CN109061198A - inhibin A detection kit and preparation method thereof - Google Patents
inhibin A detection kit and preparation method thereof Download PDFInfo
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- CN109061198A CN109061198A CN201810939724.0A CN201810939724A CN109061198A CN 109061198 A CN109061198 A CN 109061198A CN 201810939724 A CN201810939724 A CN 201810939724A CN 109061198 A CN109061198 A CN 109061198A
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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
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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
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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
- G01N2333/655—Somatostatins
Abstract
The present invention provides a kind of inhibin A detection kit and preparation method thereof, belongs to immune diagnostic technique field.Solve that existing inhibin A detection kit luminescence-producing reaction speed is slow, and sensitivity is low, and the range of linearity is narrow, problem at high cost.The kit includes: streptavidin bead suspension, the inhibin A antibody of acridinium ester label and biotin labeling inhibin A antibody.The present invention also provides a kind of preparation methods of inhibin A detection kit.It since the reagent testing principle is double-antibody method, is detected respectively by the antibody of acridinium ester label and biotin labeling, not only increases luminance while expanding order of reaction, substantially increase the specificity and detection speed of reagent.
Description
Technical field
The invention belongs to immune diagnostic technique fields, and in particular to a kind of inhibin A detection kit and preparation method thereof.
Background technique
Inhibin is by the heterodimer sugar of the Sertoli cell secretion of the granular cell and male testical of female ovary
Protein hormones.Inhibin selectively inhibits the secretion of hypophysis follicle-stimulating hormone (FSH) (FSH), while also carrying out itself in gonad
Paracrine activity.Molecular weight by the inhibin molecule handled completely is about 32kD, it include two different chains (α and
β), the two chains are covalently attached by cystine linkage.Precursor is that the high molecular mass form of an alpha subunit also appears in flowing folliculus
In liquid and serum.In addition, having also appeared free alpha subunit form, it is not connected with β subunit, and is a lack of inhibin
Bioactivity.Inhibin A (INH-A/Inhibin A) includes an alpha subunit and a β A subunit.It is some
The report delivered points out that inhibin A can be used for the research of Human Physiology genesiology, can be used as monitoring to the measurement of inhibin A
The endocrine indexes of ovarian function.
In immunoassay, the separation of Ag-Ab immune complex is often comparatively laborious, time-consuming, and often signal-to-noise ratio is low
The reason of, the generally existing detection problem that lower bound sensitivity is not high, the range of linearity is relatively narrow of kit of inhibin A is detected at present.
Summary of the invention
The purpose of the present invention is to solve existing inhibin A detection kit luminescence-producing reaction speed is slow, sensitivity is low,
The range of linearity is narrow, problem at high cost, and a kind of inhibin A detection kit is provided and preparation method thereof.
Present invention firstly provides a kind of inhibin A detection kit, which includes:
Streptavidin bead suspension
The inhibin A antibody of acridinium ester label
Biotin labeling inhibin A antibody
Preferably, the partial size of the magnetic bead in the streptavidin bead suspension is 0.05~3 μm.
Preferably, in the inhibin A antibody of the acridinium ester label, the molar ratio of acridinium ester and inhibin A antibody
For 1:(3~20).
Preferably, in the biotin labeling inhibin A antibody, the molar ratio of biotin and inhibin A antibody is
1:(5~20).
Preferably, the kit further includes Chemoluminescent substrate and cleaning solution, the Chemoluminescent substrate
Including A liquid and B liquid.
Preferably, the A liquid is nitric acid solution, and B liquid is sodium hydroxide solution, and the cleaning solution is PB.
