CN107764992B - A kind of orientation coupling method and the application of microballoon and antibody - Google Patents
A kind of orientation coupling method and the application of microballoon and antibody Download PDFInfo
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- CN107764992B CN107764992B CN201710960301.2A CN201710960301A CN107764992B CN 107764992 B CN107764992 B CN 107764992B CN 201710960301 A CN201710960301 A CN 201710960301A CN 107764992 B CN107764992 B CN 107764992B
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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
Abstract
The invention discloses the orientation coupling method of a kind of microballoon and antibody, carboxyl microballoon realizes orientation coupling with antibody F (ab ') segments in such a way that sulfydryl is coupled, and forms antibody microsphere compound.This method can effectively control direction when antibody is connect with microballoon, realize orientation coupling to improve sensitivity of the latex enhancing immune than turbid reagent, and can improve anti-interference ability of the reagent for rheumatoid factor (RF).
Description
Technical field
The present invention relates to medical sciences, more particularly to a kind of be coupled by orientation to improve emulsion reagent sensitivity and spy
Anisotropic methods and applications.
Background technology
Latex enhancing immune turbidimetry (particle-enhanced turbidimetric immunoassay, PETIA)
It is a kind of stabilization, the homogeneous immunoturbidimetry detection method of accurate body fluid albumen.PETIA methods are divided into two kinds, and one is scattering turbidimetries
Detection method；Another kind is the turbid detection method of transmittance.The basic principle of both methods is all on the surface of polymer latex microballoon
It is crosslinked polyclonal antibody or monoclonal antibody in a short time can be rapid after the microballoon and antigen binding for being crosslinked with antibody
It flocks together, changes the astigmatism performance or light transmission of reaction solution.Reaction solution astigmatism performance or light transmission (i.e. absorbance)
Change and the concentration of tested antigen have stronger correlation, can reflect the concentration of tested antigen in a certain range.PETIA
Detection method is that antigen-antibody immune response is carried out in homogeneous reaction system to measure body fluid to be detected based on this principle
The content of albumen.In addition, this method is detected using automatic clinical chemistry analyzer, high degree of automation eliminates environment etc.
The interference of factor so that this method has preferably test steady compared to the methods of enzyme linked immunosorbent assay, immunochromatographic method learning aid
Qualitative, accuracy, detection flux are high.
Currently, microspherulite diameter distribution used by PETIA detection reagents increases from 20nm~4 μm between microballoon and antibody
The bridging chemistry arm added reduces steric effect when antibody is combined with microballoon to a certain extent, on the one hand improves having for antibody
It coupling amount is imitated, is on the other hand effectively protected the three-dimensional structure of antibody, the effective protection bioactivity region of antibody.Detection
Therefore the sensitivity of reagent is greatly improved, can reach 1.0ng/ml.Although the sensitivity learned than the methods of ELISA
It is less better, but the content for existing many body fluid albumens in normal human, clinical detection can be met substantially
It is required that.
However as the rapid development of laboratory medicine, the detection that substance is detected for low concentration is more and more, and detection is dense
Degree drops to ng/ml ranks from μ g/ml ranks, even more micro pg/ml ranks, this spirit for external diagnosis reagent
Sensitivity requires also higher and higher.It is usually used in improving method of the latex enhancing immune than turbid reagent test sensitivity at present, including makes
With the antibody of more high-affinity, using grain size bigger and the better microballoon of stability etc., but for the realization of these methods, open
Degree of raising difficult questions is big, and research and development and the production cost of reagent can be increased substantially on the basis of existing, can not be tried in latex immunoturbidimetry
Popularization and application is realized in agent exploitation.
