CN108802369A - A kind of free triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof - Google Patents
A kind of free triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof Download PDFInfo
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- CN108802369A CN108802369A CN201810549781.8A CN201810549781A CN108802369A CN 108802369 A CN108802369 A CN 108802369A CN 201810549781 A CN201810549781 A CN 201810549781A CN 108802369 A CN108802369 A CN 108802369A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
- G01N21/763—Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/635—Parathyroid hormone (parathormone); Parathyroid hormone-related peptides
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2446/00—Magnetic particle immunoreagent carriers
- G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids
- G01N2446/90—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids characterised by small molecule linker used to couple immunoreagents to magnetic particles
Abstract
The present invention relates to a kind of free triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof, the kit includes：The coated magnetic particle suspension of Streptavidin, the trilute derivative of alkali phosphatase enzyme mark, the trilute antibody of biotin labeling test dilution, trilute calibration object, trilute quality-control product.The kit of the present invention is compared with available reagent box, and preparation process is simpler, and cost is lower, and detection range is wide, and stability is good.
Description
Technical field
The invention belongs to technical field of immune assay, it is related to detecting free triiodothyronine FT3 in serum
Magnetic microparticle chemiluminescence immune assay kit of content and preparation method thereof.
Background technology
3,5,3 ' triiodo thryonines（Triiodothyronine, T3）It is a kind of weight to work to various target organs
Want thyroid hormone, molecular weight 651, half-life period is 1.5 days in blood plasma, in blood plasma major part T3 be by T4 in peripheral tissues
It is metabolized through deiodination, remaining is discharged by thyroid gland, and activity is 3-8 times of T4.
T3 is the known strongest thyroid hormone of current bioactivity, in peripheral blood, 99.6%T3 by with serum/blood
Transporter in slurry in conjunction with and be transported, only 0.4% T3（FT3）It is free existing, only free T3 has metabolism
Activity, and what is combined does not have then.Main transporter includes in vivo：Thyroxine-binding globulin（TBG）, thyroxine knot
Close prealbumin and albumin.
FT3, which can be combined by cell membrane with receptor, plays physiological effect, therefore it is that physiology effect occurs for thyroid hormone
The real active part answered, and the concentration of FT3 is not influenced by the concentration of thyroxine-binding globulin in vivo, it can relatively really
Reflect thyroid functional status and other influences to function of human body with cutting.FT3 contents are to antidiastole thyroid function
It is no normal, it is hyperfunction or low significant, it is very sensitive to the diagnosis of hyperthyroidism, it is the specific index for diagnosing T3 type hyperthyroidisms.Face
On bed, TSH, FT3 and tri- joint inspections of FT4 are commonly used to confirm that hyperthyroidism or first are low, and tracking curative effect.
The main method measured for internal free triiodothyronine has radio immunoassay, enzyme linked immunological to inhale
Attached analytic approach, chemiluminescence immunoassay etc..Currently used radio immunoassay（RIA）It is to use I125Mark triiodo first
Shape gland original ammonia acid haptens is come what is realized, and synthesis technology is complicated, and the term of validity is short, has certain pollution to environment, influences detection knot
The factor of fruit is more.In recent years, chemiluminescence immunoassay technology was quickly grown, sensitivity, specificity and automation journey
Degree met or exceeded RIA level, especially the stability of marker and it is free from environmental pollution be that RIA methods are incomparable.
Current major Medium Sized Hospitals equal import automatic chemiluminescence immunoassay system, but instrument and reagent price
It is expensive.
The patent application of existing more than one piece magnetic microparticle chemiluminescence method detection trilute at present, uses
Testosterone coupled isoluminol substance markers trilute antibody, detection sensitivity is relatively low, and stability is poor.
Invention content
The chemiluminescence immunoassay that the problem to be solved in the present invention is to provide free triiodothyronine quantitatively detects examination
Agent box and preparation method thereof, the reagent term of validity that avoids radioimmunoassay is short, there are radioactive pollution, cumbersome etc.
Disadvantage, and solve that sensitivity is low, and stability is poor, defect of high cost.Simple, cost that the invention discloses a kind of preparation processes
Cheap, the good free triiodothyronine of stability chemiluminescence immunoassay immue quantitative detection reagent box and preparation method.
A kind of free triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection reagent box, including strepto- are affine
The coated magnetic particle suspension of element, the trilute derivative of alkali phosphatase enzyme mark, the triiodo of biotin labeling
Thyronine antibody tests dilution, free triiodothyronine calibration object, free triiodothyronine matter
Control product.
