CN108061798B - Detect the methods and applications of the amount of effective component in HBsAg/anti-HBs complex systems - Google Patents
Detect the methods and applications of the amount of effective component in HBsAg/anti-HBs complex systems Download PDFInfo
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- CN108061798B CN108061798B CN201610977196.9A CN201610977196A CN108061798B CN 108061798 B CN108061798 B CN 108061798B CN 201610977196 A CN201610977196 A CN 201610977196A CN 108061798 B CN108061798 B CN 108061798B
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
- G01N33/5764—Hepatitis B surface antigen
Abstract
The present invention relates to the methods of the amount of effective component in detection HBsAg/anti-HBs complex systems, comprising: 1) provides the complex systems, it includes HBsAg/anti-HBs compounds, free IgG and free HBsAg；2) affinity chromatography is carried out to the system, collects and flows through peak a containing free HBsAg；3) it provides referring to HBsAg solution, contains HBsAg identical with the HBsAg total amount of the system；Affinity chromatography is carried out to referring to HBsAg solution, collects and flows through peak b containing HBsAg；4) detection flows through the amount a for the HBsAg that dissociates in peak a, flows through the amount b of HBsAg in peak b, and dosage a, amount b, the HBsAg total amount in the system calculate the amount for the HBsAg that dissociates in the system.This method can be applied to the quality control of HBsAg/anti-HBs complex hepatitis B vaccine.
Description
Technical field
The invention belongs to biomedicine fields, in particular to a kind of detection HBsAg/anti-HBs complex systems
The methods and applications of the amount of middle effective component.
Background technique
Immune complex (immune complexes, IC) type vaccine is with corresponding specific antibody by antigen with appropriate
Ratio be mixed to form antigen antibody complex, as antigen, addition or do not add the vaccine that adjuvant is prepared.IC enhancing is exempted from
The mechanism of epidemic disease response may have the following: antibody Fc section and antigen presenting cell (antigen presenting in IC
Cell, APC) the Fc receptor on surface combines, mediates IC internalization, offering for antigenic component in IC can be improved；The Fc that IC passes through antibody
Section combines the Fc receptor on the surface Dendritic Cells (dendritic cells, DC), may act as natural adjuvant and reconciles DC maturation；
IC can direct activation effect cell, induce stronger MHC I and MHC II class " intersection is offered " to react；A small amount of IC is by lymph follicle
In follicular dendritic cell (follicular dendritic cells, FDC) capture and FDC table can be rested on for a long time
Face, this antibody response relied on T cell play an important role.
Immune complex type hepatitis B vaccine includes by HbsAg (hepatitis B surface
Antigen, HBsAg) and hepatitis b human immunoglobulin (human hepatitis B immunoglobulin, HBIG) group
At HBsAg/anti-HBs compound, easily absorbed and offered by APC, and then body can be excited to generate effective humoral immunity
And cellullar immunologic response.Immune complex type hepatitis B vaccine can induce generation more compared with commercially available saccharomyces cerevisiae hepatitis B vaccine
The AntiHBsAg antibody (anti-HBs) and cellullar immunologic response of high titre.
The preparation process of immune complex type hepatitis B vaccine are as follows: the 1) conjunction of HBsAg and HBIG are determined by " proportioning test "
Suitable ratio；2) HBsAg and HBIG is mixed according to the ratio, is incubated for the regular hour at a certain temperature, which is determined
Justice is " compound "；3) above-mentioned equivalent HBsAg is added in " compound ", continues to mix, which is defined as " adding excessive anti-
Former compound ", the solution are the stoste of immune complex type hepatitis B vaccine；4) " excessive antigenic compound will be added " and the aluminium of equivalent
Adjuvant to adsorbing again, as vaccine semi-finished product；5) ampoule dispenses, as finished product.
The effective component of immune complex type hepatitis B vaccine be HBsAg/anti-HBs compound and free HBsAg, two
The content detection of person is most important for the quality control of vaccine.It is to make that immune complex type hepatitis B vaccine, which produces HBsAg used,
The genetic engineering Purification of Recombinant Hepatitis B Virus Surface Antigen of brewer yeast expression, purity is 95% or more；HBIG be from human plasma,
The blood product prepared by cold ethanol method, IgG purity wherein remove the antibody containing anti-HBsAg 90% or more
Except anti-HBs, also containing largely for other pathogens/antigen IgG.