In latex enhancing immune than in turbid reagent preparation process, the coupling method of antibody and microballoon is numerous, but all exists
One problem, i.e. direction of the antibody on being connected to microballoon are unable to control.It is well known that the region of antibody and antigen binding is located at
In F (ab) segment of antibody, if in coupling process, what is be connected with microballoon is F (ab) segment of antibody, then flex outward
Active Fc segments are exactly not bound with antigen, it is antibodies to "wrong" antigens that this, which results in this coupled antibody,.And in antibody and microballoon
It carries out in coupling process, there are prodigious randomness, the invalid presence for connecting antibody makes the profit of antibody in the connection direction of antibody
It is not high with rate, and substantially reduce the final sensitivity of reagent.The method proposed has the orientation referred in CN106501505A
Coupling method, but still there are two disadvantages for this method.First, this results of laboratory and Liang Yangui are in its master thesis
Result of study show full length antibody reduction after purpose product yield it is very low.The purpose of reduction full length antibody is to open to be located at
Two pairs of disulfide bond between Fc sections of heavy chain obtain the antibody fragment that a heavy chain and a light chain are connected by disulfide bond, and profit
It is oriented coupling with the free sulfhydryl group of formation.But by using different types of reducing agent, different recovery time and not
Same reducing condition carries out the screening of antibody reduction scheme, and final purpose efficiency of pcr product is no more than 25%.Second is that this method is easy
The interference for causing rheumatoid factor (RF) reduces the sensitivity and specificity of reagent.
Invention content
The object of the present invention is to provide the orientation coupling method of a kind of microballoon and antibody, this method can effectively control antibody
Direction when being connect with microballoon realizes that orientation is coupled to improve sensitivity of the latex enhancing immune than turbid reagent and stability,
And reagent is improved simultaneously for the anti-interference ability of rheumatoid factor (RF) and realizes detection of the reagent to whole blood sample.The present invention
Another object be to provide application of this method in external diagnosis reagent case prepared by PETIA methodologies.
To achieve the above object, the technical solution adopted by the present invention is as follows：
The present invention provides a kind of orientation coupling method of microballoon and antibody, and carboxyl microballoon is with antibody F (ab ') segment with sulfydryl
The mode of coupling realizes orientation coupling, forms antibody microsphere compound.
Antibody F (ab ') segment of the present invention refers to by F (ab ')2Segment restores the band of the disulfide bond formation between heavy chain
There is the segment of free sulfhydryl group；The F (ab ')2Segment refers to the F (ab) comprising antibody2And a kind of antibody of antibody whole hinge area
Segment.
More specifically, the orientation coupling method of microballoon and antibody of the present invention, includes the following steps：
1) F (ab ') of antibody is prepared by proteolytic cleavage reaction for full length antibody2Segment, F (ab ')2Segment, which refers to, includes
The F (ab) of antibody2And a kind of antibody fragment of antibody whole hinge area；Restore F (ab ')2Disulfide bond formation between fragment heavy chain
Segment with free sulfhydryl group, i.e. antibody F (ab ') segment；
2) activated carboxyl microballoon makes the carboxyl microballoon after activation with antibody F (the ab ') segments described in step 1) with sulfydryl idol
The mode of connection realizes orientation coupling, forms antibody microsphere compound.
The method of the invention is realized with antibody F (ab ') segment and is coupled with the orientation of microballoon, and the profit of antibody and microballoon is improved
With rate, sensitivity and the specificity of emulsion reagent are improved, while the detection that RF is interfered and realized whole blood sample can be eliminated.
The present invention also provides the orientation coupling methods of a kind of more specifically microballoon and antibody, include the following steps：
(1) antibody F (ab ')2The preparation of segment：Protease is added into antibody and carries out endonuclease reaction, 4~40 DEG C of reactions
Reaction is terminated after 0.5~4 hour, obtains F (ab ')2Segment；
(2) antibody F (ab ')2The reduction of segment：The F (ab ') obtained to step (1)2It is added reducing agent in segment, 4~40
It is reacted 0.5~2 hour under the conditions of DEG C, purifying obtains F (ab ') segment containing free sulfhydryl group；
(3) F (ab ') segments are coupled with microballoon：Activator is added in carboxyl microballoon, and it is small that 35~38 DEG C of activation are incubated 0.5~1
When, deactivator is removed, F (ab ') segment containing free sulfhydryl group that step (2) obtains is added, is allowed in such a way that sulfydryl is coupled
Orientation is coupled on microballoon.
Protease described in step (1) of the present invention can obtain F (ab ') to be any with digestion2The enzyme of segment, such as stomach egg
White enzyme.The concentration of the protease is preferably in 30~200U/mg antibody, more preferable 50~100U/mg antibody, further preferably
60U/mg antibody.