Further, the coated magnetic particle suspension of the Streptavidin is four of surface package with Streptavidin
Fe 3 O particle be suspended in magnetic particle coating object buffer solution formed in suspension, the magnetic particle suspension it is a concentration of
0.1mg/mL~1.0mg/mL；It is 20mM~200mM Tris that magnetic particle, which is coated with object buffer solution,（Trishydroxymethylaminomethane）Buffering
Liquid, PH ranging from 6.5~8.0；
The trilute derivative of the alkali phosphatase enzyme mark is alkaline phosphatase and trilute
The conjugate of derivative is diluted in the solution formed in enzyme marker buffer solution, and wherein trilute derivative is
The sulfonated hydroxysuccinimidyl acyl of the chemical synthesis substance or N- of n-hydroxysuccinimide (NHS) and trilute is sub-
The chemical synthesis substance of amine (Sulfo-NHS) and trilute；The enzyme marker buffer solution is 20mM~200mM
Tris buffer solutions, PH ranging from 6.5~8.0；
The trilute antibody of the biotin labeling is the coupling of biotin and trilute antibody
Object is diluted in the solution formed in biotinylated derivative buffer solution；Wherein trilute antibody is mouse Dan Ke
Grand antibody, the biotinylated derivative buffer solution are 20mM~200mM Tris buffer solutions, PH ranging from 6.5~8.0.
The free triiodothyronine calibration object is the calibration object buffer solution containing 20% fetal calf serum, described free
The working concentration of trilute calibration object is respectively 0,2.50,5.00,10.00,20.00,40.00 pg/mL.
The free triiodothyronine quality-control product is the quality-control product buffer solution containing 20% fetal calf serum, described free
The working concentration of trilute quality-control product is respectively 5.00,20.00 pg/mL.
Further, the coated magnetic particle suspension of the Streptavidin is four of surface package with Streptavidin
Fe 3 O particle, grain size are 1um~1.5um, are suspended in the suspension formed in magnetic particle coating object buffer solution, the chain
A concentration of 0.5mg/mL of the mould coated magnetic particle suspension of Avidin；The magnetic particle coating object buffer solution is 100mM Tris
（Trishydroxymethylaminomethane）Buffer solution, PH 7.0.
Further, the trilute derivative of the alkali phosphatase enzyme mark is alkaline phosphatase and triiodo
The conjugate of thyronine derivative is diluted in the dilution of institute's shape in enzyme marker buffer solution, and the triiodo thyroid gland is former
Threonine derivative is the dilution of the conjugate and enzyme marker buffer solution of alkaline phosphatase and trilute derivative
Ratio is 1:400~1:2000, preferred dilution ratio is 1:800；The enzyme marker buffer solution is 20mM~200mM
Tris buffer solutions, PH ranging from 6.5~8.0, preferred a concentration of 20mM, preferred PH are 7.4.
Further, the trilute antibody of the biotin labeling is biotin and triiodo thyroid gland original ammonia
The conjugate of sour antibody is diluted in the dilution of institute's shape in biotinylated derivative buffer solution, the trilute antibody
Dilution ratio for the conjugate of biotin and trilute antibody and biotinylated derivative buffer solution is 1:200~
1:1000, preferred dilution ratio is 1:500；Biotinylated derivative buffer solution is 20mM~200mM Tris buffer solutions, PH models
It is 6.5~8.0 to enclose, and preferred a concentration of 20mM, preferred PH are 8.0；Wherein trilute antibody is that mouse is single
Clonal antibody.
Further, the free triiodothyronine calibration object is the calibration object buffer solution containing 20% fetal calf serum,
Trilute sterling is diluted to working concentration, respectively 0,2.50,5.00,10.00,20.00,40.00 pg/
mL；The calibration object buffer solution is 20mM~200mM Tris buffer solutions, and PH ranging from 6.5~8.0 is preferred a concentration of
20mM, preferred PH are 7.4；The free triiodothyronine quality-control product is the quality-control product buffering containing 20% fetal calf serum
Trilute sterling is diluted to working concentration, respectively 5.00,20.00 pg/mL by liquid；Quality-control product buffer solution is
20mM~200mM Tris buffer solutions, pH range are 6.5~8.0, and preferred a concentration of 20mM, preferred pH value is 7.4.
Further, the trilute derivative is prepared using following processing step：
S1. the preparation of trilute-succinate：It takes trilute to be added in distilled water, is added
Copper sulphate, back flow reaction 5 hours in 100 DEG C of water-baths after being cooled to room temperature, are filtered to remove solid impurity, then by filtrate ice
Bath cools to crystallization and is precipitated, and with ethyl alcohol recrystallization, obtains white flaky crystals trilute-succinate；
S2. the preparation of n-hydroxysuccinimide (NHS) and trilute synthetic：Take above-mentioned triiodo thyroid gland
Former propylhomoserin-succinate is dissolved in citric acid solution, and n-hydroxysuccinimide and 1- (3- dimethylamino-propyls) -3- is added
Ethyl-carbodiimide hydrochloride, under the conditions of being protected from light 2~8 DEG C reaction 8h, cross chromatographic column collect n-hydroxysuccinimide (NHS) with
The eluent of trilute synthetic after concentration, is recrystallized with absolute ethyl alcohol, obtains white, needle-shaped crystals N- hydroxyls
Succinimide (NHS) and trilute synthetic, i.e. trilute derivative.
