CN106053820A - Application of microdialysis sampling-flow injection chemiluminescence combined method in determination of affinity between small-molecule hapten and monoclonal antibody - Google Patents
Application of microdialysis sampling-flow injection chemiluminescence combined method in determination of affinity between small-molecule hapten and monoclonal antibody Download PDFInfo
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Abstract
The invention discloses application of a microdialysis sampling-flow injection chemiluminescence combined method in determination of the affinity between a small-molecule hapten and a monoclonal antibody and a specific determination method. A combined analysis method is used for researching affinity between the small-molecule hapten and the monoclonal antibody in a homogeneous phase system under a label-free condition and is simple, convenient, stable and sensitive. Compared with existing methods for determining the affinity of antibodies, the method has the advantages that the antibodies do not need to be labeled by virtue of signal molecules such as enzyme, and the small-molecule hapten and a carrier protein do not need to be coupled for coating, so that the tedious works of labeling, coupling and the like can be omitted; and more importantly, the real combination condition between the hapten and the antibody can be objectively reflected, expensive instruments are not required, and the cost is relatively low. According to the application and the method, a simple, rapid and objective analytical technical measure is provided for the performance evaluation of the antibodies, and meanwhile, the application field of the microdialysis sampling technique is newly extended.
Description
Technical field
The invention belongs to immunoassay field, relate to on-line microdialysis sampling-portable injection chemiluminescence combination analysis method
Application in the affinity of the monoclonal antibody for small haptens measures.
Background technology
Monoclonal antibody technique is a kind of immunological technique, and it will produce the same tumor cell of single bone-marrow-derived lymphocyte of antibody
Hybridization, it is thus achieved that can produce antibody, again can the hybridoma of infinite multiplication, and produce antibody with this.This technology is antibody
Molecules research provides brand-new means, is greatly promoted immunologic development.In recent years, monoclonal antibody technique
Application widely has been obtained in immune detection, immunization therapy, the field such as isolated and purified.Preparation and sieve in monoclonal antibody
During choosing, one of most important work is the binding affinity to specific antigen of the monoclonal antibody to different cell strain secretions
This key index is evaluated, and it has highly important directive significance for the exploitation application of monoclonal antibody.
It is presently used for measuring the method for binding constant between Ag-Ab and mainly includes competition binding method, rhodanate eluting
The different Phase Analysis Method based on antibody labeling such as method, non-competing enzyme immunoassay (EIA).In recent years, also there are some cold methods
For measuring affinity of antibody, its principle is similar with foregoing tags method, difference be antibody without with signal probe labelling,
But after antigen and antibodies, measure sensing with the non-marked method such as surface plasma body resonant vibration, QCM
Bioconjugation reaction on device interface, research antigen and the cohesive process of antibody according to this.The drawback of labelling method is, all antibody
Being required to be marked with signaling molecules such as enzymes, one to carry out labeling process loaded down with trivial details, and two carry out labelling may affect the biological activity of antibody,
The measurement result obtained is difficult to objectively respond between Ag-Ab real affinity.Regardless of whether be labelling method or non-marked
Method, is required for being fixed on antigen ELISA Plate or sensor surface.If antigen is small haptens, owing to they are difficult to
ELISA Plate or sensor surface are the most fixing, it usually needs form artificial complete antigen rear with carrier protein couplet and can be used for wrapping
Quilt, and coupling may bring problems with: first, coupling process more bothers;Secondly, coupling may destroy antigen molecule structure
In antigenic determinant, or, antigenic determinant and coupling protein hypotelorism, the association reaction between Ag-Ab is produced
Sterically hindered, all can not truly reflect the affinity of antibody.Therefore, it is necessary to development is a kind of without to small haptens and anti-
Body carries out coupling or labelling can directly carry out the homogeneous analysis that measures for the monoclonal antibody affinity of small haptens
Method.
Summary of the invention
In view of this, it is an object of the invention to provide one without small haptens and antibody are carried out coupling or mark
Note can directly carry out the homogeneous analysis method measured for the monoclonal antibody affinity of small haptens.
For reaching above-mentioned purpose, the present invention provides following technical scheme:
1. on-line microdialysis sampling-portable injection chemiluminescence combination method affinity between small haptens and monoclonal antibody
Application in mensuration.