Reaction is terminated after preferably 30~40 DEG C of step (1) endonuclease reaction reaction 0.5~1.5 hour of the present invention.
Using step of the present invention (1) the method also original antibody, Fc sections of digestion is carried out to antibody so that step (2)
F (ab ') segment rate of recovery containing free sulfhydryl group of gained is more than 90%, and antibody utilization rate is high.
After reducing agent is added in step (2) of the present invention, reacted 0.5~2 hour under the conditions of preferably 30~40 DEG C.Preferably, originally
Reducing agent described in inventive step (2) includes but not limited to three (2- carboxyethyls) phosphines (TCEP), dithiothreitol (DTT) (DTT), β-mercapto
It is one or more in base ethyl alcohol, Mercamine Cysteamine, preferred Mercamine Cysteamine.The concentration of the reducing agent is excellent in 0.2~50mM
Select 0.2~10mM, more preferable 2mM.
In step (1) and step (2) of the present invention within the scope of reaction temperature, time, enzyme and reducing agent, to antibody
Digesting efficiency, reduction efficiency and antibody activity holding best results.
The purifying of F (ab ') segment containing free sulfhydryl group can use HiTrap Desalting in step (2) of the present invention
5ml desalting columns carry out.
(3) 35~38 DEG C of activation of step of the present invention are incubated 0.5~1 hour, can greatly improve activation efficiency.
Activator described in step (3) of the present invention is 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides
(EDC) and N- (2- amino-ethyls) maleimide (NH2- MAL), it is preferred that EDC and NH2The molar ratio of-MAL is 1:1~1:
10；The carboxyl-content of the ratio and microballoon of EDC and carboxyl microballoon is related, preferred molar ratio EDC：Carboxyl=0.2:1~10:1,
More preferable 0.2:1~5:1, it can ensure latex microsphere stability within this range, improve antibody coupling efficiency, be higher or lower than
This range can all lead to the decline, agglutination and the reduction of antibody coupling efficiency of latex microsphere stability.
It is incubated by the activation of activator, NH2Amino in-MAL is reacted with the carboxyl on microballoon, by dimaleoyl imino
In group's modification to microballoon.
The method of the invention is suitable for the coupling of various different antibodies and carboxylic acid microballoon.
The present invention also provides the orientation coupling methods of the microballoon and antibody to prepare external diagnosis reagent in PETIA methodologies
Application in box.
The present invention also provides the orientation coupling methods of the microballoon and antibody in preparing immunodiagnosis or detection reagent
Using.
The present invention also provides antibody microsphere compound prepared by the method, the microballoon of the compound and the F of antibody
(ab ') segment is coupled with sulfydryl.Antibody F (ab ') segment flexes outward in compound of the present invention, may be implemented in sample
Determined antigen and antibody efficient combination.
Antibody microsphere compound of the present invention can effectively reduce interference of the RF to testing result.
The present invention also provides a kind of composition, including the antibody microsphere compound and common auxiliary material pharmaceutically.
The present invention also provides a kind of external diagnosis reagent cases including the antibody microsphere compound.
The present invention also provides the microsphere compound, compositions or agents boxes in preparing immunodiagnosis or detection reagent
Using.
The present invention also provides the microsphere compounds or composition to prepare the application in treating disease medicament.
Compared with prior art, the present invention has following advantage：
1, the method for microballoon and antibody coupling provided by the invention, the technology using orientation coupling make antibody on microballoon
It is controllable to connect direction, reduces the randomness of antibody coupling, improves the utilization rate of antibody and microballoon, this method is suitable for various
The coupling of different antibodies and latex microsphere, the technique for being easily formed scale.
2, using this method also original antibody, Fc sections of digestion has been carried out to antibody first so that last gained contains certainly
It is more than 90% by F (ab ') segment rate of recovery of sulfydryl, antibody utilization rate is high.
3, for the latex enhancing immune prepared using this method than turbid detection reagent, sensitivity can reach 0.13ng/ml, can
To realize the application in high project is required in all multipair sensitivity techniques such as cTnI, PCT.
4, the latex enhancing immune prepared using this method can effectively reduce RF to testing result than turbid detection reagent
Interference.
