CN108982482A - Inhibin B chemiluminescence immune detection reagent kit and preparation method thereof - Google Patents
Inhibin B chemiluminescence immune detection reagent kit and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of inhibin B chemiluminescence immune detection reagent kits and preparation method thereof, belong to vitro detection technical field.Kit of the invention includes the inhibin B monoclonal antibody solution of chemiluminescent labels label, Streptavidin magnetic particle, and the inhibin B monoclonal antibody solution of coupling label substance markers.Kit of the invention can complete the detection to inhibin B using Full-automatic chemiluminescence immunoassay analysis meter as detection instrument.This chemical luminescence immune analysis reagent box is mating with instrument, the time needed for shortening clinical detection.The detection accuracy of this chemical luminescence immune analysis reagent box is higher.Kit of the invention can substitute enzyme-linked immunization kit on the market in terms of accuracy, provide more accurate measurement data for client.Kit of the invention has high sensitivity, accurate quantitative analysis, "dead" risk and its detection time short, and the advantages of can detect to antigen, antibody class target substance.
Description
Technical field
The present invention relates to vitro detection technical fields, and in particular to a kind of inhibin B chemiluminescence immune detection reagent kit
And preparation method thereof.
Background technique
Inhibin (INH) belongs to transforming growth factor β family of polypeptides, and mature INH is the dimer sugar egg of 32kDa a kind of
Bai Jisu.INH is formed by connecting by one of α subunit and two kinds of β subunits (β A and β B) by disulfide bond, i.e. inhibin A (alpha-beta A) and
Inhibin B (alpha-beta B).The INH of only dimeric forms just has biological activity.
Male INHB is secreted by sustentacular cell of testis, and another fraction outside sexual gland by organizing to generate.Inhibin A is followed in male
Ring blood middle concentration is very low, is not easy to be detected, and inactive.Inhibin B has bioactivity, is males pQE30/en
Major physiological product and important form.Males pQE30/en B accounts for absolute leading position, mainly by male testical sertoli cell
It generates, is the direct mark object of male's production of sperm ability.Inhibin B can instruct male sterility analysis, assisted reproductive technology, detection
Chemicotherapy is to the damage of male's spermatogenesis, diagnosis sex premature, delayed puberty etc..
Women INHB is secreted by gonad granulocyte and thecacells, and inhibin B can instruct Ovary reserve to assess:
Inhibin B level can than bFSH value earlier, more directly react ovarian reserve situation, when inhibin B level is not enough to maintain
BFSH in the normal range when, just show as the raising of blood bFSH；It predicts premature ovarian failure (POF): in POF patient, inhibin B
Decline, and the raising earlier than FSH are had begun before amenorrhoea, E2 is only just substantially change when entering menopause；Auxiliary diagnosis is more
Capsule Ovary Syndrome (PCOS)；Auxiliary diagnosis endometriosis；Diagnostic monitoring gonad granulocyte cancer；Guidance selection auxiliary
Reproductive technology scheme etc..
The method of measurement inhibin B has radiommunoassay (RIA), EIA enzyme immunoassay (EIA), time-resolved fluorescence at present
(TRFIA) etc..But they all have the defects that some intrinsic in the application: there are radionuclide contaminations, marker for RIA method
The commodity price phase is short, complicated for operation, test result is unstable.There are the activity of enzyme to be easy inactivation for EIA method, leads to the sensitivity of EIA method
It is not high, it is difficult to accurate quantification.Although the sensitivity of TRFIA method is high compared with RIA method, there are the risks of rare heavy metal pollution.
Summary of the invention
The invention solves in the prior art the technical issues of, a kind of inhibin B chemiluminescence immunoassay detection reagent is provided
Box and preparation method thereof.Inhibin B chemiluminescence immune detection reagent kit of the invention has sensitive for measuring inhibin B
It is short to spend height, accurate quantitative analysis, "dead" risk and its detection time, and antigen, antibody class target substance can be examined
The advantages of survey.
