CN109239368A - Measure the method and kit of progesterone - Google Patents
Measure the method and kit of progesterone Download PDFInfo
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- CN109239368A CN109239368A CN201811146334.4A CN201811146334A CN109239368A CN 109239368 A CN109239368 A CN 109239368A CN 201811146334 A CN201811146334 A CN 201811146334A CN 109239368 A CN109239368 A CN 109239368A
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- progesterone
- antibody
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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
-
- 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
-
- 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
- G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
Abstract
The present invention relates to a kind of methods for measuring progesterone, comprising: 1) progesterone that sample is modified with water-soluble biological element or derivatives thereof and antiprogestin antibody is mixed incubation, wherein the antiprogestin antibody is marked through acridine chemiluminescent substance；2) Streptavidin particle is added；And 3) obtain the concentration of progesterone in sample.This method can improve the measurement effect of progesterone.The invention further relates to the kits of measurement progesterone.
Description
Technical field
The invention belongs to field of immunodetection, and in particular to a kind of for measuring the chemiluminescence immunoassay side of progesterone
Method.
Background technique
Progesterone (Progesterone) is a kind of 21 important carbons steroid hormones in human body, 314.5 dalton of molecular weight,
For the intermediate product during cholesterol metabolic, it is transformed through pregnenolone by isomerase by cholesterol.Its function is main
It is the growth and function for maintaining corpus luteum, preparation fertilization egg implantation inhibits uterus muscle contraction etc..Progesterone in blood mainly with white egg
The carriers such as white, sex hormone binding globulin, cortisol binding globulin are combined and are recycled in vivo.Normal male and women produce
Raw progesterone level is all very low.Progesterone content in blood increases rapidly on the day before the ovulation of menstrual cycle of female.It is becoming pregnant
Afterwards, corpus luteum continues to secrete progesterone, and is continued until the 12nd week of gestation；Hereafter, placenta is increasingly becoming the main next of progesterone
Source.If not becoming pregnant, atrophy of corpus iuteum, the progesterone in blood is rapidly reduced to follicular phase level.
In recent years, the measuring method of progesterone mainly has chemiluminescence immunoassay, enzyme immunoassay (EIA) and radio-immunity
Analytic approach etc., wherein acridine chemiluminescence immunoassay is the common method in the measurement of progesterone.Established at present
In acridine chemiluminescence detection system, as progesterone is commercialized in the ARCHITECT Progesterone of Abbott (Abbott Laboratories) company
The ADVIA of detection reagent and SIEMENS companyProgesterone detection reagent is commercialized in CP, is all made of derivatives of progesterone packet
Progesterone is quantitative determined come the mode of the antibody marked with the progesterone competitive binding acridine in sample by particle.
Although above-mentioned detection architecture has been widely used for the measurement of clinical progesterone sample, there are still certain to lack
It falls into.On the one hand, derivatives of progesterone is connected with particle in mentioned reagent, since particle volume, quality are much larger than progesterone antigen, institute
To be also easy to produce space steric effect when antigen is in conjunction with antibody；On the other hand, it is coated in the derivatives of progesterone one of microparticle surfaces
As be hydrophobic structure, easily because hydrophobic effect assemble prevent antibody from effectively identifying its epitope, thus leads to derivatives of progesterone not
Stable problem.
Accordingly, when using acridine chemiluminescence analysis to measure progesterone, there is improve particle in reaction process to bring
Space steric effect and progesterone or derivatives thereof stability demand；There is also the further progesterone that improves to measure effect simultaneously
Demand.
Summary of the invention
In order to achieve the above objectives, the present inventor's trial various ways are adjusted existing measuring method, such as directly
Derivatives of progesterone is marked using small molecule acridine compounds, or uses BSA bridge joint acridine and progesterone or derivatives thereof to solve
Above-mentioned steric hindrance and/or stability problem.Unfortunately, these methods fail to get a desired effect.
After numerous studies, the present inventor is found surprisingly that can effectively improve steric hindrance effect with the following method
Answer and derivatives of progesterone stability: progesterone for first modifying water-soluble biological element or derivatives thereof and the progesterone competition in sample are tied
Close acridine labelled antibody；Adding streptavidin particle, (Fig. 1 shows this hair in conjunction with the derivatives of progesterone of biotin modification
The experimental principle of bright method), then realize the present invention.