A kind of preparation method of free triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection reagent box, packet
Include following steps：
（1）The trilute derivative preparation method of alkali phosphatase enzyme mark：Sodium carbonate is added in alkaline phosphatase
In buffer solution (pH8.0), trilute derivative is added（T3-NHS）, it is 4 DEG C~37 DEG C reactions 0.5 in temperature
~24 hours, then ProteinG affinity columns (GE companies) is used to purify enzyme labelled antibody, obtains the combination of trilute enzyme
Trilute enzyme conjugates is diluted in enzyme marker buffer solution by object, dilution ratio 1:400~1:2000；
（2）The trilute preparation method for antibody of biotin labeling：By trilute antibody（Mouse,
Monoclonal）Be added in sodium carbonate buffer (pH8.0), biotin derivative be added, temperature be 4 DEG C~37 DEG C reactions 0.5~
24 hours, ProteinG affinity columns (GE companies) purifying biological element labelled antibody is then used, obtains trilute life
Object element conjugate, trilute biotin conjugate is diluted in biotinylated derivative buffer solution, dilution ratio
It is 1:200~1:1000；
（3）The preparation method of trilute calibration object：With calibration object buffer solution by trilute sterling
It is diluted to working concentration, respectively 0,2.50,5.00,10.00,20.00,40.00 pg/mL；
（4）The preparation method of trilute quality-control product：With quality-control product buffer solution by trilute sterling
It is diluted to working concentration, respectively 5.00,20.00 pg/mL；
（5）Assembling：By the trilute derivative of above-mentioned alkali phosphatase enzyme mark, the triiodo first shape of biotin labeling
Gland original ammonia acid antibody, trilute calibration object, trilute quality-control product are assembled into box, in 2~8 DEG C of items
It is preserved under part.
Further, a kind of system of free triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection reagent box
Preparation Method includes the following steps：
（1）The trilute derivative preparation method of alkali phosphatase enzyme mark：Sodium carbonate is added in alkaline phosphatase
In buffer solution (pH8.0), trilute derivative is added（T3-NHS）, after 37 DEG C are reacted 4 hours, use
ProteinG affinity columns (GE companies) purify enzyme labelled antibody, trilute enzyme conjugates are obtained, by triiodo thyroid gland
Former propylhomoserin enzyme conjugates is diluted in enzyme marker buffer solution, dilution ratio 1:1000；
（2）The trilute preparation method for antibody of biotin labeling：By trilute antibody（Mouse,
Monoclonal）It is added in sodium carbonate buffer (pH8.0), biotin derivative is added and uses ProteinG after 37 DEG C are reacted 4 hours
Affinity column (GE companies) purifying biological element labelled antibody, obtains trilute biotin conjugate, by triiodo first shape
Gland original ammonia acid biotin conjugate is diluted in biotinylated derivative buffer solution, dilution ratio 1:500；
（3）The preparation method of trilute calibration object：With calibration object buffer solution by trilute sterling
It is diluted to working concentration, respectively 0,2.50,5.00,10.00,20.00,40.00 pg/mL；
（4）The preparation method of trilute quality-control product：With quality-control product buffer solution by trilute sterling
It is diluted to working concentration, respectively 5.00,20.00 pg/mL；
（5）Assembling：By the trilute derivative of above-mentioned alkali phosphatase enzyme mark, the triiodo first shape of biotin labeling
Gland original ammonia acid antibody, trilute calibration object, trilute quality-control product are assembled into box, in 2~8 DEG C of items
It is preserved under part.
Kit Performance Evaluating Indexes of the present invention：Accuracy, line are carried out to the kit prepared using this method
Property, precision, specificity and stability are measured.
Kit reaction principle of the present invention：Using magnetic microparticles as the solid phase of immune response, chemiluminescence is utilized
Immunoassay method coordinates with chemiluminescence class analyzer, for measuring the original of the free triiodo thyroid gland in human serum/blood plasma
Histidine content.The technical principle of reaction is：Free triiodothyronine in sample to be tested, calibration object or quality-control product and alkali
The trilute Dan Ke of the trilute derivative competitive binding biotin labeling of acid phosphatase label
The magnetic particle of coating Streptavidin is then added in grand antibody, and being combined with biotin by Streptavidin makes antigen-antibody answer
Close object be connected on magnetic particle, the Direct precipitation in externally-applied magnetic field, by immune response formed compound be not associated with it is other
Substance detaches.The compound of precipitation is cleaned, enzyme-catalyzed chemical luminescence substrate is added, substrate, by catalytic pyrolysis, is formed under enzyme effect
Unstable excitation state intermediate, just sends out photon when excitation state intermediate returns to ground state, forms luminescence-producing reaction, using shining
Instrument measures the luminous intensity of reaction.Within the measurement range, luminous intensity with the trilute concentration in sample at anti-
Than it is dense that quantitatively trilute in sample to be tested can be calculated using four parameter Logistic equation models of improvement
Degree.
The free triiodothyronine magnetic microparticle chemiluminescence immunological quantitative determining kit of the present invention, preparation process
Simply, of low cost, stability is good, performance reaches the peer-level of famous foreign brand reagent.