Preferably, the kit further includes calibration object and quality-control product: calibration concentration is respectively 0pg/mL, 10pg/
mL、50pg/mL、100pg/mL、500pg/mL、1000pg/mL、2500pg/mL。
The present invention also provides a kind of preparation methods of inhibin A detection kit, this method comprises:
Step 1: the preparation of streptavidin bead suspension
1) after iron chloride and frerrous chloride being dissolved mixing in water, ammonium hydroxide is added, reacts 3-5h at 50-70 DEG C, obtains
To magnetic microsphere；
2) magnetic microsphere, PEG4000, ammonium hydroxide and ethyl orthosilicate that step 1) obtains are stirred to react at 60-65 DEG C
14h-18h obtains modified magnetic microsphere；
3) modified magnetic microsphere, ammonium hydroxide, tetramethylammonium hydroxide TMA and the 3- aminopropyl three obtained step 2)
Methoxy silane is stirred at room temperature reaction 14h-18h, obtains the magnetic microsphere of surface modification amino；
4) step 3) is obtained into the magnetic microsphere of modification amino and reaction 30-40min is stirred at room temperature in TMA, glutaraldehyde,
Then SA is being added, 37 DEG C are stirred to react 14h-18h, are being added glutamic acid and BSA the reaction was continued 1-2h, obtaining Streptavidin
Change bead suspension；
Step 2: the preparation of the inhibin A antibody of acridinium ester label
Inhibin A antibody is put into centrifuge tube and is centrifuged, carbonic acid buffer is then added, acridine ester solution is added after mixing
Centrifugation is protected from light being put into after the centrifugation seal of tube in magazine, then magazine is put into gas bath constant temperature oscillator and is mixed, closing is added
Liquid is put into gas bath constant temperature oscillator and mixes, and by the antibody closed by purifying, collection, is then placed in buffer and dilutes,
It saves；
Step 3: the preparation of biotin labelled antibodies
Inhibin A antibody is taken, is added the long-chain sulfonation biotin activated after the TRIS buffer dialysis purification of pH6.5,2
~8 DEG C of 1~3h of reaction, then it is transferred to room temperature the reaction was continued 10~30min, it is eventually adding lysine and reacts 10~30min, through pure
Change, isometric glycerol and NaN is added3Or ProClin300, obtain the inhibin A antibody of coupling label substance markers.
Preferably, the molar ratio of iron chloride and frerrous chloride is 2:1 in the step one.
Preferably, the confining liquid in the step two is lysine.
Beneficial effects of the present invention
Present invention firstly provides a kind of inhibin A detection kits and preparation method thereof, and the kit is with Streptavidin
Change bead suspension as immune response and isolated solid phase carrier, the magnetic particle of Avidin is mainly used for and biotin
The antibody of label is connected, and can capture target substance by immune response and form immune complex, in the effect of externally-applied magnetic field
Under, immune complex is detained to get off, and realizes separation, to remove the interference of sample mesostroma, reduces background value.And
And compared with the immunoassay of microwell plate, the intervention of magnetic particle not only makes the dosage of reaction reagent reduce about 1/2, and
Greatly shorten the reaction time, it is often more important that reduce background value, improve sensitivity and applicability.The kit simultaneously
Avidin-biotin system is introduced, which has the characteristics that high sensitivity, high specific, high stability and applicability.
Biotin combines easily in conjunction with the large biological molecules such as protein and nucleic acid, then with biotin derivative, and signal multistage is amplified,
It is able to maintain original bioactivity of macromolecular substances.Combination between Avidin and biotin has high affinity, reaction
In high specificity, do not increase nonspecific interference, will not be impacted because of reaction reagent concentration level, while acid, alkali, denaturation
Agent and organic solvent will not influence the binding force of Avidin and biotin, increase the stability of reagent entirety.Due to the examination
Agent testing principle is double-antibody method, is detected, is not only increased by the antibody of acridinium ester label and biotin labeling respectively
Luminance expands order of reaction simultaneously, the specificity and detection speed of reagent is substantially increased, to be clinical for detecting inhibin A
Relevant disease and scientific research provide certain technical support.
Detailed description of the invention
Fig. 1 is 1 inhibin A detection kit dose response calibration curve of the embodiment of the present invention.
Specific embodiment
Present invention firstly provides a kind of inhibin A detection kit, which includes:
Streptavidin bead suspension
The inhibin A antibody of acridinium ester label
Biotin labeling inhibin A antibody
According to the present invention, the partial size of the magnetic bead in the streptavidin bead suspension is 0.05~3 μm, more excellent
It is selected as 1 μm.
According to the present invention, in the inhibin A antibody of the acridinium ester label, acridinium ester and inhibin A antibody label
Molar ratio is preferably 1:(3~20), more preferably 1:(3~5).
According to the present invention, in the biotin labeling inhibin A antibody, the label of biotin and inhibin A antibody
Molar ratio is preferably 1:(5~20), more preferably 1:(5~10).
According to the present invention, the kit further includes Chemoluminescent substrate and cleaning solution, the chemiluminescent substrate
Liquid includes A liquid and B liquid；The A liquid is preferably nitric acid solution, and B liquid is preferably sodium hydroxide solution, cleaning solution PB.