5, for the latex enhancing immune prepared using this method than turbid detection reagent, antibody uses the side of chemical coupling with microballoon
Formula is oriented coupling, and connection is more secured, can be resistant to more surfactants, effectively avoid the addition of surfactant
The case where causing antibody to fall off from microsphere surface, therefore the detection of whole blood sample may be implemented, meet care diagnostic (POCT)
It is required that.
Description of the drawings
Fig. 1 is the front and back FP1 antibody of reduction；
Fig. 2 is the result of kit B Detection of Stability under the conditions of 37 DEG C；
Fig. 3 is that kit B whole bloods and serologic test result compare.
Specific implementation mode
Below in conjunction with the accompanying drawings by the detailed description of specific implementation mode come the present invention is furture elucidated, so that the present invention
Advantages and features can be easier to be readily appreciated by one skilled in the art, apparent clear to be made to protection scope of the present invention
Define, but be not limitation of the present invention, only illustrate.The experiment side of actual conditions is not specified in following embodiments
Method according to normal condition, or executes according to the normal condition proposed by manufacturer, no special instruction, and used reagent is equal
It is commercially available.
The preparation and purification of F (the ab ') antibody fragment of embodiment 1 containing free sulfhydryl group
1, antibody F (ab ')2The preparation of segment
(1) pepsin is weighed, mixing in 0.1M sodium citrate buffer solutions pH3.5, final concentration 1mg/ml are dissolved in；
(2) cardic fatty acid binding protein (H-FABP) monoclonal antibody FP1 is taken, with 0.1M sodium citrate buffer solutions
PH3.5 is diluted to 2mg/ml, and mixing；
(3) pepsin solution, wherein pepsin are added into the antibody after dilution：FP1=60U/mg, 37 DEG C of isothermal vibrations
Reaction 1 hour, the 3M Tris-HCl pH of buffer 9.0 that 6% volume is added terminate reaction.
2, the preparation of antibody F (ab ') segment
(1) Mercamine Cysteamine is weighed, mixing in 0.1M PBS pH7.4, final concentration 0.2mol/L are dissolved in；NEM is weighed, it is molten
The mixing in isopropanol, final concentration ask 1mol/L；
(2) Mercamine Cysteamine is diluted with PBS buffer solution, the F (ab ') of above-mentioned FP1 is added after mixing2Segment so that β-mercapto
The final concentration of 2mM of base ethamine, and F (ab ')2Segment final concentration of 1mg/ml, 37 DEG C of constant-temperature incubations 1.5 hours, is completed after incubation
After be stored in 4 DEG C, and take 1/10 volume of part NEM solution close after run glue verification reduction effect, verification result such as Fig. 1 institutes
Show.
The result shows that antibody molecule amount is 50kDa or so after reduction, it is made of the heavy chain after a light chain and digestion.Point
Two miscellaneous bands that son amount is 25kDa or so are the digestions of antibody caused by the disulfide bond between light chain and heavy chain is opened further
It is all very high with reduction efficiency.
3, the purifying of F (ab ') segment
F (ab ') fragment purification is carried out using HiTrap Desalting HP 5mlx2 (GE) desalination chromatographic column, it is slow with PBS
The 2ml/ sample introduction desalination of F (ab ') segment that above-mentioned steps are prepared after fliud flushing balance chromatographic column, and will be produced after desalting and purifying
Object is concentrated into 2mg/ml, and the rate of recovery of F (ab ') segment is more than 90%.
Sulfydryl coupling of the embodiment 2 based on carboxyl microballoon
1, the activation of carboxyl microballoon
(1) P0323 (JSR) polystyrene carboxyl microballoon is taken to be diluted to 0.1mg/ml with 20mMMES pH6.0；
(2) EDC and NH2-MAL are weighed, it is water-soluble to 1mg/ml respectively, and with 1：5 ratio is fed into above-mentioned microballoon dilution
Liquid, and microballoon is activated, 37 DEG C of isothermal vibrations are incubated 0.5 hour, and centrifugation after the completion removes supernatant, with 20mM HEPES pH 7.5
Mixing is resuspended.
