/ml, and in the coated photosensitive particulate of described Avidin, the mass ratio of Avidin and photosensitive particulate is 1:3～10; Described TA detection of particles makes by the following method:
1) mix: luminous particle and triiodo thryonine are mixed in damping fluid, and the mass ratio of luminous particle and triiodo thryonine is 10:(0.01-0.05); Described damping fluid is MES damping fluid or phosphate buffer; The particle size range of described luminous particle is 100-400nm; In mixed liquor, the concentration of luminous particle is 10-40mg/ml;
2) reaction: add the EDAC solution of damping fluid preparation mix and react, the mass ratio of luminous particle and EDAC is 25:1;
3) toward step 2) add the BSA solution of damping fluid preparation mix and react in the reactant liquor that obtains;
4) wash products, obtains the coated luminous particle of triiodo thryonine.
2. the detection kit of TA as claimed in claim 1, is characterized in that, the particle size range of described luminous particle is 150-300nm.
3. the detection kit of TA as claimed in claim 1, is characterized in that, in described TA detection of particles, the luminous quantity of luminous particle is 150,000-350,000 photon number/100 μ g luminous particle.
4. the detection kit of TA as claimed in claim 1, is characterized in that, the mass ratio of described luminous particle and triiodo thryonine is 10:0.02.
5. the detection kit of TA as claimed in claim 1, is characterized in that, the mode of the cleaning of described step 4) is that centrifuge method is cleaned.
6. the detection kit of TA as claimed in claim 1, is characterized in that, in described biotin labeled anti-TA antibody, the molecule ratio of biotin and anti-TA antibody is 30:1.
7. the detection kit of TA as claimed in claim 1, is characterized in that, in the coated photosensitive particulate of described Avidin, the mass ratio of Avidin and photosensitive particulate is 1:5.
8. the detection kit of TA as claimed in claim 1, it is characterized in that, described TA detection of particles, biotin labeled anti-TA antibody, dissociate agent and the coated photosensitive particulate independent packaging respectively of Avidin, and coated luminous particle, biotin labeled anti-TA antibody and the coated photosensitive particulate of Avidin of triiodo thryonine is suspension.
9. the detection kit of TA as claimed in claim 8, is characterized in that, the solvent of described suspension is selected from HEPES buffer system or Tris buffer system.
10. the detection kit of TA as claimed in claim 9, is characterized in that, the composition that the solvent of TA detection of particles suspension is pH8.0 is HEPES, NaCl and EDTA-Na-2H
2the HEPES damping fluid of O.
The detection kit of 11. TAs claimed in claim 9, is characterized in that, the composition that the solvent of biotin labeled anti-TA antibody suspension is pH8.0 is Tris, NaCl and EDTA-Na-2H
2the Tris damping fluid of O.
The detection kit of 12. TAs claimed in claim 9, is characterized in that, the solvent of the coated photosensitive particulate suspension of Avidin is that composition is HEPES, NaCl and EDTA-Na-2H
2the HEPES damping fluid of O.
The detection kit of 13. TAs claimed in claim 8, is characterized in that, also comprises protein protective agent, prevents one or more in stable reagent or the antiseptic of particles agglomerate in described suspension.
The detection kit of 14. TAs claimed in claim 13, is characterized in that, described protein protective agent is BSA, and the stable reagent that prevents particles agglomerate is Tween20, and antiseptic is gentamicin and Proclin300.
The detection kit of 15. TAs claimed in claim 8, is characterized in that, also comprises the TA solution that multiple concentration is known in described kit, the independent packaging respectively of the TA solution of variable concentrations.
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| CN101865916A (en) | 2010-10-20 |
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