Accordingly, in a first aspect, being wrapped the present invention provides a kind of progesterone measuring method based on direct chemoluminescence method
It includes:
1) progesterone that sample is modified with water-soluble biological element or derivatives thereof and antiprogestin antibody are mixed into incubation,
In, the antiprogestin antibody is marked through acridine chemiluminescent substance；
2) Streptavidin particle is added；
3) Streptavidin particle is separated；And
4) concentration of progesterone in sample is obtained.
In a preferred embodiment, the Streptavidin particle is the coated magnetic particle of streptavidin albumen.
In a preferred embodiment, the partial size of the streptavidin particle is 1~3 μm, more preferably 1 μm.
In a preferred embodiment, the biotin is the biotin with water-soluble Linker, water-soluble
Linker can be-PEGn(n=1~10) ,-SO3 -And/or glycine betaine.
In a particular embodiment, the acridine chemiluminescent substance is acridinium ester or derivatives thereof, preferably a word used for translation
Pyridine ester.
In a preferred embodiment, the antiprogestin antibody is source of mouse monoclonal antibody.
In a preferred embodiment, the sample can be serum, heparin blood plasma, edta plasma.
In a particular embodiment, the concentration of progesterone in sample is obtained by measurement luminous signal value.
In second aspect, the present invention provides a kind of for detecting the kit of progesterone, comprising:
Progesterone or derivatives thereof, described progesterone or derivatives thereof are modified through water-soluble biological element；
Antiprogestin antibody, the antibody are marked through acridine chemiluminescent substance；With
Streptavidin particle.
In a preferred embodiment, the Streptavidin particle is the coated magnetic particle of streptavidin albumen.
In a preferred embodiment, the partial size of the streptavidin particle is about 1~3 μm, and more preferably about 1 μm.
In a particular embodiment, the acridine chemiluminescent substance is acridinium ester or derivatives thereof, preferably a word used for translation
Pyridine ester.
In a preferred embodiment, the antiprogestin antibody is source of mouse monoclonal antibody.
The beneficial effects of the present invention are the sensitivity and stability that improve progesterone assay kit.
Detailed description of the invention
Fig. 1 shows the experimental principle of the method for the present invention；
Fig. 2 shows the Progesterone III for using Roche companyMeasure experimental principle when progesterone；
When Fig. 3 shows method (Roche-AE method) the measurement progesterone calibration object using method and comparative example 2 of the invention
Result；
Specific embodiment
Currently, being used to measure in the commercialization acridine chemical illuminating reagent of progesterone, there is be also easy to produce space steric effect
And the problem of derivatives of progesterone stability difference, these situations more or less will affect final measurement effect.In order to improve
So that the measurement effect of progesterone is even more ideal, the present inventor attempts to existing the stability of space steric effect and derivatives of progesterone
Acridine chemiluminescence analysis method be adjusted, however most of adjustment modes do not obtain more preferably effect.
On the basis of numerous studies, inventors have surprisingly discovered that " progesterone first modified with water-soluble biological element is derivative
Progesterone competitive binding acridine labelled antibody in object and sample；Streptavidin magnetic particle is added to spread out with biotin modification progesterone
This mensuration mode of bioconjugation " obtains even more ideal while improving space steric effect and derivatives of progesterone stability
Measurement effect.
In field of immunodetection, the existing analysis method using biotin and streptavidin system, for example,
The Progesterone III of Roche companyUsing a kind of electrochemiluminescence analysis method: first by sample and life
The progesterone antibody of object element combines, and adds the derivatives of progesterone reaction of streptavidin magnetic particle and tris (bipyridine) ruthenium label
(Fig. 2 shows the experimental principles of the above method).On the one hand, due to marking derivatives of progesterone using tris (bipyridine) ruthenium, thus it is theoretical
On should there is no because caused by particle space steric effect and derivatives of progesterone assemble the problem of；On the other hand, with the present invention
Method it is identical, this method is also with the progesterone competitive binding antiprogestin antibody in derivatives of progesterone and sample, i.e., both
Belong to competition law.On this basis, we attempt the tris (bipyridine) ruthenium in the progesterone measuring method of Roche company replacing with a word used for translation
Pyridine substance (hereinafter referred to Roche-AE method), and the measurement effect of expected replaced method by with method phase of the invention
Seemingly.However surprisingly: although equally using using acridinium ester luminesceence analysis, the experimental principle based on competition law and equally
Streptavidin and biotin system, method of the invention are but substantially better than Roche-AE method in terms of sensitivity, stability
(embodiment 3~4 as detailed below).
Accordingly, the present invention provides a kind of method and kit for measuring progesterone, which overcome in current commercial reagents
The problem of existing space steric effect and derivatives of progesterone are assembled, while this method and reagent achieve unexpected measurement
Effect.