The technical effects of the invention are that：
（1）The trilute derivative preparation method of alkali phosphatase enzyme mark is using trilute
Derivative（T3-NHS）Coupling phosphatase directly is carried out, other patents that compare or producer are trilutes-
BSA derivatives（T3-CMO-BSA）Or trilute-OVA derivatives（T3-CMO-OVA）, this method cost is lower,
Technique is simpler, more controllably.There may be the interference of certain steric hindrance, shadows for the high molecular weight protein derivative of trilute
The accuracy of sound test result, and trilute derivative of the present invention（T3-NHS）It is and triiodo thyroid gland
The similar small-molecule substance of former propylhomoserin structure, the steric hindrance interference effectively avoided the problem that.
（2）The preparation method of biotin labelled antibodies
It is directly coupled with antibody using the biotin through overactivation, it is simpler without adding coupling agents, the techniques such as EDC NHS
Single, cost is lower.
Specific implementation mode
It is further illustrated the present invention with reference to example, the advantages and features of the present invention becomes apparent from what is be described.But
It is to be understood that this example is only a kind of example of the present invention, any restrictions can't be done to the scope of the present invention.
The technical solution adopted by the present invention is：A kind of free triiodothyronine magnetic microparticle chemiluminescence immune quantitative
Detection kit, including：The coated magnetic particle suspension of Streptavidin, the trilute of alkali phosphatase enzyme mark
Derivative, the trilute antibody of biotin labeling test dilution, free triiodothyronine calibration
Product, free triiodothyronine quality-control product.
The coated magnetic particle suspension of Streptavidin of the present invention is four of surface package with Streptavidin
Fe 3 O, grain size are 1um~1.5um, are suspended in magnetic particle coating object buffer solution, a concentration of 0.1mg/mL~1.0mg/
ML, preferred a concentration of 0.5mg/mL；It is 20mM~200mM Tris that magnetic particle, which is coated with object buffer solution,（Trihydroxy methyl amino first
Alkane）Buffer solution, PH ranging from 6.5~8.0, preferred a concentration of 100mM, preferred PH are 7.0.
The trilute derivative of alkali phosphatase enzyme mark of the present invention is alkaline phosphatase and triiodo
The conjugate of thyronine derivative, wherein trilute derivative are n-hydroxysuccinimide (NHS)
With the sulfonated HOSu NHS of chemical synthesis substance or N- (Sulfo-NHS) and triiodo first of trilute
The chemical synthesis substance of shape gland original ammonia acid.It is diluted in enzyme marker buffer solution, dilution ratio 1:400~1:2000, preferably
Dilution ratio be 1:800；Enzyme marker buffer solution is 20mM~200mM Tris buffer solutions, and PH ranging from 6.5~8.0 is excellent
A concentration of 20mM of choosing, preferred PH are 7.4.
The trilute derivative is n-hydroxysuccinimide (NHS) and trilute
Chemical synthesis substance, chemical structural formula is as follows：
Or the trilute derivative is the sulfonated HOSu NHSs of N- (Sulfo-NHS) and triiodo first
The chemical synthesis substance of shape gland original ammonia acid, chemical structural formula are as follows：
The preparation process of trilute derivative of the present invention：
（1）The preparation of trilute-succinate：Take trilute（It purchases from Sigma companies）
3.5g is in 200mL distilled water, addition copper sulphate 800mg, back flow reaction 5 hours in 100 DEG C of water-baths, after being cooled to room temperature,
It is filtered to remove solid impurity, then has within 2 hours crystallization to be precipitated the cooling of filtrate ice bath, with ethyl alcohol recrystallization, obtains white plates
Crystallize trilute-succinate 2.6g.
（2）The preparation of n-hydroxysuccinimide (NHS) and trilute synthetic：Take the triiodo first of 1.2g
Shape gland original ammonia acid-succinate is dissolved in 50mM citric acid solutions（PH4.0）In, n-hydroxysuccinimide (NHS) is added
600mg and 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC) 200mg, 2~8 DEG C of reactions under the conditions of being protected from light
8h crosses the eluent that chromatographic column collects n-hydroxysuccinimide (NHS) and trilute synthetic, after concentration,
It is recrystallized with absolute ethyl alcohol, obtains white, needle-shaped crystals n-hydroxysuccinimide (NHS) and synthesized with trilute
Object 160mg is to get T3-NHS.
It is biotin and trilute the present invention relates to the trilute antibody of biotin labeling
The conjugate of antibody, wherein trilute antibody are mouse monoclonal antibody.It is diluted in biotinylated derivative buffering
In liquid, dilution ratio 1:200~1:1000, preferred dilution ratio is 1:500；Biotinylated derivative buffer solution be 20mM~
200mM Tris buffer solutions, PH ranging from 6.5~8.0, preferred a concentration of 20mM, preferred PH are 8.0.