2. on-line microdialysis sampling-portable injection chemiluminescence combination method measures small haptens and monoclonal antibody
Between the method for affinity, comprise the following steps: in the small haptens homogeneous immunoreation system with monoclonal antibody, adopt
Obtain the dialysis solution containing the free small haptens of part by the on-line microdialysis method of sampling, then use flow injection chemistry to send out
Light method measures the concentration of free small haptens in dialysis solution, and the transmitance in conjunction with the microdialysis method of sampling tries to achieve homogeneous immunity
The concentration of free small haptens in reaction system, calculates further in homogeneous immunoreation system and ties with monoclonal antibody
The concentration of the small haptens closed, analyzes or/and Klotz analysis meter calculates monoclonal antibody finally according to Scatchard
Affinity constant.
Preferably, described method specifically includes following steps:
(1) prepare the small haptens solution of a series of known variable concentrations, use Flow Injection Chemiluminescence to survey respectively
Its chemiluminescence intensity fixed, maps to obtain to the concentration of small haptens working curve with chemiluminescence intensity;
(2) the small haptens solution of concentration known use the on-line microdialysis method of sampling obtain containing the little molecule of part half
The dialysis solution of antigen, uses the chemiluminescence intensity of flow-injection chemiluminescence method dialysis solution, according to step (1) gained work
Extrapolate the haptenic concentration of dialysis solution small molecular as curve, calculate the transmitance of the microdialysis method of sampling further;
(3) concentration of immobilized monoclonal antibody, by it, small haptens with a series of known variable concentrations is tied respectively
Close reaction, obtain a series of homogeneous immunoreation system, use the on-line microdialysis method of sampling to obtain a series of containing portion respectively
Dividing the dialysis solution of free small haptens, the chemistry using Flow Injection Chemiluminescence to measure this serial dialysis liquid respectively is sent out
Light intensity, extrapolates the concentration of free small haptens in this serial dialysis liquid respectively according to step (1) gained working curve,
The transmitance of integrating step (2) the gained microdialysis method of sampling tries to achieve free little molecule half in the homogeneous immunoreation system of this series
The concentration of antigen, calculates the small haptens being combined with monoclonal antibody in the homogeneous immunoreation system of this series further
Concentration, finally according to Scatchard analyze or/and Klotz analysis meter calculates the affinity constant of monoclonal antibody.
As an instantiation, described monoclonal antibody is terbutaline monoclonal antibody, and small haptens is special
Bu Talin.
As a preferred implementation of examples detailed above, on-line microdialysis sampling-portable injection chemiluminescence combination method
Measure the method for affinity between small haptens and monoclonal antibody particularly as follows: little molecule half resists described in step (1) and (2)
Described in original solution and step (3), the solvent of homogeneous immunoreation system is 0.010 M pH 7.4 PBS;Step
(1) specific as follows to Flow Injection Chemiluminescence described in (3): luminous agent is 1.0 × 10-5M luminol and 1.0 × 10-4
The M potassium ferricyanide, the luminol of described concentration is by 1.0 × 10-2M sodium hydroxide dissolves and prepares;The flow velocity of current-carrying and luminous agent is equal
It is 2.2 mL/min;Described in step (2) and (3), the on-line microdialysis method of sampling is specific as follows: sample temperature is 37 DEG C;Fill
Flow liquid is 0.010 M pH 7.4 PBS, and perfusion rate is 4.0 μ L/min.
The beneficial effects of the present invention is: the invention provides the combination of on-line microdialysis sampling-portable injection chemiluminescence
Analytic process application in the affinity of the monoclonal antibody for small haptens measures and concrete assay method, this connection
It is under the conditions of cold, in homogeneous system, study the affinity between small haptens and monoclonal antibody by analytic process,
Method is simple, convenient, stable, sensitive.Compared with traditional marking type method, it is without carrying out with signaling molecule antagonists such as enzymes
Labelling, can save this tedious work of labelling, can more objectively reflect the situation that is truly combined between antigen with antibody.With
Non-marked method is compared, and it is without being used for being coated after carrier protein couplet by small haptens, can save coupling this
Tedious work, the true affinity reflecting antibody, and need not the instrument of costliness, cost is relatively low.The present invention is not only antibody
Performance evaluation provide a kind of analytical technology means simple, quick, objective, be also that microdialysis Sampling techniques are applied simultaneously
The new expansion in field.