In order to solve the above-mentioned technical problem, technical solution of the present invention is specific as follows:
A kind of inhibin B chemiluminescence immune detection reagent kit, comprising: R1 reagent, R2 reagent and R3 reagent；
The R1 reagent is to be with the pH value being made of 50mM MES, 0.05% Tween-20 and 0.05%Proclin300
The diluted Streptavidin magnetic granule solid reagent of 6.5 buffer；
The R2 reagent is to be with the pH value being made of 50mM MES, 0.05% Tween-20 and 0.05%Proclin300
The inhibin B monoclonal antibody solution of the diluted chemiluminescent labels label of 6.5 buffer；
The R3 reagent be coupling label substance markers inhibin B monoclonal antibody solution, glycerol and NaN3 or
The mixed solution of ProClin300；The inhibin B monoclonal antibody solution of the coupling label substance markers is identical with glycerol volume,
The volume of the NaN3 or ProClin300 is the inhibin B monoclonal antibody solution and glycerol two of the coupling label substance markers
The 0.1% of the sum of person's volume.
In the above-mentioned technical solutions, the magnetic bead concentration of the Streptavidin magnetic granule solid reagent is 0.01%~1%.
In the above-mentioned technical solutions, the partial size of the Streptavidin magnetic particle is 0.05~3 μm.
In the above-mentioned technical solutions, the suppression in the inhibin B monoclonal antibody solution of the chemiluminescent labels label
The molar ratio for making element B monoclonal antibody and chemiluminescent labels is 1:3~20.
In the above-mentioned technical solutions, the inhibin B in the inhibin B monoclonal antibody solution of the coupling label substance markers
Monoclonal antibody and the molar ratio of coupling marker are 1:5~20.
In the above-mentioned technical solutions, the change in the inhibin B monoclonal antibody solution of the chemiluminescent labels label
Luminous marker is acridinium ester, luminol, different luminol or tris (bipyridine) ruthenium.
In the above-mentioned technical solutions, the coupling mark in the inhibin B monoclonal antibody solution of the coupling label substance markers
Note object is the derivative of biotin, antigen or the two.
In the above-mentioned technical solutions, the inhibin B chemiluminescence immune detection reagent kit further includes chemiluminescence bottom
Thing liquid, the Chemoluminescent substrate include A liquid and B liquid, and the A liquid is nitric acid solution, and the B liquid is sodium hydroxide solution.
In the above-mentioned technical solutions, the inhibin B chemiluminescence immune detection reagent kit further includes inhibin B calibration
Product, concentration are respectively 0.0pg/mL, 20.0pg/mL, 100.0pg/mL, 250.0pg/mL, 500.0pg/mL, 1300.0pg/
The inhibin B solution of mL and 1500.0pg/mL.
A kind of preparation method of inhibin B chemiluminescence immune detection reagent kit, comprising the following steps:
(1) preparation of R1 reagent
It after taking Streptavidin magnetic particle solution, addition TBST solution to mix well, is placed on magnetic separator, until upper
Clearly without muddiness, supernatant is abandoned, leaves and takes magnetic particle, after repeated washing 3 times, with by 50mM MES, 0.05% Tween-20 and 0.05%
Streptavidin magnetic granule solid reagent is configured to after the buffer dilution that the pH value of Proclin300 composition is 6.5；
(2) preparation of R2 reagent
After inhibin B monoclonal antibody is put into centrifuge tube bottom position, carbonate buffer solution is added, mixes well, mixes
The DMF solution of chemiluminescent labels is added after even, is centrifuged under room temperature with centrifuge, it is close then will to be centrifuged effective sealed membrane
It is honored as a queen to be put into and be protected from light in magazine, magazine is put into gas bath constant temperature oscillator mixes later, add lysine confining liquid, then
It is put into after gas bath constant temperature oscillator middling speed mixes and is closed；By 250 column purification of sephadex G on the antibody closed, use
The elution of PB buffer, fraction collection, with the pH value being made of 50mM MES, 0.05% Tween-20 and 0.05%Proclin300
For the inhibin B monoclonal antibody solution for being configured to chemiluminescent labels label after 6.5 buffer dilution；
(3) preparation of R3 reagent
Inhibin B monoclonal antibody is taken, the coupling mark activated is added after the TRIS buffer dialysis purification for being 6.5 with pH
Remember object, after 2~8 DEG C of reactions, then is transferred to room temperature the reaction was continued, being eventually adding lysine, the reaction was continued, reaction solution desalting column
Purifying, is added isometric glycerol and 0.1%NaN3Or 0.1%ProClin300, the inhibin B for obtaining coupling label substance markers are mono-
Clonal antibody solution.