Free triiodothyronine calibration object of the present invention is the calibration object buffer solution containing 20% fetal calf serum,
Trilute sterling is diluted to working concentration, respectively 0,2.50,5.00,10.00,20.00,40.00 pg/
mL；Calibration object buffer solution is 20mM~200mM Tris buffer solutions, and PH ranging from 6.5~8.0, preferred a concentration of 20mM are excellent
The PH of choosing is 7.4.
Free triiodothyronine quality-control product of the present invention is the quality-control product buffer solution containing 20% fetal calf serum,
Trilute sterling is diluted to working concentration, respectively 5.00,20.00 pg/mL；Quality-control product buffer solution is
20mM~200mM Tris buffer solutions, pH range are 6.5~8.0, and preferred a concentration of 20mM, preferred pH value is 7.4.
Embodiment 1
One, the preparation of kit：
（1）Magnetic particle is coated with object buffer solution and prepares：
| Material | Dosage |
| Trishydroxymethylaminomethane | 2.42g |
| Sodium chloride | 18.00g |
| Tween-20 | 0.50g |
| Bovine serum albumin(BSA) | 50.00g |
| Proclin300 | 1.00g |
Above-mentioned material is added in 1000mL deionized waters, dissolving is sufficiently stirred, adjusts PH to 7.00 ± 0.10.
（2）It is prepared by enzyme marker buffer solution：
| Material | Dosage |
| Trishydroxymethylaminomethane | 2.42g |
| Sodium chloride | 18.00g |
| Tween-20 | 1.00g |
| Bovine serum albumin(BSA) | 50.00g |
| Proclin300 | 1.00g |
Above-mentioned material is added in 1000mL deionized waters, dissolving is sufficiently stirred, adjusts PH to 7.40 ± 0.10.
（3）It is prepared by biotinylated derivative buffer solution：
| Material | Dosage |
| Trishydroxymethylaminomethane | 2.42g |
| Sodium chloride | 4.50g |
| Tween-20 | 1.00g |
| Bovine serum albumin(BSA) | 10.00g |
| Proclin300 | 1.00g |
Above-mentioned material is added in 1000mL deionized waters, dissolving is sufficiently stirred, adjusts PH to 8.00 ± 0.10.
（4）Dilution is tested to prepare：
| Material | Dosage |
| Trishydroxymethylaminomethane | 2.42g |
| Sodium chloride | 4.50g |
| Tween-20 | 1.00g |
| Bovine serum albumin(BSA) | 10.00g |
| Proclin300 | 1.00g |
Above-mentioned material is added in 1000mL deionized waters, dissolving is sufficiently stirred, adjusts PH to 7.40 ± 0.10.
（5）It is prepared by calibration object buffer solution：
| Material | Dosage |
| Trishydroxymethylaminomethane | 2.42g |
| Sodium chloride | 18.00g |
| Tween-20 | 1.00g |
| Fetal calf serum | 200mL |
| Proclin300 | 1.00g |
Above-mentioned material is added in 800mL deionized waters, dissolving is sufficiently stirred, adjusts PH to 7.40 ± 0.10.
（6）It is prepared by quality-control product buffer solution：
| Material | Dosage |
| Trishydroxymethylaminomethane | 2.42g |
| Sodium chloride | 18.00g |
| Tween-20 | 2.00g |
| Fetal calf serum | 200mL |
| Proclin300 | 1.00g |
Above-mentioned material is added in 800mL deionized waters, dissolving is sufficiently stirred, adjusts PH to 7.40 ± 0.10.
（1）The coated magnetic particle suspension manufacturing methods of Streptavidin：
By the coated magnetic particle mother liquor of the Streptavidin of commercialization（It purchases in Nanjing Pan Gu's gene nano Science and Technology Ltd.）
It is 0.5mg/mL with magnetic bead coating object buffer solution diluted concentration.
（2）The trilute derivative preparation method of alkali phosphatase enzyme mark：
100ug alkaline phosphatases are added in 1mL10mM sodium carbonate buffers (pH8.0), 1ug triiodo thyroid gland original ammonia is added
Acid derivative（T3-NHS, it is company that buying is refined in Shenzhen）, it is 4 DEG C~37 DEG C in temperature and reacts 0.5~24 hour, then uses
ProteinG affinity columns (GE companies) purify enzyme labelled antibody, obtain trilute enzyme conjugates.It is diluted in enzyme label
In object buffer solution, dilution ratio 1:1000.
（3）The trilute preparation method for antibody of biotin labeling：
By 20ug trilute antibody（Mouse, monoclonal are purchased in Meridian companies of the U.S.）1mL10mM is added
In sodium carbonate buffer (pH8.0), 50ug biotin derivatives are added, being 4 DEG C~37 DEG C in temperature reacts 0.5~24 hour,
Then ProteinG affinity columns (GE companies) purifying biological element labelled antibody is used, trilute biotin knot is obtained
Close object.It is diluted in biotinylated derivative buffer solution, dilution ratio 1:500.