Accompanying drawing explanation
Fig. 1 is on-line microdialysis sampling-portable injection chemiluminescence combined analysis device (MP: micro-injection pump;M: micro-
Analysis probe;P1: current-carrying transmitting device;P2: luminous agent transmitting device;V: injection valve;F: flow cell;D: chemiluminescence detector;
W: waste liquid;A: current-carrying;B: luminous agent 1;C: luminous agent 2;Anti-TEB: terbutaline monoclonal antibody;Terbutaline: special
Bu Talin;PBS:PBS buffer).
Fig. 2 is the working curve of Flow Injection Chemiluminescence detection terbutaline sulphate.
Fig. 3 is the Scatchard curve that terbutaline interacts with its monoclonal antibody.
Fig. 4 is the Klotz curve that terbutaline interacts with its monoclonal antibody.
Detailed description of the invention
In order to make the object, technical solutions and advantages of the present invention clearer, below in conjunction with excellent to the present invention of accompanying drawing
Embodiment is selected to be described in detail.
Fig. 1 is on-line microdialysis sampling-portable injection chemiluminescence combined analysis device.As it can be seen, this analytical equipment
Including microdialysis sampling system and portable injection chemiluminescence system;Described microdialysis sampling system includes micro-injection pump and micro-
Dialysis probe;Described portable injection chemiluminescence system includes current-carrying transmitting device, luminous agent transmitting device, injection valve, circulation
Pond and chemiluminescence detector;The liquid outlet of described microdialysis probe is with the injection valve in portable injection chemiluminescence system even
Logical.This analytical equipment is carried out automatic programming control by computer.
When using above-mentioned analytical equipment to carry out affinity mensuration between small haptens and monoclonal antibody, first by little molecule
The homogeneous immunoreation system of hapten and monoclonal antibody carries out the incubation of certain time, makes immunoreation reach balance, then
Microdialysis probe is placed in the immunoreation system reaching balance, utilizes micro-injection pump that perfusate pushes to microdialysis and visit
At pin, making perfusate carry out dialysis exchange through microdialysis probe and immunoreation system, the dialysis solution after exchange is (containing free little point
Sub-hapten) enter in the sampling ring of injection valve, brought after mix into generation chemiluminescence reaction, chemistry with luminous agent by current-carrying
Photomultiplier tube collection in optical signals chemiluminescence detector.
Embodiment 1 on-line microdialysis sampling-portable injection chemiluminescence combination method measures terbutaline monoclonal antibody
Affinity
1, the drafting of the working curve of Flow Injection Chemiluminescence detection terbutaline sulphate
Prepare the terbutaline sulphate solution (solvent is 0.010 M pH 7.4 PBS) of a series of concentration known.By micro-
Amount syringe pump is joined directly together (not passing through microdialysis probe), respectively by above-mentioned with the injection valve in portable injection chemiluminescence system
Series terbutaline sulphate solution is sucked in micro-injection pump, micro-injection pump control the speed with 4.0 μ L/min direct
Push in sampling ring, use Flow Injection Chemiluminescence to be measured, record chemiluminescence signal.Flow injection chemistry is sent out
Light method is specific as follows: luminous agent is 1.0 × 10-5M luminol is (by 1.0 × 10-2M sodium hydroxide dissolves and prepares) and 1.0 ×
10-4The M potassium ferricyanide;The flow velocity of current-carrying and luminous agent is 2.2 mL/min;The running voltage of photomultiplier tube is-700
V;Within every 8 minutes, measure once (7 points of samplings in 30 seconds, sample size is 30 μ L).Each concentration replication 4 times.Strong with chemiluminescence
Spend working curve that terbutaline sulphate concentration is mapped to obtain, as shown in Figure 2.
2, the mensuration of microdialysis probe transmitance (R)
Use on-line microdialysis sampling-portable injection chemiluminescence combined analysis device as shown in Figure 1.Microdialysis probe is put
Enter in the terbutaline sulphate solution (solvent is 0.010 M pH 7.4 PBS) of concentration known, 37 DEG C of constant temperature water baths.