Since antigen-antibody reaction is that reversible dynamic equilibrium is reacted, vaccinogen liquid (" adding excessive antigenic compound ") body
Contain following several ingredients in system: HBsAg/anti-HBs compound, free HBsAg, free IgG are (a large amount of non-specific
IgG, micro anti-HBs).The characteristics of this dynamic equilibrium, separation method is limited, by each component separation and accurate quantification is examined
Survey is the difficult point of immune complex type production of vaccine Quality Control.For a long time, in immune complex type hepatitis B vaccine effectively
The accurate detection of ingredient is to perplex the problem of the researcher and technical staff of related fields, is still lacked for immune in the prior art
The detection method efficiently and accurately of each effective component of complex vaccine.
Summary of the invention
To solve above-mentioned problems of the prior art, the present invention provides a kind of detection HBsAg/anti-HBs is compound
The methods and applications of the amount of effective component in objects system.
Specifically, the present invention provides:
(1) a kind of method for detecting the amount of effective component in HBsAg/anti-HBs complex systems, comprising:
1) HBsAg/anti-HBs complex systems are provided, which includes HBsAg/anti-HBs compound, free IgG
With free HBsAg；The effective component is the free HBsAg and the HBsAg/anti-HBs compound；
2) affinity chromatography is carried out to the HBsAg/anti-HBs complex systems, wherein the medium energy of the affinity chromatography
Enough adsorb HBsAg/anti-HBs compound and free IgG；It collects and flows through peak a containing free HBsAg；
3) it provides referring to HBsAg solution, it is described to contain and the HBsAg/anti-HBs composite body referring to HBsAg solution
The identical HBsAg of HBsAg total amount of system；The affinity chromatography is carried out referring to HBsAg solution to described, collection contains HBsAg's
Flow through peak b；
4) specifically detection flows through the amount a of the free HBsAg in peak a, and flows through the amount b of the HBsAg in peak b, uses
The HBsAg total amount in a, amount b and the HBsAg/anti-HBs complex systems is measured, the HBsAg/anti-HBs is calculated
The amount of free HBsAg in complex systems.
(2) method according to (1), wherein the method also includes:
5) affinity chromatography is carried out to the HBsAg/anti-HBs complex systems, wherein the medium energy of the affinity chromatography
Enough adsorb HBsAg/anti-HBs compound and free HBsAg；The HBsAg/anti-HBs being adsorbed on chromatography media is compound
Object and free HBsAg are eluted, and collect the eluting peak a containing the HBsAg/anti-HBs compound and the free HBsAg；
6) protein content in the eluting peak a is detected, the HBsAg/anti-HBs obtained by the protein content and step 4) is compound
The amount of free HBsAg in objects system, the HBsAg/anti-HBs calculated in the HBsAg/anti-HBs complex systems are multiple
Close the amount of object.
(3) method according to (1) or (2), wherein the HBsAg/anti-HBs complex systems are compound to be immunized
Object type hepatitis B vaccine stoste.
(4) method according to (1) or (2), wherein step 2) and 3) described in affinity chromatography be the affine layer of albumin A
Analysis.
(5) method according to (2), wherein affinity chromatography described in step 5) is heparin affinity chromatography.
(6) method according to (1) or (2), wherein specifically detecting institute using immunological method in step 4)
The amount of the stating a and amount b.
(7) method according to (1) or (2), wherein in step 4), the ratio of calculation amount a proportion b, and calculating should
The product of HBsAg total amount in ratio and the HBsAg/anti-HBs complex systems, thus obtains the HBsAg/anti-
The amount of free HBsAg in HBs complex systems.
(8) method according to (2), wherein subtracting step 4) with the protein content in the eluting peak a in step 6)
The amount of free HBsAg in gained HBsAg/anti-HBs complex systems, it is compound thus to obtain the HBsAg/anti-HBs
The amount of HBsAg/anti-HBs compound in objects system.
(9) method according to (2), wherein elution described in step 5) is using phosphate buffer as eluent.
(10) method according to (2), wherein the method also includes i) providing referring to HBIG solution, reference HBIG
Solution contains IgG identical with the IgG total amount of the HBsAg/anti-HBs complex systems；Reference HBIG solution is carried out
Affinity chromatography described in step 5) will be washed referring to non-specific adsorption in HBIG solution in the IgG on the medium of the affinity chromatography
It takes off, collects the eluting peak b containing the IgG, detect the protein content in eluting peak b；And
Wherein in step 6), with the protein content in the eluting peak b, the protein content in the eluting peak a and step
4) amount of the free HBsAg in gained HBsAg/anti-HBs complex systems, calculates the HBsAg/anti-HBs compound
The amount of HBsAg/anti-HBs compound in system.