The beneficial effects of the present invention are:
1, chemiluminescent labels used in inhibin B chemiluminescence immune detection reagent kit provided by the invention have excitation
It is direct chemiluminescence that state, which returns to the energy jump generated when ground state, does not need the participation of enzyme, saves time and cost.
2, inhibin B chemiluminescence immune detection reagent kit provided by the invention is using Streptavidin magnetic particle and coupling
The analog of marker can be firmly combined together, and reduced non-specific adsorption, improved the accuracy of test sample, be resisted dry
It is strong to disturb ability.
3, inhibin B chemiluminescence immune detection reagent kit provided by the invention is full automatic measurement sample, and directly
Numerical value is provided, reduces manual operation error, and realize unattended.
4, chemiluminescent labels used in inhibin B chemiluminescence immune detection reagent kit provided by the invention are most preferably
For acridinium ester, the chemiluminescence immunoassay system range of linearity is wide.
5, inhibin B chemiluminescence immune detection reagent kit and instrument provided by the invention form closed system, and system is missed
Difference is small.
6, inhibin B chemiluminescence immune detection reagent kit provided by the invention can be immune point with Full-automatic chemiluminescence
Analyzer is detection instrument, completes the detection to inhibin B.This chemical luminescence immune analysis reagent box and instrument are mating, shorten
Time needed for clinical detection.The detection accuracy of this chemical luminescence immune analysis reagent box is higher.
7, inhibin B chemiluminescence immune detection reagent kit provided by the invention can substitute on the market in terms of accuracy
Enzyme-linked immunization kit provides more accurate measurement data for client.
8, inhibin B chemiluminescence immune detection reagent kit of the invention is for measuring inhibin B, have high sensitivity,
Accurate quantitative analysis, "dead" risk and its detection time are short, and can be detected to antigen, antibody class target substance excellent
Point.
Detailed description of the invention
Invention is further described in detail with reference to the accompanying drawings and detailed description.
Fig. 1 is the development process figure of inhibin B chemiluminescence immune detection reagent kit provided by the invention.
Fig. 2 is the INHB canonical plotting that embodiment 3 obtains.
Specific embodiment
Invention thought of the invention are as follows: referring to Fig. 1, the present invention determines solid-phase magnetic beads by screening raw material, liquid phase marker,
Be coupled marker, then by deploying to each phase antibody concentration and being formulated adjustment after, and then providing a kind of includes chemiluminescence mark
Remember the inhibin B monoclonal antibody solution of substance markers, the inhibin B Dan Ke of Streptavidin magnetic particle and coupling label substance markers
The inhibin B chemiluminescence immune detection reagent kit of grand antibody-solutions.Inhibin B chemiluminescence immunoassay detection provided by the invention
Kit for measuring inhibin B compared to traditional detection method, the combination of antigen, antibody be under similar fluid conditions into
Row, reaction are more sensitive rapidly；On the other hand, inhibin B chemiluminescence immune detection reagent kit and chemiluminescence immunoassay detect
Instrument forms closed system, and reagent, the addition of sample and Detection task are automatically performed by instrument, reduces the mistake of manual operation
Difference improves the sensitivity and accuracy of whole system.