According to the present invention, the kit further includes calibration object and quality-control product, the same school of quality-control product preparation method
Quasi- product, the calibration object can be prepared by the following method to obtain:
1. the composition of calibration object buffer: by the sodium chloride of PB, 0.15mol/L of 0.1mol/L, 1% bovine serum albumin
The tween-20 of bletilla 0.05% is formed, pH value 6.5, and 2-8 DEG C of storage is spare；
2. taking inhibin Staphylococal Protein A with 1. step gained calibration object buffer is configured to concentration is respectively 0pg/mL, 10pg/mL,
The calibration object of 50pg/mL, 100pg/mL, 500pg/mL, 1000pg/mL, 2500pg/mL, equivalent are distributed into concentration and are respectively
7 bottles of calibration objects of 0pg/mL, 10pg/mL, 50pg/mL, 100pg/mL, 500pg/mL, 1000pg/mL, 2500pg/mL.
The present invention also provides a kind of preparation methods of inhibin A detection kit, this method comprises:
Step 1: the preparation of streptavidin bead suspension
1) after iron chloride and frerrous chloride being dissolved mixing in water, ammonium hydroxide is added, reacts 3-5h at 50-70 DEG C, obtains
To magnetic microsphere；The molar ratio of the iron chloride and frerrous chloride is preferably 2:1；
2) it is scattered in 50% ethyl alcohol again after washing the magnetic microsphere that step 1) obtains and sequentially adds 0.5%
PEG4000,0.1% ammonium hydroxide and 0.1% ethyl orthosilicate are stirred to react 14h-18h at 60-65 DEG C, and it is micro- to obtain modified magnetism
Ball；The volume ratio of the PEG4000, ammonium hydroxide and ethyl orthosilicate are preferably 1:5:1；
3) it is resuspended in after alternately washing modified magnetic microsphere ultrapure water and dehydrated alcohol that step 2) obtains
The 3- aminopropyl three of 0.05% ammonium hydroxide, 0.5% tetramethylammonium hydroxide TMA and 2.5% is separately added into 50% ethyl alcohol again
Methoxy silane is stirred at room temperature reaction 14h-18h, obtains the magnetic microsphere of surface modification amino；The ammonium hydroxide, tetramethyl hydrogen
The volume ratio of amine-oxides TMA and 3- aminopropyl trimethoxysilane is preferably 5:2:4；
4) by step 3) obtain modification amino magnetic microsphere cleaned with PBS buffer solution and be resuspended add 0.1%TMA,
Reaction 30-40min is stirred at room temperature in 2.5% glutaraldehyde, is cleaned with carbonic acid buffer and is pressed after ultrasonic disperse is resuspended later
SA is added in 20ug/mg magnetic microsphere, and 37 DEG C are stirred to react 14h-18h, is being added 0.1% glutamic acid and 1%BSA the reaction was continued 1-
2h, PBS buffer solution are resuspended in 1%BSA ultrasonic disperse up to SA magnetic microsphere after washing 3 times containing 0.05% tween-20,
Magnetic particle is added and saves liquid (the Tris buffer containing 0.5%BSA), 2~8 DEG C of preservations obtain streptavidin magnetic bead.Institute
The TMA that states, glutaraldehyde, SA, glutamic acid, BSA volume ratio be preferably 2:3:10:1:1；
Step 2: the preparation of the inhibin A antibody of acridinium ester label
Inhibin A antibody is put into centrifuge tube and is centrifuged, preferably at room temperature be centrifuged 10s~30s, guarantee antibody be located at from
Heart bottom of the tube position, is then added carbonic acid buffer, and the centrifugation of acridine ester solution is added after mixing, and the centrifuging temperature is preferably
Room temperature, centrifugation time are preferably 0.5min~3min；It is protected from light being put into after the centrifugation seal of tube in magazine, magazine is then put into gas
It bathes in constant temperature oscillator (25 DEG C) and mixes, the mixing time is preferably 2-4h, and confining liquid is added, is put into gas bath constant temperature oscillation
Closing is mixed in device, the off-period is preferably 1-2h, by 250 column purification of sephadex G, use on the antibody closed
The elution of PB buffer, collects, is then placed in buffer and dilutes, and saves；
The quality (μ g) of the inhibin A antibody: the volume (μ l) of acridine ester solution is preferably 100:1；The a word used for translation
The concentration of pyridine ester solution is preferably 2mg/mL；The confining liquid is preferably lysine, and mass fraction is preferably 20%；Described
Buffer is 50mM MES, 0.05% Tween-20,0.05%Proclin300, pH6.5；
Step 3: the preparation of biotin labelled antibodies
Inhibin A antibody is taken, is added the long-chain sulfonation biotin activated after the TRIS buffer dialysis purification of pH6.5,2
~8 DEG C of 1~3h of reaction, then it is transferred to room temperature the reaction was continued 10~30min, it is eventually adding lysine and reacts 10~30min, through pure
Change, isometric glycerol and 0.1%NaN3 or 0.1%ProClin300 is added, the inhibin A for obtaining coupling label substance markers is anti-
Body.