（4）The preparation method of free triiodothyronine calibration object：
Trilute sterling is diluted to working concentration, respectively 0 with calibration object buffer solution, 2.50,5.00,
10.00,20.00,40.00 pg/mL.
（5）The preparation method of free triiodothyronine quality-control product：
Trilute sterling is diluted to working concentration, respectively 5.00,20.00 pg/ with quality-control product buffer solution
mL。
（6）Assembling：Mentioned reagent component is assembled into box, is preserved under the conditions of 2~8 DEG C.
Two, the test method of kit：
（1）Sample-adding and incubation process：Draw free triiodothyronine calibration object, quality-control product or fresh patient's sample 50uL
It is added in reaction tube, the trilute derivative 50uL and biotin labeling of alkali phosphatase enzyme mark is then added
Trilute antibody 50uL, 37 DEG C of incubation reactions 10 minutes；Then it is outstanding that the coated magnetic particle of Streptavidin is added
Supernatant liquid 50uL, 37 DEG C of incubation reactions 10 minutes；
（2）Magneto separate cleaning process：Reaction tube after the completion of incubation reaction is placed on Magneto separate frame and stands 1 minute, in removing
Clear liquid；Magnetic bead is added for the first time and is coated with object buffer solution 300uL, is placed on Magneto separate frame and stands 1 minute, remove supernatant；Second
Secondary addition magnetic bead is coated with object buffer solution 300uL, is placed on Magneto separate frame and stands 1 minute, removes supernatant；Magnetic bead is added in third time
It is coated with object buffer solution 300uL, is placed on Magneto separate frame and stands 1 minute, removes supernatant；
（3）Luminescence process：530 substrate solutions of Lumi-Phos are added（It purchases in Lumigen companies of the U.S.）200uL, 37 DEG C are protected from light
It is incubated after five minutes, luminous value is measured with 9507 semi-automatic Chemiluminescence Apparatus of shore pine.
Three, the performance test results of kit：
（1）The range of linearity is 0~40.00 pg/mL, linear coefficient：r≥0.9900；
（2）Imprecision is no more than 6% in batch；
（3）Accuracy：The rate of recovery is between 90%~110%；
（4）Minimum detectability：Test result is not higher than 0.4 pg/mL；
（5）Specificity：The iodo- l-tyrosine of 3- of 1000ng/mL, 3,5-, the bis- iodo- l-tyrosine of 1000ng/mL, 1000ng/mL
Thyroxine（TT4）, test result is not higher than 0.4 pg/mL：
| Chaff interferent | Concentration | Test result | Conclusion |
| The iodo- l-tyrosine of 3- | 1000ng/mL | 0.26pg/mL | 0.4 pg/mL of < |
| 3,5- bis- iodo- l-tyrosine | 1000ng/mL | 0.32pg/mL | 0.4 pg/mL of < |
| Thyroxine | 1000ng/mL | 0.17pg/mL | 0.4 pg/mL of < |
（6）Stability：37 DEG C accelerate 7 days in 2~8 DEG C of ± 10% ranges of reagent test result error；
| Sample | Concentration | 37 DEG C of acceleration, 7 days relative deviations |
| Low value Quality Control | 5.00 pg/mL | -1.93% |
| High level Quality Control | 20.00 pg/mL | -3.24% |
It can be seen that by above result：Kit of the present invention is compared with foreign same type kit, performance test knot
Fruit is very close to reaching good results, trilute magnetic microparticle chemiluminescence immune quantitative detection reagent of the invention
Box has good applicability and advance.
Embodiment described above is only the preferred embodiment of the present invention.It should be pointed out that dilution ratio of the present invention
Refer to that ratio diluted in mass ratio is not departing from the technology of the present invention side for those skilled in the art
Under the premise of case, some improvements and modifications can also be made, these improvement and modification also should be regarded as protection scope of the present invention, this
The available prior art of each section being not known in embodiment is realized.
Claims (9)
1. a kind of free triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection reagent box, it is characterised in that：It
Including the coated magnetic particle suspension of Streptavidin, the trilute derivative of alkali phosphatase enzyme mark, biology
The trilute antibody of element label tests dilution, free triiodothyronine calibration object, triiodo first of dissociating
Shape gland original ammonia acid quality-control product.