The present invention provides a kind of inhibin B chemiluminescence immune detection reagent kit, comprising: R1 reagent, R2 reagent and R3 examination
Agent；It is 6.5 that the R1 reagent, which is with the pH value being made of 50mM MES, 0.05% Tween-20 and 0.05%Proclin300,
The diluted Streptavidin magnetic granule solid reagent of buffer；The R2 reagent be with by 50mM MES, 0.05% Tween-20 and
The inhibin B Dan Ke of the diluted chemiluminescent labels label of the buffer that the pH value of 0.05%Proclin300 composition is 6.5
Grand antibody-solutions；The R3 reagent be coupling label substance markers inhibin B monoclonal antibody solution, glycerol and NaN3 or
The mixed solution of ProClin300；The inhibin B monoclonal antibody solution of the coupling label substance markers is identical with glycerol volume,
The volume of the NaN3 or ProClin300 is the inhibin B monoclonal antibody solution and glycerol two of the coupling label substance markers
The 0.1% of the sum of person's volume.It is preferred that the magnetic bead concentration of the Streptavidin magnetic granule solid reagent is 0.01%~1%.It is excellent
The partial size for selecting the Streptavidin magnetic particle is 0.05~3 μm.It is preferred that the inhibin B of the chemiluminescent labels label is mono-
The molar ratio of inhibin B monoclonal antibody and chemiluminescent labels in clonal antibody solution is 1:3~20.It is preferred that the idol
The molar ratio of inhibin B monoclonal antibody and coupling marker in the inhibin B monoclonal antibody solution of connection label substance markers
For 1:5~20.It is preferred that the chemiluminescent labels in the inhibin B monoclonal antibody solution of the chemiluminescent labels label
For acridinium ester, luminol, different luminol or tris (bipyridine) ruthenium.It is preferred that the inhibin B monoclonal of the coupling label substance markers is anti-
Coupling marker in liquid solution is the derivative of biotin, antigen or the two.It is preferred that the inhibin B chemiluminescence
Immunity detection reagent further includes Chemoluminescent substrate, and the Chemoluminescent substrate includes A liquid and B liquid, and the A liquid is nitre
Acid solution, the B liquid are sodium hydroxide solution.It is preferred that the inhibin B chemiluminescence immune detection reagent kit further includes suppression
Make element B calibration object, concentration be respectively 0.0pg/mL, 20.0pg/mL, 100.0pg/mL, 250.0pg/mL, 500.0pg/mL,
The inhibin B solution of 1300.0pg/mL and 1500.0pg/mL.
The present invention also provides a kind of preparation methods of inhibin B chemiluminescence immune detection reagent kit, comprising the following steps:
(1) preparation of R1 reagent
It after taking Streptavidin magnetic particle solution, addition TBST solution to mix well, is placed on magnetic separator, until upper
Clearly without muddiness, supernatant is abandoned, leaves and takes magnetic particle, after repeated washing 3 times, with by 50mM MES, 0.05% Tween-20 and 0.05%
Streptavidin magnetic granule solid reagent is configured to after the buffer dilution that the pH value of Proclin300 composition is 6.5；
(2) preparation of R2 reagent
After inhibin B monoclonal antibody is put into centrifuge tube bottom position, carbonate buffer solution is added, mixes well, mixes
The DMF solution of chemiluminescent labels is added after even, is centrifuged under room temperature with centrifuge, it is close then will to be centrifuged effective sealed membrane
It is honored as a queen to be put into and be protected from light in magazine, magazine is put into gas bath constant temperature oscillator mixes later, add lysine confining liquid, then
It is put into after gas bath constant temperature oscillator middling speed mixes and is closed；By 250 column purification of sephadex G on the antibody closed, use
The elution of PB buffer, fraction collection, with the pH value being made of 50mM MES, 0.05% Tween-20 and 0.05%Proclin300
For the inhibin B monoclonal antibody solution for being configured to chemiluminescent labels label after 6.5 buffer dilution；
(3) preparation of R3 reagent
Inhibin B monoclonal antibody is taken, the coupling mark activated is added after the TRIS buffer dialysis purification for being 6.5 with pH
Remember object, after 2~8 DEG C of reactions, then is transferred to room temperature the reaction was continued, being eventually adding lysine, the reaction was continued, reaction solution desalting column
Purifying, is added isometric glycerol and 0.1%NaN3Or 0.1%ProClin300, the inhibin B for obtaining coupling label substance markers are mono-
Clonal antibody solution.