Controlling to carry out low speed perfusion by micro-injection pump by perfusate, perfusate is carried out with terbutaline sulphate solution through microdialysis probe
Dialysis exchange, dialysis solution (containing part of sulfuric acid terbutaline) enters in the sampling ring of injection valve, is entered flow injection by current carrying strap
Chemiluminescence system, reacts with luminous agent, measures chemiluminescence signal.On-line microdialysis sampling-portable injection chemiluminescence joins
Usage is specific as follows: microdialysis probe is the MD-2310 probe of BASi company of the U.S.;Perfusate is 0.010 M pH 7.4 PBS
Buffer, perfusion rate is 4.0 μ L/min;Luminous agent is 1.0 × 10-5M luminol is (by 1.0 × 10-2M sodium hydroxide
Dissolve and prepare) and 1.0 × 10-4The M potassium ferricyanide;The flow velocity of current-carrying and luminous agent is 2.2 mL/min;Photomultiplier tube
Running voltage is-700 V;Within every 8 minutes, measure once (7 points of samplings in 30 seconds, sample size is 30 μ L).Extrapolate according to Fig. 2
The concentration of terbutaline sulphate in analysis liquid, further according to microdialysis probe transmitance computing formula: R=C d /C m (C d For dialysis
The concentration of terbutaline sulphate in liquid,C m For the total concentration of terbutaline sulphate around probe), calculate microdialysis probe
Transmitance R be 26.2%.
3, the affinity of terbutaline monoclonal antibody measures
Use on-line microdialysis sampling-portable injection chemiluminescence combined analysis device as shown in Figure 1.Fixing homogeneous immunity is anti-
Answering the concentration of terbutaline monoclonal antibody in system is 7.0 × 10-7M, the concentration of terbutaline sulphate is respectively 1.0 × 10-7 M、3.0×10-7 M、5.0×10-7 M、7.0×10-7M and 9.0 × 10-7M, cumulative volume is 1.0 mL, and solvent is
0.010 M pH 7.4 PBS.This homogeneous immunoreation system is put 37 DEG C of water-baths are stirred continuously mixing, balance 30
After minute, microdialysis probe is put in immunoreation system, controlled to carry out low speed perfusion, perfusion by micro-injection pump by perfusate
Liquid carries out dialysis exchange through microdialysis probe and immunoreation system, and dialysis solution (containing part free sulfuric acid terbutaline) enters
In the sampling ring of injection valve, entered to flow injected chemical luminescent system by current carrying strap, react with luminous agent, measure chemiluminescence letter
Number.On-line microdialysis sampling-portable injection chemiluminescence combination method is the most the same.Each concentration replication 4 times.According to Fig. 2
Extrapolating the concentration of free terbutaline sulphate in dialysis solution, the transmitance in conjunction with microdialysis probe tries to achieve free sulphur around probe
The concentration of acid terbutaline, calculates the concentration of the terbutaline sulphate being combined with terbutaline monoclonal antibody further.So
Afterwards by Scatchard analyze (r /C u =nK –rK,rRepresent the terbutaline sulphate and terbutaline list combined in system
The ratio of clonal antibody molar concentration,C u Represent the concentration of unconjugated terbutaline sulphate,nRepresent the combination of monoclonal antibody molecule
Number of sites,KRepresent the affinity constant of monoclonal antibody;Withr /C u RightrMapping can obtain a straight line, and slope and intercept by straight line can
Try to achieveKWithn) (Fig. 3) and Klotz analysis (1/r = 1/n + (1/nK)(1/C u ),rRepresent the sulphuric acid combined in system special
Bu Talin and the ratio of terbutaline monoclonal antibody molar concentration,C u Represent the concentration of unconjugated terbutaline sulphate,n
Represent the binding site number of monoclonal antibody molecule,KRepresent the affinity constant of monoclonal antibody;With 1/rTo 1/C u Mapping can obtain a straight line, by
The slope of straight line and intercept can be tried to achieveKWithn) (Fig. 4), try to achieve the affinity constant of terbutaline monoclonal antibodyKIt is respectively
4.9×106 M-1With 4.9 × 106 M-1。
Finally illustrating, above example is only in order to illustrate technical scheme and unrestricted, although by ginseng
According to the preferred embodiments of the present invention, invention has been described, it should be appreciated by those of ordinary skill in the art that can
In the form and details it is made various change, the present invention limited without departing from appended claims
Spirit and scope.