(11) method according to (10), wherein being subtracted with the protein content in the eluting peak a: in the eluting peak b
Protein content and the step 4) obtained by free HBsAg in HBsAg/anti-HBs complex systems amount, thus obtain
The amount of HBsAg/anti-HBs compound in the HBsAg/anti-HBs complex systems.
(12) method according to (10) is wherein eluted in described in step i) identical with the elution described in step 5)
Under the conditions of carry out.
(13) method according to (2), wherein further include collecting to flow through peak c containing free IgG in step 5),
It detects this and flows through the protein content in peak c, to obtain the amount of the free IgG in the HBsAg/anti-HBs complex systems.
(14) quality control of the method according to any one of (1)-(13) in immune complex type hepatitis B vaccine
Application in system.
Compared with the prior art, the present invention has the following advantages and good effect:
The present invention is chromatographed by two steps, is realized in the case where not changing solution system, respectively to HBsAg/anti-
The detection of HBs compound, free HBsAg, free IgG content.Method concept of the invention is ingenious, step is simple and convenient to operate
Quickly, and verified, this method repeatability, precision are good.Therefore, it is suitably applied immune complex type hepatitis B vaccine
Quality control.
Detailed description of the invention
Fig. 1 is MabSelect SuRe affinity chromatography map.A is that HBsAg solution chromatographs map；B is HBIG solution chromatography
Map；C is plus excessive antigenic compound chromatographs map.
Fig. 2 is CaptoTMHeparin chromatographs map.A is that HBsAg solution chromatographs map；B is that HBIG solution chromatographs map；
C is plus excessive antigenic compound chromatographs map.
Specific embodiment
Description below by way of specific embodiment and the invention will be further described referring to attached drawing, but it is pair that this, which is not,
Limitation of the invention, those skilled in the art's basic thought according to the present invention, can make various improvement, as long as but not taking off
From basic thought of the invention, it is all within the scope of the present invention.
The purpose of the present invention is to provide the amounts of effective component in a kind of detection HBsAg/anti-HBs complex systems
Method is controlled with being applied to the quality of complex hepatitis B vaccine.
As described above, since antigen-antibody reaction is that reversible dynamic equilibrium is reacted, to immune complex type second
The accurate quantification detection of effective component HBsAg/anti-HBs compound and free HBsAg in liver vaccine is a difficult point.
By the further investigation to immune complex type hepatitis B vaccine system, and to various method for separating and analyzing
It carefully studies and gropes with many experiments, the inventors found that:
HBsAg exists in the solution in the form of virus-like particle (virus like particle, VLP), average molecular
Amount is 2,400kD, and diameter is 22.8 ± 0.4nm.The relative molecular weight of anti-HBs is 150kD, and diameter is 5~10nm.Due to
The molecular weight and granular size difference of free HBsAg and HBsAg/anti-HBs compound in vaccinogen liquid be not significant, because
This can not be efficiently separated HBsAg/anti-HBs compound and free HBsAg using the gel permeation chromatography of molecular sieve principle.
In addition, the rate of recovery of gel chromatography is lower, the quality control of vaccine is not suitable for.
When being separated using the spatial exclusion chromatography of HPLC, separating degree can not will resist less than 1.5 as the result is shown
Each component is kept completely separate in original antibody complex systems.It is also contemplated that HBsAg/anti-HBs compound has with free HBsAg
The methods of the difference of sedimentation coefficient, therefore be utilized respectively PEG precipitating and density gradient centrifugation, it is intended to it is isolated.However,
HBsAg/anti-HBs compound and the sedimentation coefficient difference of free HBsAg are unobvious, and existence range is overlapping, therefore is difficult to accomplish
It is precisely separated.
Based on the above discovery, the present inventor is further it has surprisingly been found that carry out group using the method that two steps chromatograph
Detection is closed, the accurate quantification to HBsAg/anti-HBs complex content and free HBsAg content may be implemented.