The molar ratio of the system element A antibody, the long-chain sulfonation biotin and lysine that have activated is preferably 1:15:100；
The source of the long-chain sulfonation biotin activated be it is commercially available, be selected from Thermo company.
Further detailed description is done to the present invention combined with specific embodiments below.
Embodiment 1
(1) streptavidin bead suspension is prepared
Iron chloride and frerrous chloride are weighed according to molar ratio 2:1, it is dense to end that ammonium hydroxide is added after dissolution mixes in ultrapure water
Degree is 0.5%, and 60 DEG C are stirred to react 4h, obtain magnetic microsphere；
1mL 0.5% is sequentially added by being scattered in 50% ethyl alcohol again after obtained magnetic microsphere milli-Q water
PEG4000, the ammonium hydroxide of 5mL 0.1% and the ethyl orthosilicate of 1mL 0.1%, 60 DEG C are stirred to react 16h, obtain modified
Magnetic microsphere；
It is resuspended in 50% ethyl alcohol again after the above modified magnetic microsphere ultrapure water and dehydrated alcohol are alternately washed
It is separately added into the 3- aminopropyl three of the ammonium hydroxide of 5mL 0.05%, the tetramethylammonium hydroxide TMA and 4mL 2.5% of 2mL 0.5%
Reaction 16h is stirred at room temperature to get the magnetic microsphere of surface modification amino in methoxy silane；
It takes the 8mg amino-magnetic microballoon to be cleaned and be resuspended with PBS buffer solution and adds 2mL 0.1%TMA and 3mL 2.5%
Glutaraldehyde, be stirred at room temperature reaction 30min after cleaned and be resuspended micro- by 20ug/mg magnetism after ultrasonic disperse with carbonic acid buffer
SA is added in ball, and 37 DEG C are stirred to react 16h, and the reaction was continued for the glutamic acid and the BSA of 1mL 1% for being then respectively adding 1mL 0.1%
1h, PBS buffer solution are resuspended in 1%BSA ultrasonic disperse up to SA magnetic microsphere after washing 3 times containing 0.05% tween-20,
Magnetic particle is added and saves liquid (the Tris buffer containing 0.5%BSA), magnetic bead final concentration of 0.072%, 2~8 DEG C of preservations.
(2) preparation of the inhibin A antibody of acridinium ester label
500ug inhibin A antibody is put into centrifuge tube, guarantees that antibody is located at centrifuge tube bottom position (centrifuge room temperature
Centrifugation 20s) carbonate buffer solution is added afterwards, it mixes well, is added after 5uL acridinium ester and is centrifuged 0.5 under room temperature with centrifuge
Minute, it will be centrifuged after the sealing of effective sealed membrane to be put into and be protected from light in magazine, magazine is put into gas bath constant temperature oscillator (25 DEG C) later,
It mixes 4 hours, 20% lysine confining liquid of 1mL is added, is put into gas bath constant temperature oscillator (25 DEG C), middling speed mixes, off-period
50 column purification of sephadex G on the antibody closed is eluted, Fraction collection with PB buffer for 1h, it is anti-by what is gathered
Liquid solution is placed in 2~8 DEG C of preservations；By concentrated solution 100mM, the pH6.5 TRIS buffer dilution of inhibin A antibody label
To final concentration of 0.2ug/ml, 2~8 DEG C of preservations.