Claims (5)
1. on-line microdialysis sampling-portable injection chemiluminescence combination method affinity between small haptens and monoclonal antibody
Application in mensuration.
2. on-line microdialysis sampling-portable injection chemiluminescence combination method measures between small haptens and monoclonal antibody affine
The method of power, it is characterised in that comprise the following steps: in the homogeneous immunoreation system of small haptens Yu monoclonal antibody
In, use the on-line microdialysis method of sampling to obtain the dialysis solution containing the free small haptens of part, then use flow injection
The concentration of free small haptens in chemiluminescence determination dialysis solution, the transmitance in conjunction with the microdialysis method of sampling is tried to achieve all
The concentration of free small haptens in phase immunoreation system, calculate further in homogeneous immunoreation system with monoclonal
The concentration of the small haptens of antibodies, analyzes or/and Klotz analysis meter calculates monoclonal finally according to Scatchard
The affinity constant of antibody.
It is anti-that on-line microdialysis sampling-portable injection chemiluminescence the most according to claim 2 combination method measures little molecule half
The method of affinity between former and monoclonal antibody, it is characterised in that specifically include following steps:
(1) prepare the small haptens solution of a series of known variable concentrations, use Flow Injection Chemiluminescence to survey respectively
Its chemiluminescence intensity fixed, maps to obtain to the concentration of small haptens working curve with chemiluminescence intensity;
(2) the small haptens solution of concentration known use the on-line microdialysis method of sampling obtain containing the little molecule of part half
The dialysis solution of antigen, uses the chemiluminescence intensity of flow-injection chemiluminescence method dialysis solution, according to step (1) gained work
Extrapolate the haptenic concentration of dialysis solution small molecular as curve, calculate the transmitance of the microdialysis method of sampling further;
(3) concentration of immobilized monoclonal antibody, by it, small haptens with a series of known variable concentrations is tied respectively
Close reaction, obtain a series of homogeneous immunoreation system, use the on-line microdialysis method of sampling to obtain a series of containing portion respectively
Dividing the dialysis solution of free small haptens, the chemistry using Flow Injection Chemiluminescence to measure this serial dialysis liquid respectively is sent out
Light intensity, extrapolates the concentration of free small haptens in this serial dialysis liquid respectively according to step (1) gained working curve,
The transmitance of integrating step (2) the gained microdialysis method of sampling tries to achieve free little molecule half in the homogeneous immunoreation system of this series
The concentration of antigen, calculates the small haptens being combined with monoclonal antibody in the homogeneous immunoreation system of this series further
Concentration, finally according to Scatchard analyze or/and Klotz analysis meter calculates the affinity constant of monoclonal antibody.
4. it is combined method according to the on-line microdialysis sampling-portable injection chemiluminescence described in Claims 2 or 3 and measures little molecule half
The method of affinity between antigen and monoclonal antibody, it is characterised in that described monoclonal antibody is terbutaline monoclonal antibody,
Small haptens is terbutaline.
It is anti-that on-line microdialysis sampling-portable injection chemiluminescence the most according to claim 4 combination method measures little molecule half
The method of affinity between former and monoclonal antibody, it is characterised in that small haptens solution described in step (1) and (2) and
Described in step (3), the solvent of homogeneous immunoreation system is 0.010 M pH 7.4 PBS;Step (1) to (3)
Described in Flow Injection Chemiluminescence specific as follows: luminous agent is 1.0 × 10-5M luminol and 1.0 × 10-4M ferrum cyanogen
Changing potassium, the luminol of described concentration is by 1.0 × 10-2M sodium hydroxide dissolves and prepares;The flow velocity of current-carrying and luminous agent is 2.2
mL/min;Described in step (2) and (3), the on-line microdialysis method of sampling is specific as follows: sample temperature is 37 DEG C;Perfusate is
0.010 M pH 7.4 PBS, perfusion rate is 4.0 μ L/min.
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CN1158166A (en) * | 1994-09-14 | 1997-08-27 | Cma微透析股份公司 | Method for analysis and device for carrying out the method |
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