The present invention provides a kind of sides of the amount of effective component in detection HBsAg/anti-HBs complex systems as a result,
Method, comprising:
1) HBsAg/anti-HBs complex systems are provided, which includes HBsAg/anti-HBs compound, free IgG
With free HBsAg；The effective component is the free HBsAg and the HBsAg/anti-HBs compound；
2) affinity chromatography is carried out to the HBsAg/anti-HBs complex systems, wherein the medium energy of the affinity chromatography
Enough adsorb HBsAg/anti-HBs compound and free IgG；It collects and flows through peak a containing free HBsAg；
3) it provides referring to HBsAg solution, it is described to contain and the HBsAg/anti-HBs composite body referring to HBsAg solution
The identical HBsAg of HBsAg total amount of system；The affinity chromatography is carried out referring to HBsAg solution to described, collection contains HBsAg's
Flow through peak b；
4) specifically detection flows through the amount a of the free HBsAg in peak a, and flows through the amount b of the HBsAg in peak b, uses
The HBsAg total amount in a, amount b and the HBsAg/anti-HBs complex systems is measured, the HBsAg/anti-HBs is calculated
The amount of free HBsAg in complex systems；
In order to further detect the amount of the HBsAg/anti-HBs compound in HBsAg/anti-HBs complex systems, institute
State method further include:
5) affinity chromatography is carried out to HBsAg/anti-HBs complex systems, wherein the medium of the affinity chromatography can be inhaled
Attached HBsAg/anti-HBs compound and free HBsAg；By the HBsAg/anti-HBs compound being adsorbed on chromatography media and
Free HBsAg is eluted, and collects the eluting peak a containing the HBsAg/anti-HBs compound and the free HBsAg；
6) protein content in the eluting peak a is detected, the HBsAg/anti-HBs obtained by the protein content and step 4) is compound
The amount of free HBsAg in objects system, the HBsAg/anti-HBs calculated in the HBsAg/anti-HBs complex systems are multiple
Close the amount of object.
In step 1), HBsAg/anti-HBs complex systems can be prepared according to known methods.In the compound
In system, the total amount of HBsAg and the total amount of HBIG are commonly known, usage amount when as preparing.Hereinafter,
" HBsAg/anti-HBs complex systems " are also referred to as " complex systems ".
In immune complex type hepatitis B vaccine, the vaccine semifinished or finished goods after aluminium adjuvant absorption are gone in use
After dirty agent desorption, antigen antibody complex can be dissociated, and HBsAg particle can dissociate, and the active constituent content detected at this time is simultaneously
It is unable to its actual content in vaccine of actual response.Therefore, present invention preferably employs vaccinogen liquids (" to add excessive antigen multiple
Close object ") the HBsAg/anti-HBs complex systems are used as, to HBsAg/anti-HBs compound therein and dissociate
The content of HBsAg detect, and is more conducive to carrying out vaccine quality control in this way.
In step 2) of the invention, HBsAg/anti-HBs complex systems are carried out using affinity chromatography medium affine
Chromatography, free IgG and HBsAg/anti- using characteristic of the chromatography media in conjunction with IgG, in the complex solution system
HBs compound can be adsorbed on chromatography media, and free HBsAg is then present in and flows through in peak.Due to flowing through in peak except free
Except HBsAg, it is also possible to there are in HBIG in addition to IgG ingredient interference, therefore can not by directly detect protein content come
Measure free content of the HBsAg in the system.
Due in HBsAg/anti-HBs complex systems, (such as can be 120 μ g/ml) known to the total amount of HBsAg,
Therefore the present invention carries out the reference solution containing same amount of same batch HBsAg and the same affine layer of the complex systems
Analysis, collection flow through peak.Then, specifically the HBsAg antigen flowed through in peak of the two is quantified using immunological method
Detection.In this way, flowing through HBsAg in peak with the amount (amount a) for flowing through HBsAg in peak of resulting complex systems, referring to solution
Amount (amount b) and complex systems in HBsAg total amount, can be accurately calculated free in complex systems
The amount of HBsAg.
In one embodiment, in step 4), the ratio of calculation amount a proportion b, and calculate the ratio with it is described
Thus the product of HBsAg total amount in HBsAg/anti-HBs complex systems obtains the HBsAg/anti-HBs compound
The amount of free HBsAg in system.
Preferably, step 2) and 3) described in affinity chromatography be protein A affinity chromatography.