Below in conjunction with specific embodiment and embodiment, it is specifically described the present invention, advantages of the present invention and various effects
It thus will clearly present.It will be understood by those skilled in the art that these specific embodiments and embodiment are for illustrating
The present invention is not intended to limit the present invention.
Throughout the specification, unless otherwise specified, terms used herein are interpreted as usual in this field
Used meaning.Therefore, unless otherwise defined, all technical and scientific terms used herein has leads with belonging to the present invention
The identical meaning of the general understanding of field technique personnel.Contradiction if it exists, this specification are preferential.
Streptavidin particle
In the present invention, " streptavidin particle " is also referred to as " streptavidin particle " and " streptavidin pearl ",
Refer to Streptavidin particle be the coated particle of streptavidin albumen, specifically by streptavidin albumen covalent coupling in
On the acylated particle of toluene Huang or carboxyl particle, partial size is usually in 1.0~3.0 μ ms.
Common streptavidin particle can be the coated magnetic particle of streptavidin albumen, such as purchase as Thermo
Fisher'sMyOneTMStreptavidin T1/C1,M-280 Streptavidin；Purchase for
The Magnosphere of JSR Life Sciences companyTMMS300/Streptavidin, however, the present invention is not limited thereto.
The progesterone or derivatives thereof of water-soluble biological element modification
In the present invention, " progesterone or derivatives thereof of water-soluble biological element modification " refers to water soluble group (such as water
Dissolubility Linker) the progesterone of biotin modification or derivatives thereof, wherein water-soluble Linker can be-PEGn(n=1~
10)、-SO3 -, glycine betaine etc..
In the present invention, " derivatives of progesterone " refers to the progesterone containing the active group that can be used for being coupled, institute on molecule
The active group of modification has no effect on its immunoreactivity.Currently, derivatives of progesterone is widely used in progesterone measurement field,
Such as the ADVIA of SIEMENS companyThe ARCHITECT of progesterone detection reagent and Abbott is commercialized in CP
It includes derivatives of progesterone in progesterone detection reagent that Progesterone, which is commercialized,.Illustrative derivatives of progesterone includes progesterone-
3-CMO (Progesterone-3-CMO), progesterone -6-CMO (Progesterone-6-CMO), -11 α of progesterone-hemisuccinic acid ester
(Progesterone-11 alpha-Hemisuccinate), however, the present invention is not limited thereto.
Antiprogestin antibody
Term " antiprogestin antibody " refers to the immunoglobulin molecules of specific binding progesterone, including but not limited to inosculating antibody
Body, humanized antibody, human antibody, CDR grafted antibody and antibody construct, such as scFv (scFv) or Antibody Fusion egg
It is white；In addition, further relating to antibody recombinantly or synthetically.
" segment of antiprogestin antibody " generally includes the antigen binding domain of antiprogestin antibody, light chain and/or heavy chain variable region,
At least part of one or more (such as six) CDR retains at least certain binding specificity of parental generation antibody.Antibody piece
The example of section includes but is not limited to Fab, Fab', F (ab')2With Fv segment；Homodimer；Linear antibodies；Single-chain antibody molecules,
For example, sc-Fv；And the multi-specificity antibody formed by antibody fragment.In general, when based on mole come expression activity, segment
Retain at least 50% combination activity to progesterone.Preferably compared with parental generation antibody, segment retain at least 60%, 70%,
80%, 90%, the 95% or 100% combination activity to progesterone.
Preferably, antibody fragment refers to antigen binding domain, light chain and the heavy chain variable region or six CDR of antibody.
In some embodiments, antibody sources can be rabbit source, source of mouse, goat source and/or sheep source etc..
In a preferred embodiment, antibody can be source of mouse monoclonal antibody.
Acridine chemiluminescent substance
In the present invention, " acridine chemiluminescent substance " refers to acridinium ester and its derivative, acridine sulfonamide chemistry hair
Stimulative substance and lucigenin, it is suitable for chemiluminescence immunoassay systems.In alkaline H2O2In solution, molecule is by hydrogen peroxide
When ion attack, unstable dichloroethane is generated, this dichloroethane is decomposed into CO2With the N- methylacridine of excited electronic state
Ketone issues photon when it returns to ground state.