2. a kind of free triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection examination according to claim 1
Agent box, it is characterised in that：The coated magnetic particle suspension of Streptavidin is four of surface package with Streptavidin
Fe 3 O particle be suspended in magnetic particle coating object buffer solution formed in suspension, the magnetic particle suspension it is a concentration of
0.1mg/mL~1.0mg/mL；It is 20mM~200mM Tris that magnetic particle, which is coated with object buffer solution,（Trishydroxymethylaminomethane）Buffering
Liquid, PH ranging from 6.5~8.0；
The trilute derivative of the alkali phosphatase enzyme mark is alkaline phosphatase and trilute
The conjugate of derivative is diluted in the solution formed in enzyme marker buffer solution, and wherein trilute derivative is
The sulfonated hydroxysuccinimidyl acyl of the chemical synthesis substance or N- of n-hydroxysuccinimide (NHS) and trilute is sub-
The chemical synthesis substance of amine (Sulfo-NHS) and trilute；The enzyme marker buffer solution is 20mM~200mM
Tris buffer solutions, PH ranging from 6.5~8.0；
The trilute antibody of the biotin labeling is the coupling of biotin and trilute antibody
Object is diluted in the solution formed in biotinylated derivative buffer solution；Wherein trilute antibody is mouse Dan Ke
Grand antibody, the biotinylated derivative buffer solution are 20mM~200mM Tris buffer solutions, PH ranging from 6.5~8.0；
The free triiodothyronine calibration object is the calibration object buffer solution containing 20% fetal calf serum, the free triiodo
The working concentration of thyronine calibration object is respectively 0,2.50,5.00,10.00,20.00,40.00 pg/mL；
The free triiodothyronine quality-control product is the quality-control product buffer solution containing 20% fetal calf serum, the free triiodo
The working concentration of thyronine quality-control product is respectively 5.00,20.00 pg/mL.
3. a kind of free triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection examination according to claim 2
Agent box, it is characterised in that：The coated magnetic particle suspension of Streptavidin is four of surface package with Streptavidin
Fe 3 O particle, grain size are 1um~1.5um, are suspended in the suspension formed in magnetic particle coating object buffer solution, the chain
A concentration of 0.5mg/mL of the mould coated magnetic particle suspension of Avidin；The magnetic particle coating object buffer solution is 100mM Tris
（Trishydroxymethylaminomethane）Buffer solution, PH 7.0.
4. a kind of free triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection examination according to claim 2
Agent box, it is characterised in that：The trilute derivative of the alkali phosphatase enzyme mark is alkaline phosphatase and triiodo
The conjugate of thyronine derivative is diluted in the dilution of institute's shape in enzyme marker buffer solution, and the triiodo thyroid gland is former
Threonine derivative is the dilution of the conjugate and enzyme marker buffer solution of alkaline phosphatase and trilute derivative
Ratio is 1:400~1:2000, preferred dilution ratio is 1:800；The enzyme marker buffer solution is 20mM~200mM
Tris buffer solutions, PH ranging from 6.5~8.0, preferred a concentration of 20mM, preferred PH are 7.4.
5. a kind of free triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection examination according to claim 2
Agent box, it is characterised in that：The trilute antibody of the biotin labeling is biotin and triiodo thyroid gland original ammonia
The conjugate of sour antibody is diluted in the dilution of institute's shape in biotinylated derivative buffer solution, the trilute antibody
Dilution ratio for the conjugate of biotin and trilute antibody and biotinylated derivative buffer solution is 1:200~
1:1000, preferred dilution ratio is 1:500；Biotinylated derivative buffer solution is 20mM~200mM Tris buffer solutions, PH models
It is 6.5~8.0 to enclose, and preferred a concentration of 20mM, preferred PH are 8.0；Wherein trilute antibody is that mouse is single
Clonal antibody.
6. a kind of free triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection examination according to claim 2
Agent box, it is characterised in that：The free triiodothyronine calibration object is the calibration object buffer solution containing 20% fetal calf serum,
Trilute sterling is diluted to working concentration, respectively 0,2.50,5.00,10.00,20.00,40.00 pg/
mL；The calibration object buffer solution is 20mM~200mM Tris buffer solutions, and PH ranging from 6.5~8.0 is preferred a concentration of
20mM, preferred PH are 7.4；The free triiodothyronine quality-control product is the quality-control product buffering containing 20% fetal calf serum
Trilute sterling is diluted to working concentration, respectively 5.00,20.00 pg/mL by liquid；Quality-control product buffer solution is
20mM~200mM Tris buffer solutions, pH range are 6.5~8.0, and preferred a concentration of 20mM, preferred pH value is 7.4.
7. a kind of free triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection examination according to claim 2
Agent box, it is characterised in that：The trilute derivative is prepared using following processing step：
S1. the preparation of trilute-succinate：It takes trilute to be added in distilled water, is added
Copper sulphate, back flow reaction 5 hours in 100 DEG C of water-baths after being cooled to room temperature, are filtered to remove solid impurity, then by filtrate ice
Bath cools to crystallization and is precipitated, and with ethyl alcohol recrystallization, obtains white flaky crystals trilute-succinate；
S2. the preparation of n-hydroxysuccinimide (NHS) and trilute synthetic：Take above-mentioned triiodo thyroid gland
Former propylhomoserin-succinate is dissolved in citric acid solution, and n-hydroxysuccinimide and 1- (3- dimethylamino-propyls) -3- is added
Ethyl-carbodiimide hydrochloride, under the conditions of being protected from light 2~8 DEG C reaction 8h, cross chromatographic column collect n-hydroxysuccinimide (NHS) with
The eluent of trilute synthetic after concentration, is recrystallized with absolute ethyl alcohol, obtains white, needle-shaped crystals N- hydroxyls
Succinimide (NHS) and trilute synthetic, i.e. trilute derivative.