Inhibin B chemiluminescence immune detection reagent kit provided by the invention is for when detecting inhibin B, using full-automatic
Chemical illumination immunity analysis instrument (the auspicious CM180 of enlightening) detects inhibin B calibration object, draws standard curve, is built in computer
Software；Then clinical sample is tested according to demand, and the concentration of inhibin B is calculated according to the light quantity subnumber of sample；Finally to inhibition
Plain B automatic chemiluminescence immunoassay system carries out the evaluation of performance (sensitivity, linear, anti-interference/specificity).
Embodiment 1: the preparation of inhibin B chemiluminescence immune detection reagent kit:
(1) preparation of Streptavidin magnetic granule solid reagent:
Taking concentration is 0.5 milliliter of the Streptavidin magnetic particle solution (50mg) of 100mg/mL, and the TBST that 10mL is added is molten
Liquid mixes well after ten minutes, is placed on magnetic separator, until supernatant abandons supernatant, leave and take magnetic particle without muddiness.Repeated washing
Being made into magnetic bead concentration in 50mM MES, 0.05% tween, 0.05%Proclin300, the buffer of pH6.5 after 3 times is
0.05% solid-phase reagent, 2~8 DEG C of preservations.
(2) preparation of the inhibin B monoclonal antibody solution of acridinium ester label:
500 μ g inhibin B monoclonal antibodies are put into centrifuge tube, guarantee that antibody is located at the (centrifugation of centrifuge tube bottom position
Machine room temperature is centrifuged 20s) carbonate buffer solution is added afterwards, it mixes well, the DMF that 1mL 2mg/mL acridinium ester is added after mixing is molten
Liquid is centrifuged 0.5 minute under room temperature with centrifuge.It will be centrifuged to be put into after effective sealed membrane seals and be protected from light in magazine, later will
Magazine is put into gas bath constant temperature oscillator (25 DEG C), is mixed 4 hours.20% lysine confining liquid of 1mL is added, is put into gas bath constant temperature
Oscillator (25 DEG C), middling speed mix, off-period 1h.By 250 column purification of sephadex G on the antibody closed, PB is used
Buffer elution, fraction collection.The antibody-solutions gathered are placed in 2~8 DEG C of preservations.By inhibin after purification when use
B antibody concentrated solution 50mM MES, 0.05% Tween-20,0.05%Proclin300, the buffer of pH6.5 are diluted to dense eventually
Degree is 0.1 μ g/mL, 2~8 DEG C of preservations.
(3) preparation of the inhibin B monoclonal antibody solution of biotin labeling:
Inhibin B monoclonal antibody is taken, has been activated 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, finally
The lysine reaction 30min of 100 times of molar ratios is added, isometric glycerol and 0.1%NaN is added in reaction solution desalination column purification3
Or 0.1%ProClin300.
Embodiment 2: inhibin B chemiluminescence immune detection reagent kit detection method:
With Full-automatic chemiluminescence immunoassay analysis meter (CM180) for detection instrument, methodology mode is double antibody sandwich method,
That is the instrument sample that sequentially adds 20 μ L, the inhibin B monoclonal antibody solution of the acridinium ester label of 50 μ L embodiments 1 preparation,
The inhibin B monoclonal antibody solution of the biotin labeling of 50 μ L embodiments 1 preparation and the strepto- of 40 μ L embodiments 1 preparation are affine
Biscuit porcelain granule solid reagent.Reaction after twenty minutes, carries out Magneto separate.Reactant is sent into darkroom by instrument, primary that the bottom that shines is added
Thing liquid A liquid (HNO3Solution) and B liquid (NaOH solution) reacted, last recording light quantum number.