Acridine ester derivant is well-known in chemiluminescence immune assay field, such as Chinese patent
The acridine ester derivant (Formulas I) and preparation method thereof for chemiluminescence immune assay is described in 201510090045.7；Law
Deng (Novel poly-substituted aryl acridinium esters and their use in
immunoassay,J Biolumin Chemilumin.1989Jul；4 (1): 88-98) it has synthesized and a series of can be used for immune point
The acridine ester analogs of analysis.
Measuring method
The present invention provides a kind of methods for measuring progesterone, comprising:
1) sample obtained from subject is at least mixed and is incubated for following two reagent: modified through water-soluble biological element
Progesterone or derivatives thereof, the antiprogestin antibody that is marked through acridine chemiluminescent substance；
2) streptavidin particle, reaction a period of time are added into the mixture obtained by step 1)；
3) chemiluminescence signal value is obtained.
In a kind of modification of measuring method of the present invention, comprising:
1) by the sample obtained from subject at least with the antiprogestin antibody and chain that are marked through acridine chemiluminescent substance
Enzyme Avidin particle is mixed and is incubated for；
2) progesterone or derivatives thereof modified through water-soluble biological element is added into the obtained mixture of step 1)；
3) chemiluminescence signal value is obtained.
In a preferred embodiment, the acridine chemiluminescent substance can be acridinium ester and its derivative.
In a preferred embodiment, the streptavidin particle can be 1 μm of streptavidin magnetic particle.
In a preferred embodiment, the antiprogestin antibody marked through acridine chemiluminescent substance is anti-for source of mouse monoclonal
Body.
In a preferred embodiment, further include the steps that contacting sample with dissociation agent in 1).For example, dissociation agent can be with
It is pre-mixed to be used as a reagent with the progesterone or derivatives thereof of water-soluble biological element modification；Or can with through acridine
The antibody of chemiluminescent substance label is pre-mixed as a reagent；Or to can be used as individual reagent pre- for dissociation agent
Elder generation and sample contact.
In a particular embodiment, it may further include between step 2) and step 3) described in cleaning and/or separation
The step of streptavidin particle.Wherein cleaning can be by unbonded progesterone through biotin modification or derivatives thereof, not
In conjunction with antiprogestin antibody and other materials removal.
In a particular embodiment, by the way that substrate solution is added chemiluminescence reaction can occur for step 3), wherein described
Substrate solution is preferably NaOH and H2O2；In step 3) concentration of progesterone can be obtained by reading luminous signal value.
Kit
The present invention provides a kind of for measuring the kit of progesterone, comprising:
The progesterone or derivatives thereof of water-soluble biological element modification；
Antiprogestin antibody, the antibody are marked through acridine chemiluminescent substance；With
The particle modified through streptavidin.
It will be appreciated by those skilled in the art that the kit can also include other for measuring other reagents of progesterone
Or component, such as the dissociation agent for stripping down the progesterone in sample from the carriers such as sex hormone binding globulin；For
Draw the calibration object of standard curve；Quality-control product for quality control；For the substrate solution of chemiluminescence reaction to occur；Cleaning is slow
Fliud flushing and Sample dilution etc., as long as these reagents are used to constitute the kit of measurement progesterone.
In a particular embodiment, the concentration of progesterone of the water-soluble biological element modification or derivatives thereof is about 1ng/
ML~1000ng/mL, dissociation agent concentration is about 0.01 μ of μ g/mL~50 g/mL.
In a particular embodiment, the concentration of the antiprogestin antibody is about 1ng/mL~1000ng/mL.
In a particular embodiment, the concentration of the streptavidin magnetic particle is about 10 μ of μ g/mL~1000 g/mL.
The present invention is described in more detail by the following examples, but the present invention is not limited to these Examples.
Embodiment 1
Preparation method:
1) it prepares streptavidin magnetic particle: taking 1mL streptavidin magnetic particle (Thermo Fisher) with 20mM pH
After 7.4PBS+0.1%BSA+0.05%Tween 20+0.05%PC-300 is washed 3 times, it is diluted to 50mL.
2) it prepares biotinylation progesterone: successively weighing -11 α of 50mg progesterone-hemisuccinic acid ester (Sichuan mikey biological material
Technology Co., Ltd.), 45.0mg EDC.HCl (1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate),
16.0mg HOBT (I-hydroxybenzotriazole), 36.0mg TEA (triethylamine) are in 1.0mL DMF (N,N-dimethylformamide)
In, it is vortexed after mixing, room temperature is protected from light 1h.161.0mg biotin-PEG is then added4-NH2(Sichuan mikey biological material
Technology Co., Ltd.), room temperature is protected from light for 24 hours.Products therefrom is spare after high-efficient liquid phase chromatogram purification.By the dilution of 1:2000
Than by the 20mM pH 7.4PBS+0.1%BSA+2% sucrose+0.05%Tween 20+ of biotinylation progesterone after purification
0.05%PC-300 is diluted to working concentration.It is eventually adding dissociation agent and reaches drawing azoles to final concentration of 2 μ g/mL.