(3) preparation of biotin labelled antibodies
500ug inhibin A antibody is taken, has been activated with 15 times of molar ratios are added after the TRIS buffer dialysis purification of pH6.5
Long-chain sulfonation biotin (Sulfo-NHS-LC-Biotin), 2~8 DEG C of reaction 2h, then be transferred to room temperature the reaction was continued 30min,
It is eventually adding the lysine reaction 30min of 100 times of molar ratios, isometric glycerol and 0.1% is added in reaction solution desalination column purification
NaN3Or 0.1%ProClin300.
Fig. 1 is the inhibin A detection kit dose response calibration curve that the embodiment of the present invention 1 is prepared；Have in figure
Body relevant parameter is as shown in table 1: i.e. by calibration object concentration be 0pg/mL, 10pg/mL, 50pg/mL, 100pg/mL, 500pg/mL,
1000pg/mL, 2500pg/mL make standard curve, obtain dependent equation are as follows: y=1453x+8552. linearly dependent coefficient r is greater than
0.999。
Table 1
| Calibration point | Calibration object concentration | Light quantity |
| 1 | 0 | 622 |
| 2 | 10 | 14507 |
| 3 | 50 | 72533 |
| 4 | 100 | 145066 |
| 5 | 500 | 755328 |
| 6 | 1000 | 1490656 |
| 7 | 2500 | 3626640 |
Embodiment 2
(1) streptavidin bead suspension is prepared
Iron chloride and frerrous chloride are weighed according to molar ratio 2:1, it is dense to end that ammonium hydroxide is added after dissolution mixes in ultrapure water
Degree is 0.5%, and 60 DEG C are stirred to react 3h, obtain magnetic microsphere；
1mL 0.5% is sequentially added by being scattered in 50% ethyl alcohol again after obtained magnetic microsphere milli-Q water
PEG4000, the ammonium hydroxide of 5mL 0.1% and the ethyl orthosilicate of 1mL 0.1%, 60 DEG C are stirred to react 14h, obtain modified
Magnetic microsphere；
It is resuspended in 50% ethyl alcohol again after the above modified magnetic microsphere ultrapure water and dehydrated alcohol are alternately washed
It is separately added into the 3- aminopropyl three of the ammonium hydroxide of 5mL 0.05%, the tetramethylammonium hydroxide TMA and 4mL 2.5% of 2mL 0.5%
Reaction 14h is stirred at room temperature to get the magnetic microsphere of surface modification amino in methoxy silane；
It takes the 8mg amino-magnetic microballoon to be cleaned and be resuspended with PBS buffer solution and adds 2mL 0.1%TMA and 3mL 2.5%
Glutaraldehyde, be stirred at room temperature reaction 35min after cleaned and be resuspended micro- by 20ug/mg magnetism after ultrasonic disperse with carbonic acid buffer
SA is added in ball, and 37 DEG C are stirred to react 14h, and the reaction was continued for the glutamic acid and the BSA of 1mL 1% for being then respectively adding 1mL 0.1%
1.5h, it is micro- up to SA magnetism that PBS buffer solution is resuspended in ultrasonic disperse in 1%BSA after washing 3 times containing 0.05% tween-20
Ball is added magnetic particle and saves liquid (the Tris buffer containing 0.5%BSA), magnetic bead final concentration of 0.072%, 2~8 DEG C of preservations.
(2) preparation of the inhibin A antibody of acridinium ester label
500ug inhibin A antibody is put into centrifuge tube, guarantees that antibody is located at centrifuge tube bottom position (centrifuge room temperature
Centrifugation 10s) carbonate buffer solution is added afterwards, it mixes well, is centrifuged 2 points under room temperature with centrifuge after 5uL acridinium ester is added
Clock, will be centrifuged after the sealing of effective sealed membrane to be put into and is protected from light in magazine, and magazine is put into gas bath constant temperature oscillator (25 DEG C) later, is mixed
Even 2 hours, 20% lysine confining liquid of 1mL is added, is put into gas bath constant temperature oscillator (25 DEG C), middling speed mixes, and off-period is
2h elutes 50 column purification of sephadex G on the antibody closed, Fraction collection, the antibody that will be gathered with PB buffer
Solution is placed in 2~8 DEG C of preservations；Concentrated solution 100mM, the pH6.5TRIS buffer that inhibin A antibody marks is diluted to end
Concentration is 0.2ug/ml, 2~8 DEG C of preservations.