Embodiment 3: inhibin B chemiluminescence immune detection reagent kit performance evaluation
(1) linear detection: by calibration object concentration be 0.0pg/mL, 20.0pg/mL, 100.0pg/mL, 250.0pg/mL,
500.0pg/mL, 1300.0pg/mL and 1500.0pg/mL make standard curve, obtain linearly dependent coefficient r=0.998, linear model
It encloses for 10-1300pg/mL.Referring to table 1 and Fig. 2.
Table 1
| Concentration | Light quantity subnumber |
| 0 | 1790 |
| 20 | 3450 |
| 100 | 9260 |
| 250 | 13379 |
| 500 | 26620 |
| 1300 | 60074 |
| 1500 | 69485 |
(2) Calculation of Sensitivity
The definition of sensitivity for analysis is that 20 light quantity subnumber measurements are carried out to zero calibration object, its average value is taken to subtract twice
Standard deviation, it is sensitivity obtained by standard curve that gained light quantity subnumber, which is brought into,；Calculate the detection examination of inhibin B chemiluminescence immunoassay
The sensitivity of agent box is 11.97pg/mL, and see Table 2 for details.
Table 2
(3) anti-interference specificity experiments:
Taking pure blood to add interfering substance clearly includes: bilirubin, hemoglobin, triglycerides, biotin, respectively measurement addition
The pooled serum of interfering substance and simple serum, calculate anti-interference rate, with ± 10% for boundary.The results show that interfering substance is not
The measurement of inhibin B can be significantly affected.
The attached anti-interference specificity experiments data of table 3:
Embodiment 4: the preparation of inhibin B chemiluminescence immune detection reagent kit:
(1) preparation of Streptavidin magnetic granule solid reagent:
Taking concentration is 1.5 milliliters of the Streptavidin magnetic particle solution (75mg) of 50mg/mL, and the TBST solution of 15mL is added
It after mixing well 15 minutes, is placed on magnetic separator, until supernatant abandons supernatant, leave and take magnetic particle without muddiness.Repeated washing 3
Being made into magnetic bead concentration in 50mM MES, 0.05% tween, 0.05%Proclin300, the buffer of pH6.5 after secondary is
0.01% solid-phase reagent, 2~8 DEG C of preservations.
(2) preparation of the inhibin B monoclonal antibody solution of acridinium ester label:
1000 μ g inhibin B monoclonal antibodies are put into centrifuge tube, guarantee that antibody is located at the (centrifugation of centrifuge tube bottom position
Machine room temperature is centrifuged 30s) carbonate buffer solution is added afterwards, it mixes well, the DMF that 1.5mL 2mg/mL acridinium ester is added after mixing is molten
Liquid is centrifuged 1 minute under room temperature with centrifuge.It will be centrifuged to be put into after effective sealed membrane seals and be protected from light in magazine, it later will be dark
Box is put into gas bath constant temperature oscillator (26 DEG C), is mixed 5 hours.20% lysine confining liquid of 2mL is added, is put into the vibration of gas bath constant temperature
It swings device (26 DEG C), middling speed mixes, off-period 2h.It is slow with PB by 250 column purification of sephadex G on the antibody closed
Fliud flushing elution, fraction collection.The antibody-solutions gathered are placed in 2~8 DEG C of preservations.By inhibin B after purification when use
Antibody concentrated solution 50mM MES, 0.05% Tween-20,0.05%Proclin300, the buffer of pH6.5 are diluted to final concentration
For 0.1 μ g/mL, 2~8 DEG C of preservations.
(3) preparation of the inhibin B monoclonal antibody solution of biotin labeling:
Inhibin B monoclonal antibody is taken, has been activated 5 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 10min, finally
The lysine reaction 20min of 100 times of molar ratios is added, isometric glycerol and 0.1%NaN is added in reaction solution desalination column purification3
Or 0.1%ProClin300.