3) it prepares the antiprogestin antibody of acridinium ester label: 0.1mg progesterone antibody (Medix Biochemica) being taken to use 10mM
Acridinium ester be marked by the molar ratio of 1:10, desalination column purification.Isometric glycerol vortex is eventually adding to mix.Use 50mM
PH 7.4Tris+0.1%BSA+2% sucrose+0.05%Tween 20+0.05%PC-300 is by above-mentioned marker by 1:4000's
Volume ratio is diluted to working concentration.
4) it prepares calibration object: accurately weighing 1~2mg progesterone sterling in DMSO, be configured to the intermediate fluid of 4 μ g/mL, then
With 20mM pH 7.4PBS+10%BSA+2% sucrose+0.05%Tween 20+0.05%PC-300 be diluted to 0.0ng/mL,
Eight concentration ladders of 0.2ng/mL, 1.0ng/mL, 4.0ng/mL, 10.0ng/mL, 20.0ng/mL, 40.0ng/mL, 60.0ng/mL
Degree.
Measuring method:
Reagent preparation, calibration object and quality-control product in embodiment 1 are placed in 3000 automatic chemiluminescence immunoassay of I (to step
Gram Biological Co., Ltd.) on, and be measured by instrumentation explanation.
Take the antiprogestin antibody of 20 μ L samples (or calibration object, Quality Control), 50 μ L biotinylation progesterone, 50 μ L acridinium ester labels
It is incubated for 10min under the conditions of 37 DEG C, 50 μ L streptavidin magnetic particles are then added and are incubated for 10min again at 37 DEG C.Washing, magnetic
Substrate solution is added after separation to shine.Luminous signal and the inversely proportional relationship of testing concentration.
Comparative example 1
Prepare the coated magnetic particle of progesterone: amino magnetic bead used isM-270 Amine (is purchased from Thermo
Fisher), it is coated with according to its method recorded in specification, wherein progesterone (- 11 α of progesterone-hemisuccinic acid ester, Sichuan
Mikey biology new material technology Co., Ltd) coating ratio be 5 μ g/mg, with 20mM pH 7.4PBS+0.1%BSA+0.05%
1:50 is diluted to working concentration to Tween 20+0.05%PC-300 by volume, is stored in 2~8 DEG C.
Acridine labelled antibody, calibration objectDeng preparation with embodiment 1.
Measuring method: take 20 μ L samples (or calibration object, Quality Control), the 50 coated magnetic particles of μ L progesterone (containing reaching for 2 μ g/mL
That azoles), 50 μ L acridines label antibody be incubated for 20min under the conditions of 37 DEG C.Substrate solution is added after washing, Magneto separate to shine.
The measurement of embodiment 2 calibration object and quality-control product
Using the measuring method and ratio of this programme in embodiment 1 compared with 1 in measuring method to progesterone calibration object Cal 1~
8(0.0ng/mL、0.2ng/mL、1.0ng/mL、4.0ng/mL、10.0ng/mL、20.0ng/mL、40.0ng/mL、60.0ng/
ML) and quality-control product (QC1, indicate concentration 0.50ng/mL；QC2 indicates concentration 7.40ng/ml；QC3 indicates concentration 20.60ng/
ML), experimental result is as shown in table 1 below:
Table 1
As shown in Table 1, (derivatives of progesterone is coated in the scheme on the magnetic particle in the prior art) Quality Control of comparative example 1 is surveyed
Determine result and be apparently higher than mark concentration and the solution of the present invention (embodiment 1), and signal value is reduced with the extension of standing time.
Its reason may be that cannot efficiently identify, since the steric hindrance of magnetic particle causes antibody in conjunction with derivatives of progesterone, in turn
Cause measurement result higher；On the other hand, due to the hydrophobic property of derivatives of progesterone, easily constantly assemble and draw on magnetic particle surface
Playing signal reduces.
2 Roche-AE method of comparative example
In progesterone measuring method disclosed at present, the measuring method of Roche Progesterone III kit with
The solution of the present invention is closest, and in order to verify the effect of Roche scheme on acridine luminesceence analysis body platform, we are by Ru
(bpy)3 2+The progesterone of label replaces with the progesterone of acridine label, to obtain the combination of reagent used in comparative example 2.