2, antibody coupling
(1) it takes 20mM HEPES pH 7.5 to dilute FP1-F (ab '), dilution is added in the microballoon of mixing in step 1- (2)
In good antibody, 37 DEG C of isothermal vibrations are incubated 1 hour, and the closing of 5% bovine serum albumin solution, 37 DEG C of perseverances are added in the completed
Temperature concussion is incubated 1 hour；
(2) it takes reagent centrifugation after above-mentioned closing to remove supernatant, be used in combination 10mM HEPES pH7.0 resuspension mixings, antibody after mixing
Microsphere compound is in 4 DEG C of preservations.
Embodiment 3 prepares platelet-activating factor acetylhydro-lase (Lp-PLA2) detection kit (glue using orientation coupling method
Breast enhancing immunoturbidimetry)
1, prepared by kit
The preparation of two plants of pairing antibody F (ab ') segments of anti-human Lp-PLA2 and its antibody after being coupled respectively with microballoon
The preparation of microsphere compound is identical as the process being previously mentioned in Examples 1 and 2, by two kinds of antibody microsphere compounds after the completion of preparation
1:1 mixing is the anti-human Lp-PLA2 antibody latexs microsphere compound in R2 reagents.
The specific ingredient of platelet-activating factor acetylhydro-lase (Lp-PLA2) detection kit is formulated as follows：
Reagent R1：
Reagent R2：
Boric acid-sodium tetraborate buffer solution (pH 8.5) 0.02M
Anti-human Lp-PLA2 antibody latexs microsphere compound 0.1g/L
Proclin 300 0.2%
2, sensitivity technique
The patients serum that 9 Lp-PLA2 test results are positive is collected, diagnostic methodology detects for enzyme process kit, 1
Normal human serum, source are Nanjing drum tower hospital；
(it is purchased from Diazyme, enzyme linked immunosorbent assay claims kit in this experiment with Lp-PLA2 mass methods detection kit
A this 10 serum difference assignment) are given, and chooses wherein an example high level sample and carries out gradient dilution to 1ng/ml with PBS buffer solution；
The Lp-PLA2 detection kits made from step 1 (latex enhancing immune turbidimetry) (claim kit B) in this experiment
The sample of this 10 samples and gradient dilution is detected respectively, and each test sample 3 times is averaged, continuous monitoring 10 days, day
Between CV less than 20% critical concentration be reagent sensitivity.Select commercially available certain producer Lp-PLA2 detection kit (latex simultaneously
Enhance immunoturbidimetry) (claiming kit C in this experiment), kit carried out parallel test as a contrast.
1 kit A of table CV between sample assignment information and kit B and C test days
By table 1, it can be seen that, through the invention, Lp-PLA2 detection kits, i.e., (latex enhancing immune is than turbid by kit B
Method) sensitivity of 1ng/ml can be reached, and existing commercially available Lp-PLA2 detection kits, i.e. kit C sensitivity are only capable of reaching
To 4ng/ml.
3, RF Interference Detections
The anti-RF interference performances of detection kit B as follows：
(1) the close no haemolysis of measured value of relatively low range, the human serum sample (drum tower of jaundice, chyle, turbid phenomenon are collected
Hospital), it is mixed, which is measured, the value for obtaining its Lp-PLA2 is 78.23ng/ml；
(2) according to shown in the following table 1, table 2, the RF chaff interferents of various concentration gradient are added in mixed human serum；
(3) blank determination is carried out to the serum sample for being added to different RF unit concentrations using kit B, C respectively, repeated
3 times, calculate mean value, CV and deviation, deviation range be considered as within ± 10% it is noiseless, deviation range be more than ± 10% be considered as it is dry
It disturbs.
The result of the serum sample of 2 kit B detection addition various concentration RF chaff interferents of table
The result of the serum sample of 3 kit C detection addition various concentration RF chaff interferents of table
Table 2 the results show that range with RF concentration from 0 to 2000, the variation of the detected value deviation of kit B compared with
It is small, detected value and when being not added with interference detected value deviation within ± 10%, be determined as that there is no interference.
Table 3 is the results show that range with RF concentration from 0 to 2000, the detected value deviation of kit C also increase therewith
Greatly, when RF concentration is more than 500IU/ml, detected value and when being not added with interference detected value deviation more than 10%, be determined to have
Interference.