(3) preparation of biotin labelled antibodies
500ug inhibin A antibody is taken, has been activated with 15 times of molar ratios are added after the TRIS buffer dialysis purification of pH6.5
Long-chain sulfonation biotin (Sulfo-NHS-LC-Biotin), 2~8 DEG C of reaction 3h, then be transferred to room temperature the reaction was continued 10min,
It is eventually adding the lysine reaction 10min of 100 times of molar ratios, isometric glycerol and 0.1% is added in reaction solution desalination column purification
NaN3Or 0.1%ProClin300.
Embodiment 3
(1) streptavidin bead suspension is prepared
Iron chloride and frerrous chloride are weighed according to molar ratio 2:1, it is dense to end that ammonium hydroxide is added after dissolution mixes in ultrapure water
Degree is 0.5%, and 60 DEG C are stirred to react 5h, obtain magnetic microsphere；
1mL 0.5% is sequentially added by being scattered in 50% ethyl alcohol again after obtained magnetic microsphere milli-Q water
PEG4000, the ammonium hydroxide of 5mL 0.1% and the ethyl orthosilicate of 1mL 0.1%, 60 DEG C are stirred to react 18h, obtain modified
Magnetic microsphere；
It is resuspended in 50% ethyl alcohol again after the above modified magnetic microsphere ultrapure water and dehydrated alcohol are alternately washed
It is separately added into the 3- aminopropyl three of the ammonium hydroxide of 5mL 0.05%, the tetramethylammonium hydroxide TMA and 4mL 2.5% of 2mL 0.5%
Reaction 18h is stirred at room temperature to get the magnetic microsphere of surface modification amino in methoxy silane；
It takes the 8mg amino-magnetic microballoon to be cleaned and be resuspended with PBS buffer solution and adds 2mL 0.1%TMA and 3mL 2.5%
Glutaraldehyde, be stirred at room temperature reaction 40min after cleaned and be resuspended micro- by 20ug/mg magnetism after ultrasonic disperse with carbonic acid buffer
SA is added in ball, and 37 DEG C are stirred to react 18h, and the reaction was continued for the glutamic acid and the BSA of 1mL 1% for being then respectively adding 1mL 0.1%
1h, PBS buffer solution are resuspended in 1%BSA ultrasonic disperse up to SA magnetic microsphere after washing 3 times containing 0.05% tween-20,
Magnetic particle is added and saves liquid (the Tris buffer containing 0.5%BSA), magnetic bead final concentration of 0.072%, 2~8 DEG C of preservations.
(2) preparation of the inhibin A antibody of acridinium ester label
500ug inhibin A antibody is put into centrifuge tube, guarantees that antibody is located at centrifuge tube bottom position (centrifuge room temperature
Centrifugation 30s) carbonate buffer solution is added afterwards, it mixes well, is centrifuged 3 points under room temperature with centrifuge after 5uL acridinium ester is added
Clock, will be centrifuged after the sealing of effective sealed membrane to be put into and is protected from light in magazine, and magazine is put into gas bath constant temperature oscillator (25 DEG C) later, is mixed
Even 3 hours, 20% lysine confining liquid of 1mL is added, is put into gas bath constant temperature oscillator (25 DEG C), middling speed mixes, and off-period is
2h elutes 50 column purification of sephadex G on the antibody closed, Fraction collection, the antibody that will be gathered with PB buffer
Solution is placed in 2~8 DEG C of preservations；Concentrated solution 100mM, pH6.5 the TRIS buffer that inhibin A antibody marks is diluted to
Final concentration of 0.2ug/ml, 2~8 DEG C of preservations.
(3) preparation of biotin labelled antibodies
500ug inhibin A antibody is taken, has been activated with 15 times of molar ratios are added after the TRIS buffer dialysis purification of pH6.5
Long-chain sulfonation biotin (Sulfo-NHS-LC-Biotin), 2~8 DEG C of reaction 1h, then be transferred to room temperature the reaction was continued 30min, most
The lysine that 100 times of molar ratios are added afterwards reacts 20min, and isometric glycerol and 0.1% is added in reaction solution desalination column purification
NaN3Or 0.1%ProClin300.