Embodiment 5: the preparation of inhibin B chemiluminescence immune detection reagent kit:
(1) preparation of Streptavidin magnetic granule solid reagent:
Taking concentration is 2 milliliters of the Streptavidin magnetic particle solution (400mg) of 200mg/mL, and the TBST solution of 10mL is added
It mixes well after twenty minutes, is placed on magnetic separator, until supernatant abandons supernatant, leave and take magnetic particle without muddiness.Repeated washing 4
Being made into magnetic bead concentration after secondary in 50mM MES, 0.05% tween, 0.05%Proclin300, the buffer of pH6.5 is 1%
Solid-phase reagent, 2~8 DEG C of preservations.
(2) preparation of the inhibin B monoclonal antibody solution of acridinium ester label:
2000 μ g inhibin B monoclonal antibodies are put into centrifuge tube, guarantee that antibody is located at the (centrifugation of centrifuge tube bottom position
Machine room temperature is centrifuged 60s) carbonate buffer solution is added afterwards, it mixes well, the DMF that 20mL 2mg/mL acridinium ester is added after mixing is molten
Liquid is centrifuged 2 minutes under room temperature with centrifuge.It will be centrifuged to be put into after effective sealed membrane seals and be protected from light in magazine, it later will be dark
Box is put into gas bath constant temperature oscillator (27 DEG C), is mixed 6 hours.20% lysine confining liquid of 4mL is added, is put into the vibration of gas bath constant temperature
It swings device (27 DEG C), middling speed mixes, off-period 4h.It is slow with PB by 250 column purification of sephadex G on the antibody closed
Fliud flushing elution, fraction collection.The antibody-solutions gathered are placed in 2~8 DEG C of preservations.By inhibin B after purification when use
Antibody concentrated solution 50mM MES, 0.05% Tween-20,0.05%Proclin300, the buffer of pH6.5 are diluted to final concentration
For 0.1 μ g/mL, 2~8 DEG C of preservations.
(3) preparation of the inhibin B monoclonal antibody solution of biotin labeling:
Inhibin B monoclonal antibody is taken, has been activated 20 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 60min, finally
The lysine reaction 50min of 100 times of molar ratios is added, isometric glycerol and 0.1%NaN is added in reaction solution desalination column purification3
Or 0.1%ProClin300.
Chemiluminescent labels acridinium ester used in above-described embodiment is replaced with into luminol, different luminol or three respectively
Coupling marker biotin is replaced with antigen, it can R2, R3 containing above-mentioned label substance markers is prepared by bipyridyl ruthenium
The kit of reagent.
Inhibin B chemiluminescence immune detection reagent kit prepared in the above embodiments has sensitive for measuring inhibin B
It is short to spend height, accurate quantitative analysis, "dead" risk and its detection time, and antigen, antibody class target substance can be examined
The advantages of survey.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (10)
1. a kind of inhibin B chemiluminescence immune detection reagent kit, comprising: R1 reagent, R2 reagent and R3 reagent；Its feature exists
In,
It is 6.5 that the R1 reagent, which is with the pH value being made of 50mM MES, 0.05% Tween-20 and 0.05%Proclin300,
The diluted Streptavidin magnetic granule solid reagent of buffer；
It is 6.5 that the R2 reagent, which is with the pH value being made of 50mM MES, 0.05% Tween-20 and 0.05%Proclin300,
The inhibin B monoclonal antibody solution of the diluted chemiluminescent labels label of buffer；
The R3 reagent is inhibin B monoclonal antibody solution, glycerol and the NaN of coupling label substance markers3Or ProClin300
Mixed solution；The inhibin B monoclonal antibody solution of the coupling label substance markers is identical with glycerol volume, the NaN3Or
The volume of ProClin300 is the sum of both inhibin B monoclonal antibody solution and the glycerol of coupling label substance markers volume
0.1%.
2. inhibin B chemiluminescence immune detection reagent kit according to claim 1, which is characterized in that the strepto- parent
Magnetic bead concentration with biscuit porcelain granule solid reagent is 0.01%~1%.