This experiment shows that compared to existing latex enhancing immune, than purifying method kit, method provided by the present invention is bright
It is aobvious to improve anti-interference ability of the reagent to RF.
4, Detection of Stability
The stability of detection kit B as follows：
It is prepared in kit B and completes to be placed on 37 DEG C of constant incubators, and respectively at the 1st, 2,3,5,7,14,21,30 day
Portion of reagent is taken out to same quality-control product (low value quality-control product：150.0 ± 15.0ng/mL, high level quality-control product：350.0±
It 35.0ng/mL) is tested, replication 3 times is averaged (M), and calculates n-th day test mean value MnIt was tested with the 1st day
Mean value M1Relative deviation R, relative deviation≤15% is considered as stabilization of kit, and test results are shown in figure 2.
As seen from the figure, kit B can stablize at least 30 days under 37 DEG C of constant temperature.
5, whole blood sample detects
160 Lp-PLA2 whole blood samples and its corresponding serum sample (source is Nanjing drum tower hospital) are collected, use is above-mentioned
Kit B detects whole blood sample and serum sample simultaneously, and the result of test is carried out correlation analysis, relative coefficient R2≥
When 0.96, then it is assumed that test result correlation is good.
From the figure 3, it may be seen that the relative coefficient R of kit B test serum and whole blood sample2>=0.96, it can be determined that reagent
Box B can accurately test whole blood sample.
Embodiment 4 prepares Troponin I (cTnI) detection kit (latex enhancing immune is than turbid using orientation coupling method
Method)
1, prepared by kit
The preparation of two plants of pairing antibody F (ab ') segments of anti-human cTnI and its antibody after being coupled respectively with microballoon are micro-
The preparation of ball compound is identical as the process being previously mentioned in Examples 1 and 2, by two kinds of antibody microsphere compounds 1 after the completion of preparation:
1 mixing is the anti-human cTnI antibody latexs microsphere compound in R2 reagents.
The specific ingredient of Troponin I (cTnI) detection kit is formulated as follows：
Reagent R1：
Reagent R2：
2, sensitivity technique
The patients serum that 9 cTnI test results are positive is collected, diagnostic methodology detects for chemoluminescence method, and source is
Nanjing drum tower hospital, and choose wherein an example high level sample and carry out gradient dilution to 0.06ng/ml with PBS buffer solution；
The cTnI detection kits made from step 1 (latex enhancing immune turbidimetry) (claiming kit D in this experiment) point
The sample of this 10 samples and gradient dilution is not detected, and each test sample 3 times is averaged, continuous monitoring 10 days, between day
Critical concentrations of the CV less than 20% is reagent sensitivity.Select commercially available certain producer cTnI detection kit (latex intensifieds simultaneously
Immunoturbidimetry) (claiming kit F in this experiment), kit carried out parallel test as a contrast.
CV between 4 kit D and F test days of table
By table 4, it can be seen that, through the invention, cTnI detection kits (latex enhancing immune turbidimetry) can reach
The sensitivity of 0.13ng/ml, and existing commercially available cTnI detection kits sensitivity is only capable of reaching 0.52ng/ml.
Claims (14)
1. the orientation coupling method of a kind of microballoon and antibody, which is characterized in that activator, 35~38 DEG C of activation are added in carboxyl microballoon
It is incubated 0.5~1 hour, removes deactivator, then realize orientation coupling, shape in such a way that sulfydryl is coupled with antibody F (ab ') segments
At antibody microsphere compound；The activator is 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and N- (2-
Amino-ethyl) maleimide, and 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and N- (2- amino-ethyls)
The molar ratio of maleimide is 1:1~1:10.
2. orientation coupling method according to claim 1, which is characterized in that include the following steps：
1) F (ab ') of antibody is prepared by proteolytic cleavage reaction for full length antibody2Segment, F (ab ')2Segment refers to comprising antibody
F (ab)2And a kind of antibody fragment of antibody whole hinge area；Restore F (ab ')2Disulfide bond formation between fragment heavy chain carries
The segment of free sulfhydryl group, i.e. antibody F (ab ') segment；
2) carboxyl microballoon be added activator, 35~38 DEG C activation be incubated 0.5~1 hour, remove deactivator, then with antibody F
(ab ') segment realizes orientation coupling in such a way that sulfydryl is coupled, and forms antibody microsphere compound.