It is further preferred that specifically detecting the amount a and the amount b using immunological method in step 4).Immunology
Method carries out specific detection based on the specific reaction between antigen-antibody comprising but be not limited to chemoluminescence method, enzyme-linked exempt from
Epidemic disease absorption method, Magnetism particulate immuno chemistry luminescence method etc..
Further, it is chromatographed by second step, it can also be to the HBsAg/ in HBsAg/anti-HBs complex systems
Anti-HBs compound carries out accurate quantitative analysis.
Therefore, further, method of the invention further includes that use can adsorb free HBsAg and HBsAg/anti-HBs
The chromatography media of compound chromatographs complex systems, and free HBsAg and HBsAg/anti-HBs compound can be adsorbed in
On chromatography media, free IgG is flowed through.It is washed by HBsAg the and HBsAg/anti-HBs compound that will be adsorbed on chromatography media
It takes off, and protein content detection is carried out to eluting peak, it is free in the complex systems obtained by the protein content and step 4)
The amount of HBsAg can accurately calculate the amount of the HBsAg/anti-HBs compound in the complex systems.
In one embodiment, in step 6), step 3) gained HBsAg/anti-HBs is subtracted with the protein content
Thus the amount of free HBsAg in complex systems obtains the HBsAg/ in the HBsAg/anti-HBs complex systems
The amount of anti-HBs compound.
Preferably, affinity chromatography described in step 5) is heparin (Heparin) affinity chromatography.Heparin contains sulfate
Group, can be in conjunction with the outer membrane protein of virus, and using the property, heparin affinity chromatography has adsorbed the HBsAg/ in complex systems
Anti-HBs compound and free HBsAg, and flow through free IgG.
Since in HBsAg/anti-HBs complex systems, the total amount of HBIG is also known, it is therefore preferred that being
Obtain more accurate as a result, the invention also includes i) by reference HBIG solution and institute containing same amount of same batch HBIG
State complex systems carry out with the same affinity chromatography of step 5), can be with condition identical with step 5) by non-specific adsorption
It is eluted in the IgG on chromatography media, collects eluting peak, and protein content detection is carried out to get to can to the eluting peak
Non-specific adsorption is in the amount of the IgG on chromatography media.Protein content in the eluting peak a measured by step 6) subtracts non-specific
Property absorption IgG amount, then with resulting value calculate complex systems in HBsAg/anti-HBs compound amount, thus eliminate
The interference of the free IgG of non-specific adsorption in complex systems.
The IgG of HBsAg and HBsAg/anti-HBs compound and non-specific adsorption is washed from affinity chromatography medium
It takes off and phosphate buffer can be used, such as the 2M NaCl buffer prepared with 10mMPB.
In the above-mentioned steps 5 of the method for the present invention) in, it also collects and flows through peak, and carry out protein content detection to peak is flowed through,
It can be obtained the content for the IgG that dissociates in complex systems.
Another aspect of the present invention provides method of the present invention in immune complex type hepatitis B vaccine
Application in quality control.
The content of present invention is further explained and described in mode by the following examples, but these embodiments are not understood that
For limiting the scope of the invention.
Embodiment
The source of embodiment material therefor and instrument is as follows:
MabSelect SuRe affinity chromatography medium derives from GE Healthcare
AKTA prime Plus chromatograph derives from GE Healthcare
XK16/20 chromatographic column derives from GE Healthcare
HBsAg derives from Beijing Tiantan Biological Products Co.ltd
HBIG derives from Rongsheng Pharmaceutical Co., Ltd., Chengdu
Anti-human igg (Fc specificity) Peroxidase Antibody derives from Sigma company
AKTApurifier protein chromatography system derives from GE Healthcare
CaptoTMHeparin chromatography media derives from GE Healthcare
Lowry method low protein content assay kit derives from Shanghai Li Da Biotechnology Co., Ltd
Hepatitis b virus s antigen immue quantitative detection reagent box (chemoluminescence method) derives from Zhengzhou Antu bioengineering stock
Part Co., Ltd
Solution used in embodiment is formulated as follows:
1.PBS (pH7.4,0.02M): 0.593g NaH2PO4·2H2O, 5.8gNa2HPO4·12H2O and 8.775g
NaCl adds pure water to be completely dissolved and is settled to 1L, 0.45 μm of membrane filtration, room temperature preservation.