3. inhibin B chemiluminescence immune detection reagent kit according to claim 1, which is characterized in that the strepto- parent
Partial size with biscuit porcelain particle is 0.05~3 μm.
4. inhibin B chemiluminescence immune detection reagent kit according to claim 1, which is characterized in that the chemistry hair
Inhibin B monoclonal antibody and chemiluminescent labels in the inhibin B monoclonal antibody solution of signal substance markers are rubbed
You are than being 1:3~20.
5. inhibin B chemiluminescence immune detection reagent kit according to claim 1, which is characterized in that the coupling mark
The molar ratio for remembering inhibin B monoclonal antibody and the coupling marker in the inhibin B monoclonal antibody solution of substance markers is 1:5
~20.
6. inhibin B chemiluminescence immune detection reagent kit according to claim 1, which is characterized in that the chemistry hair
Chemiluminescent labels in the inhibin B monoclonal antibody solution of signal substance markers are acridinium ester, luminol, different luminol
Or tris (bipyridine) ruthenium.
7. inhibin B chemiluminescence immune detection reagent kit according to claim 1, which is characterized in that the coupling mark
Remember that the coupling marker in the inhibin B monoclonal antibody solution of substance markers is the derivative of biotin, antigen or the two.
8. inhibin B chemiluminescence immune detection reagent kit according to claim 1, which is characterized in that further include chemistry
Luminous substrate liquid, the Chemoluminescent substrate include A liquid and B liquid, and the A liquid is nitric acid solution, and the B liquid is sodium hydroxide
Solution.
9. inhibin B chemiluminescence immune detection reagent kit according to claim 1, which is characterized in that further include inhibiting
Plain B calibration object, concentration be respectively 0.0pg/mL, 20.0pg/mL, 100.0pg/mL, 250.0pg/mL, 500.0pg/mL,
The inhibin B solution of 1300.0pg/mL and 1500.0pg/mL.
10. the preparation method of inhibin B chemiluminescence immune detection reagent kit described in a kind of claim 1-7 any one,
Characterized by comprising the following steps:
(1) preparation of R1 reagent
Take Streptavidin magnetic particle solution, be added after TBST solution mixes well, be placed on magnetic separator, until supernatant without
Muddiness abandons supernatant, leaves and takes magnetic particle, after repeated washing 3 times, with by 50mM MES, 0.05% Tween-20 and 0.05%
Streptavidin magnetic granule solid reagent is configured to after the buffer dilution that the pH value of Proclin300 composition is 6.5；
(2) preparation of R2 reagent
After inhibin B monoclonal antibody is put into centrifuge tube bottom position, carbonate buffer solution is added, mixes well, after mixing
The DMF solution of chemiluminescent labels is added, is centrifuged under room temperature with centrifuge, after being then centrifuged effective sealed membrane sealing
It is put into and is protected from light in magazine, magazine is put into gas bath constant temperature oscillator mixes later, add lysine confining liquid, be subsequently placed into
Gas bath constant temperature oscillator middling speed is closed after mixing；It is slow with PB by 250 column purification of sephadex G on the antibody closed
Fliud flushing elution, fraction collection are 6.5 with the pH value being made of 50mM MES, 0.05% Tween-20 and 0.05%Proclin300
Buffer dilution after be configured to chemiluminescent labels label inhibin B monoclonal antibody solution；
(3) preparation of R3 reagent
Inhibin B monoclonal antibody is taken, the coupling activated is added after the TRIS buffer dialysis purification for being 6.5 with pH and marks
Object after 2~8 DEG C of reactions, then is transferred to room temperature the reaction was continued, and being eventually adding lysine, the reaction was continued, and reaction solution desalting column is pure
Change, isometric glycerol and 0.1%NaN is added3Or 0.1%ProClin300, obtain the inhibin B Dan Ke of coupling label substance markers
Grand antibody-solutions.
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Application publication date: 20181211 |