3. orientation coupling method according to claim 2, which is characterized in that include the following steps：
(1) antibody F (ab ')2The preparation of segment：Protease is added into antibody and carries out endonuclease reaction, 4~40 DEG C of reactions 0.5~4
Reaction is terminated after hour, obtains F (ab ')2Segment；
(2) antibody F (ab ')2The reduction of segment：The F (ab ') obtained to step (1)2Reducing agent, 4~40 DEG C of items are added in segment
It is reacted 0.5~2 hour under part, purifying obtains F (ab ') segment containing free sulfhydryl group；
(3) F (ab ') segments are coupled with microballoon：Activator is added in carboxyl microballoon, and 35~38 DEG C of activation are incubated 0.5~1 hour, remove
F (ab ') segment containing free sulfhydryl group that step (2) obtains is added in deactivator, is allowed to orient idol in such a way that sulfydryl is coupled
It is linked on microballoon.
4. orientation coupling method according to claim 3, which is characterized in that the protease described in step (1) is stomach cardia
Enzyme；A concentration of 30~200U/mg antibody of protease.
5. orientation coupling method according to claim 4, which is characterized in that protease described in step (1) it is a concentration of
50~100U/mg antibody.
6. orientation coupling method according to claim 5, which is characterized in that protease described in step (1) it is a concentration of
60U/mg antibody.
7. orientation coupling method according to claim 3, which is characterized in that the reducing agent described in step (2) includes but not
It is limited to one or more in TCEP, DTT, beta -mercaptoethanol, Mercamine Cysteamine；The concentration of the reducing agent is in 0.2~50mM.
8. orientation coupling method according to claim 7, which is characterized in that the reducing agent described in step (2) is β-sulfydryl
Ethamine；The concentration of the reducing agent is in 0.2~10mM.
9. orientation coupling method according to claim 8, which is characterized in that a concentration of 2mM of the reducing agent.
10. orientation coupling method according to claim 1, which is characterized in that 1- (3- dimethylamino-propyls) -3- ethyl carbon
The molar ratio of diimmonium salt hydrochlorate and carboxyl microballoon is 0.2:1~10:1.
11. the antibody microsphere compound that orientation coupling method described in claim 1 is formed.
12. a kind of composition, including antibody microsphere compound described in claim 11 and common auxiliary material pharmaceutically.
13. the antibody microsphere compound described in claim 1~6 any one of them method, claim 11 or claim
Application of the composition in preparing immunodiagnosis or detection reagent described in 12.
14. the antibody microsphere compound described in claim 1~6 any one of them method, claim 11 or claim
Composition described in 12 is preparing the application in treating disease medicament.
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| CN106324251B (en) * | 2016-08-08 | 2019-03-29 | 上海睿康生物科技有限公司 | The preparation method and beta 2-microglobulin detecting kit of small fragment BMG antibody |
| CN110922476A (en) * | 2019-12-16 | 2020-03-27 | 蓝怡科技集团股份有限公司 | Biotin coupled antibody and preparation method and application thereof |
| CN112034159B (en) * | 2020-09-06 | 2023-08-29 | 武汉生之源生物科技股份有限公司 | Gelating immunomagnetic bead and preparation method and application thereof |
| CN112326953A (en) * | 2020-11-03 | 2021-02-05 | 广东海洋大学深圳研究院 | Method for directionally labeling polybiotin by using antibody |
| CN113030461B (en) * | 2021-03-12 | 2022-04-22 | 北京华科泰生物技术股份有限公司 | IgG antibody marked by amino group or carboxyl group-containing substance, human IgG magnetic particle chemical detection kit and preparation method thereof |
| CN113024669B (en) * | 2021-03-12 | 2022-06-14 | 北京华科泰生物技术股份有限公司 | RBP antibody marked by amino-group or carboxyl-group-containing substance, human RBP immunochromatography detection kit and preparation method thereof |
| CN113777312B (en) * | 2021-09-03 | 2024-02-02 | 普十生物科技(北京)有限公司 | Preparation method of hepatitis B antibody fragment, kit and application |
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