8. a kind of preparation method of free triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection reagent box, special
Sign is, includes the following steps：
（1）The trilute derivative preparation method of alkali phosphatase enzyme mark：Sodium carbonate is added in alkaline phosphatase
In buffer solution (pH8.0), trilute derivative is added（T3-NHS）, it is 4 DEG C~37 DEG C reactions 0.5 in temperature
~24 hours, then ProteinG affinity columns (GE companies) is used to purify enzyme labelled antibody, obtains the combination of trilute enzyme
Trilute enzyme conjugates is diluted in enzyme marker buffer solution by object, dilution ratio 1:400~1:2000；
（2）The trilute preparation method for antibody of biotin labeling：By trilute antibody（Mouse,
Monoclonal）Be added in sodium carbonate buffer (pH8.0), biotin derivative be added, temperature be 4 DEG C~37 DEG C reactions 0.5~
24 hours, ProteinG affinity columns (GE companies) purifying biological element labelled antibody is then used, obtains trilute life
Object element conjugate, trilute biotin conjugate is diluted in biotinylated derivative buffer solution, dilution ratio
It is 1:200~1:1000；
（3）The preparation method of trilute calibration object：With calibration object buffer solution by trilute sterling
It is diluted to working concentration, respectively 0,2.50,5.00,10.00,20.00,40.00 pg/mL；
（4）The preparation method of trilute quality-control product：With quality-control product buffer solution by trilute sterling
It is diluted to working concentration, respectively 5.00,20.00 pg/mL；
Assembling：By the trilute derivative of above-mentioned alkali phosphatase enzyme mark, the triiodo thyroid gland of biotin labeling
Former propylhomoserin antibody, trilute calibration object, trilute quality-control product are assembled into box, in 2~8 DEG C of conditions
Lower preservation.
9. a kind of free triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection examination according to claim 8
The preparation method of agent box, which is characterized in that include the following steps：
（1）The trilute derivative preparation method of alkali phosphatase enzyme mark：Sodium carbonate is added in alkaline phosphatase
In buffer solution (pH8.0), trilute derivative is added（T3-NHS）, after 37 DEG C are reacted 4 hours, use
ProteinG affinity columns (GE companies) purify enzyme labelled antibody, trilute enzyme conjugates are obtained, by triiodo thyroid gland
Former propylhomoserin enzyme conjugates is diluted in enzyme marker buffer solution, dilution ratio 1:1000；
（2）The trilute preparation method for antibody of biotin labeling：By trilute antibody（Mouse,
Monoclonal）It is added in sodium carbonate buffer (pH8.0), biotin derivative is added and uses ProteinG after 37 DEG C are reacted 4 hours
Affinity column (GE companies) purifying biological element labelled antibody, obtains trilute biotin conjugate, by triiodo first shape
Gland original ammonia acid biotin conjugate is diluted in biotinylated derivative buffer solution, dilution ratio 1:500；
（3）The preparation method of trilute calibration object：With calibration object buffer solution by trilute sterling
It is diluted to working concentration, respectively 0,2.50,5.00,10.00,20.00,40.00 pg/mL；
（4）The preparation method of trilute quality-control product：With quality-control product buffer solution by trilute sterling
It is diluted to working concentration, respectively 5.00,20.00 pg/mL；
（5）Assembling：By the trilute derivative of above-mentioned alkali phosphatase enzyme mark, the triiodo first shape of biotin labeling
Gland original ammonia acid antibody, trilute calibration object, trilute quality-control product are assembled into box, in 2~8 DEG C of items
It is preserved under part.
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Cited By (5)
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| CN109580934A (en) * | 2018-11-23 | 2019-04-05 | 深圳天辰医疗科技有限公司 | A kind of detection reagent and its preparation method and application |
| CN110286237A (en) * | 2019-06-29 | 2019-09-27 | 山东博科诊断科技有限公司 | Five chemiluminescence detection kits of first function |
| CN113621679A (en) * | 2021-08-04 | 2021-11-09 | 北京康思润业生物技术有限公司 | Homocysteine kit and preparation method thereof |
| CN114878840A (en) * | 2022-07-12 | 2022-08-09 | 昆明思安生物科技有限公司 | Kit and method for measuring triiodothyronine by magnetic particle chemiluminescence |
| CN115043898A (en) * | 2021-03-08 | 2022-09-13 | 迈克生物股份有限公司 | Biotinylated antigen derivatives, related kit and use |
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| CN115043898A (en) * | 2021-03-08 | 2022-09-13 | 迈克生物股份有限公司 | Biotinylated antigen derivatives, related kit and use |
| CN113621679A (en) * | 2021-08-04 | 2021-11-09 | 北京康思润业生物技术有限公司 | Homocysteine kit and preparation method thereof |
| CN114878840A (en) * | 2022-07-12 | 2022-08-09 | 昆明思安生物科技有限公司 | Kit and method for measuring triiodothyronine by magnetic particle chemiluminescence |
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Application publication date: 20181113 |