Performance Evaluation is carried out to the kit that embodiment is prepared below
1. minimum detection limit detects
It uses zero-dose calibration object or Sample dilution to be detected as sample, replication 20 times, obtains 20 detections
As a result RLU value (relative light unit) calculates its average value (M) and standard deviation (SD), obtains M+2SD, calibrated according to zero-dose
Concentration-RLU value result between product and adjacent calibration object carries out two o'clock regression fit and obtains linear function, by the RLU of M+2SD
Value brings above-mentioned equation into, finds out corresponding concentration value, as minimum detection limit, and as a result should meet should be less than 1.0pg/mL.As a result
It is as shown in table 2:
2. minimum detection limit data of table
2. linearity test
At least five kinds of concentration will be diluted in proportion close to the high level sample of the range of linearity upper limit, wherein low value concentration samples
It must be close to the lower limit of the range of linearity.It is carried out according to kit specification operation, the sample of each concentration is repeated to detect 2 times, meter
Average value is calculated, result average value and dilution ratio are subjected to straight line fitting with least square method, and calculate linearly dependent coefficient r,
As a result should meet the range of linearity is 1.0ng/mL~2500ng/mL, and linearly dependent coefficient r answers >=0.9900.As a result such as 3 institute of table
Show:
3. linear data of table
3. repeatability detection
Detection 10 times is repeated with the sample that concentration is respectively 150ng/mL ± 30ng/mL and 800ng/mL ± 160ng/mL,
Calculate the average value of 10 measurement resultsWith standard deviation SD, the coefficient of variation (CV) is calculated according to formula (2), as a result should meet change
Different coefficient (CV) answers≤8.0%.
In formula: the CV-coefficient of variation；
SD-measurement result standard deviation；
The average value of-measurement result.
The results are shown in Table 4:
The repeated data of table 4.
| Measure number | Repeated sample 1 | Repeated sample 2 |
| Rep1 | 148.27 | 824.03 |
| Rep2 | 142.12 | 841.48 |
| Rep3 | 146.17 | 815.82 |
| Rep4 | 143.08 | 830.46 |
| Rep5 | 145.94 | 824.42 |
| Rep6 | 146.00 | 843.20 |
| Rep7 | 144.94 | 825.47 |
| Rep8 | 148.47 | 847.90 |
| Rep9 | 141.99 | 834.58 |
| Rep10 | 149.74 | 842.49 |
| Measure mean value | 145.67 | 832.99 |
| SD | 2.69 | 10.56 |
| CV | 1.84% | 1.27% |
Show that kit involved in the present invention detects linear correlation coefficient r >=0.9999, i.e. linearity test by testing result
It is qualified；Minimum detection limit is 0.0937pg/mL；Repeated testing result meets the coefficient of variation (CV) and answers≤8.0% requirement, i.e., heavy
Renaturation detection is qualified.This kit has the characteristics that high sensitivity, the range of linearity are wide, stability is good.
Claims (10)
1. a kind of inhibin A detection kit, which is characterized in that the kit includes:
Streptavidin bead suspension
The inhibin A antibody of acridinium ester label
Biotin labeling inhibin A antibody.
2. a kind of inhibin A detection kit according to claim 1, which is characterized in that the streptavidin
The partial size of magnetic bead in bead suspension is 0.05~3 μm.
3. a kind of inhibin A detection kit according to claim 1, which is characterized in that the acridinium ester label
In inhibin A antibody, the molar ratio of acridinium ester and inhibin A antibody is 1:(3~20).
4. a kind of inhibin A detection kit according to claim 1, which is characterized in that the biotin labeling suppression
It makes in element A antibody, the molar ratio of biotin and inhibin A antibody is 1:(5~20).
5. a kind of inhibin A detection kit according to claim 1, which is characterized in that the kit further includes
Chemoluminescent substrate and cleaning solution, the Chemoluminescent substrate include A liquid and B liquid.
6. a kind of inhibin A detection kit according to claim 1, which is characterized in that the A liquid is nitric acid solution, B
Liquid is sodium hydroxide solution, and the cleaning solution is PB.