Preparation method:
Prepare the coated particle of streptavidin: method is the same as embodiment 1；
Prepare biotinylation antiprogestin antibody: take the progesterone antibody (Medix Biochemica) of 0.1mg 1mg/mL in
In 2mL centrifuge tube.It is marked with the biotin ester (Biotin-LC-NHS, Thermo Fisher) of 10mM by the mole of 20:1
Note, desalination column purification.Isometric glycerol vortex is eventually adding to mix.With 50mM pH 7.4Tris+0.1%BSA+2% sucrose
+ 0.05%Tween 20+0.05%PC-300 is diluted the antiprogestin antibody of above-mentioned biotin modification by the volume ratio of 1:4000
To working concentration.
Prepare the progesterone of acridine label: taking -11 α of progesterone-hemisuccinic acid ester of 100 μ L 50mM, (mikey biology in Sichuan is new
Materials Technology Ltd., DMSO dissolution), acridine hydrazides (NSP-SA-ADH, Xiamen He Lisen) is added in 1:5 in molar ratio, room
Warm roller bearing is protected from light for 24 hours.Then by products therefrom through high performance liquid chromatography separation, purifying.With 50mM pH 7.4Tris+
The progesterone that 0.1%BSA+2% sucrose+0.05%Tween 20+0.05%PC-300 marks above-mentioned acridine by 1:4000 body
Product ratio is diluted to working concentration.
Measuring method:
Take 20 μ L samples (or calibration object, Quality Control), 50 μ L biotinylation antiprogestin antibody be incubated at 37 DEG C 10min, with
The progesterone of 50 μ L acridines label is added afterwards, streptavidin magnetic particle is incubated for 10min under the conditions of 37 DEG C.Washing, Magneto separate
Substrate solution is added afterwards to shine.
3 sensitivity test of embodiment
Progesterone calibration object Cal is measured using the method (Roche-AE method) in the method and comparative example 2 in embodiment 1 respectively
1~8, experimental result is as shown in Table 2 and Fig. 3.
Table 2
It can be seen that the sensitivity of the method for the present invention is better than comparative example 2 (i.e. Roche-AE method).
4 heat stability testing of embodiment
It is placed in when by reagent combination of the invention in embodiment 1 with reagent set contract in comparative example 2 (Roche-AE method)
37 DEG C of water-bath 7 days, 14 days, the signal retention rate after calculating heat treatment, experimental result was as shown in table 3.
Table 3
As can be seen that the thermal stability of reagent of the present invention is significantly better than the reagent in comparative example 2.
Claims (10)
1. a kind of method for measuring progesterone, comprising:
1) progesterone that sample is modified with water-soluble biological element or derivatives thereof and antiprogestin antibody are mixed into incubation, wherein institute
Antiprogestin antibody is stated to mark through acridine chemiluminescent substance；
2) Streptavidin particle is added；And
3) concentration of progesterone in sample is obtained.
2. according to the method described in claim 1, wherein, the Streptavidin particle is streptavidin magnetic particle, preferably
, partial size is in 1~3 μ m.
3. according to the method described in claim 1, wherein, the acridine chemiluminescent substance is acridinium ester and its derivative.
4. according to the method described in claim 1, wherein, water-soluble biological element is with-PEGn-、-SO3 -And/or glycine betaine
Biotin, wherein n=1~10.
5. according to the method described in claim 1, wherein, the sample is serum or blood plasma.
6. method according to any one of claims 1 to 5, wherein obtained in sample by measurement luminous signal value
The concentration of progesterone.
7. kit, comprising:
Progesterone or derivatives thereof, described progesterone or derivatives thereof are modified through water-soluble biological element；
Antiprogestin antibody, the antibody are marked through acridine chemiluminescent substance；With
Streptavidin particle.
8. kit according to claim 7, wherein the Streptavidin particle is streptavidin magnetic particle, excellent
Choosing, partial size is in 1~3 μ m.
9. kit according to claim 7, wherein the acridine chemiluminescent substance is acridinium ester or its derivative
Object.
10. the kit according to any one of claim 7~9, wherein the water-soluble biological element be with-
PEGn-、-SO3 -And/or the biotin of glycine betaine, wherein n=1~10.
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| CN115494233A (en) * | 2022-09-21 | 2022-12-20 | 深圳市国赛生物技术有限公司 | Combined reagent for detecting alpha-fetoprotein heteroplasmon and detection method |
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