7. a kind of inhibin A detection kit according to claim 1, which is characterized in that the kit further includes
Calibration object and quality-control product: calibration concentration is respectively 0pg/mL, 10pg/mL, 50pg/mL, 100pg/mL, 500pg/mL, 1000pg/
mL、2500pg/mL。
8. a kind of preparation method of inhibin A detection kit according to weighing and require 1, which is characterized in that this method comprises:
Step 1: the preparation of streptavidin bead suspension
1) after iron chloride and frerrous chloride being dissolved mixing in water, ammonium hydroxide is added, reacts 3-5h at 50-70 DEG C, obtains magnetic
Property microballoon；
2) magnetic microsphere, PEG4000, ammonium hydroxide and ethyl orthosilicate that step 1) obtains are stirred to react 14h- at 60-65 DEG C
18h obtains modified magnetic microsphere；
3) modified magnetic microsphere, ammonium hydroxide, tetramethylammonium hydroxide TMA and 3- the aminopropyl trimethoxy obtained step 2)
Base silane is stirred at room temperature reaction 14h-18h, obtains the magnetic microsphere of surface modification amino；
4) step 3) is obtained into the magnetic microsphere of modification amino and reaction 30-40min is stirred at room temperature in TMA, glutaraldehyde, then
SA is being added, 37 DEG C are stirred to react 14h-18h, are being added glutamic acid and BSA the reaction was continued 1-2h, obtaining streptavidin magnetic
Pearl suspension；
Step 2: the preparation of the inhibin A antibody of acridinium ester label
Inhibin A antibody is put into centrifuge tube and is centrifuged, carbonic acid buffer is then added, after mixing be added acridine ester solution from
The heart is protected from light being put into after the centrifugation seal of tube in magazine, then magazine is put into gas bath constant temperature oscillator and is mixed, closing is added
Liquid is put into gas bath constant temperature oscillator and mixes, and by the antibody closed by purifying, collection, is then placed in buffer and dilutes,
It saves；
Step 3: the preparation of biotin labelled antibodies
Inhibin A antibody is taken, is added the long-chain sulfonation biotin activated after the TRIS buffer dialysis purification of pH6.5,2~8
DEG C 1~3h of reaction, then it is transferred to room temperature the reaction was continued 10~30min, it is eventually adding lysine and reacts 10~30min, it is purified, add
Enter isometric glycerol and NaN3Or ProClin300, obtain the inhibin A antibody of coupling label substance markers.
9. a kind of preparation method of inhibin A detection kit according to weighing and require 8, which is characterized in that the step
The molar ratio of iron chloride and frerrous chloride is 2:1 in one.
10. a kind of preparation method of inhibin A detection kit according to weighing and require 8, which is characterized in that the step
Confining liquid in two is lysine.
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| CN201810939724.0A CN109061198A (en) | 2018-08-17 | 2018-08-17 | inhibin A detection kit and preparation method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110261626A (en) * | 2019-07-31 | 2019-09-20 | 宁波奥丞生物科技有限公司 | A kind of PLGF magnetic microparticle chemiluminescence kit and its detection method |
| CN111157725A (en) * | 2020-01-09 | 2020-05-15 | 南京拂晓生物科技有限公司 | Human Legumain chemiluminescence detection kit and application thereof |
| CN113495153A (en) * | 2020-03-20 | 2021-10-12 | 郑州达诺生物技术有限公司 | System reaction liquid, inhibin A quantitative detection kit containing system reaction liquid and using method |
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|---|---|---|---|---|
| CN102565405A (en) * | 2011-08-24 | 2012-07-11 | 苏州长光华医生物试剂有限公司 | Method for immunological detection by combining acridinium ester labeling technology with general magnetic particles |
| CN103357359A (en) * | 2013-05-16 | 2013-10-23 | 英科新创(厦门)科技有限公司 | Complex immunity magnetic particle and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102565405A (en) * | 2011-08-24 | 2012-07-11 | 苏州长光华医生物试剂有限公司 | Method for immunological detection by combining acridinium ester labeling technology with general magnetic particles |
| CN103357359A (en) * | 2013-05-16 | 2013-10-23 | 英科新创(厦门)科技有限公司 | Complex immunity magnetic particle and preparation method thereof |
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| DANIEL S.MCCONNELL ET AL: "Development of a two-site solid-phase immunochemiluminescent", 《CLINICAL CHEMISTRY》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110261626A (en) * | 2019-07-31 | 2019-09-20 | 宁波奥丞生物科技有限公司 | A kind of PLGF magnetic microparticle chemiluminescence kit and its detection method |
| CN111157725A (en) * | 2020-01-09 | 2020-05-15 | 南京拂晓生物科技有限公司 | Human Legumain chemiluminescence detection kit and application thereof |
| CN113495153A (en) * | 2020-03-20 | 2021-10-12 | 郑州达诺生物技术有限公司 | System reaction liquid, inhibin A quantitative detection kit containing system reaction liquid and using method |
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