2. sample treatment liquid (diethanol amine-Triton X-100):
20% diethanol amine (DEA) mother liquor: 2mL DEA is added 8mL PBS (pH7.4,0.02M) and mixes, and room temperature is kept away
Light saves；
10%Triton X-100 mother liquor: it is mixed that 9mL PBS (pH7.4,0.02M) is added in 1mL Triton X-100
It is even, room temperature preservation；
Sample treatment liquid: 20%DEA mother liquor 1.25mL, 10%Triton X-100 mother liquor 0.2mL, PBS (pH7.4,
0.02M) 8.55mL is mixed, and room temperature is protected from light storage.
3.10mM PB:
0.2M NaH2PO4: 31.2g NaH2PO4·2H2O, pure water, which is completely dissolved, is settled to 1000mL, 0.45 μm of filter membrane mistake
Filter, room temperature preservation；
0.2M Na2HPO4: 71.6g Na2HPO4·12H2O, pure water, which is completely dissolved, is settled to 1000mL, 0.45 μm of filter membrane mistake
Filter, room temperature preservation；
10mM PB (pH 7.4): 0.2M NaH is taken2PO419mL, 0.2M Na2HPO489mL, pure water are settled to 2000mL.
" 4. adding excessive antigenic compound ":
According to ratio determined by " proportioning test ", the HBsAg of 20mL is mixed with the HBIG of 13.9mL, 37 DEG C are incubated for 1
Hour, 2-8 DEG C is overnight.It 20ml HBsAg is added, adjusts HBsAg concentration to 120 μ g/mL with physiological saline, at this time HBIG
Concentration is 37.14mg/mL.
Embodiment 1. " adding excessive antigenic compound " and individually HBsAg affinity chromatography
Using MabSelect SuRe affinity chromatography medium, AKTA prime Plus chromatograph, XK16/20 chromatographic column into
Row affinity chromatography.By test sample " adding excessive antigenic compound ", (HBsAg total amount is that 120 μ g/mL, HBIG total amounts are 37.14mg/
ML) loading, with 2mL loading ring, loading volume 2mL, combination buffer PBS, chromatographic flow rates are 2mL/ minutes；Collection flows through peak
90mL returns to baseline to ultraviolet detection peak, is eluted with 0.1M pH3.0 glycine-HCl solution.Reference is simple HBsAg
Normal saline solution (HBsAg concentration is 120 μ g/mL), loading flows through peak with " adding excessive antigenic compound ", collection.
Embodiment 2. flows through HBsAg antigenic content in peak and detects
Using chemoluminescence method (hepatitis b virus s antigen immue quantitative detection reagent box (chemoluminescence method)) respectively into
Row " adding excessive antigenic compound " and simple HBsAg flow through the detection of HBsAg antigenic content in peak: test sample and standard items take
0.2mL is added 0.2mL sample treatment liquid (diethanol amine-Triton X-100), and room temperature is protected from light processing 30 minutes；With 2%BSA,
Simple HBsAg is flowed through into peak and carries out 200 times of dilutions, adds excessive antigenic compound to flow through peak and carries out 100 times of dilutions, standard items carry out
It is serially diluted (160ng/mL, 80ng/mL, 40ng/mL, 20ng/mL, 10ng/mL, 5ng/mL, 2.5ng/mL and 1.25ng/
ML), standard curve is from 160ng/mL to 1.25ng/mL.Measuring samples after 50 μ L dilution and standard items are added in ELISA Plate, are pressed
It is detected according to kit specification.Straight line fitting is done with the logarithm of the logarithm of standard concentration and corresponding luminous value, obtains standard song
Line, the luminous value logarithm of sample is according to finding out respective concentration logarithm in standard curve, then carries out antilogarithm and acquire test sample
Concentration.
" free HBsAg " content of embodiment 3. calculates
Free HBsAg content (μ g/mL)=plus excessive antigenic compound flow through peak HBsAg content/reference HBsAg solution stream
Wear peak HBsAg content × 100% × 120 μ g/mL=40.35 μ g/mL/147.20 μ g/mL × 100% × 120 μ g/mL=
32.89μg/mL
Embodiment 4. " free HBsAg content " detection method specificity verifying
(1) MabSelect SuRe affinity column loading HBsAg: will be with batch HBsAg protein concentration with physiological saline
120 μ g/mL are diluted to, MabSelect SuRe affinity chromatography is carried out.Collect it is all flow through peak, accurate quantification flows through solution body
Product, chemoluminescence method detect HBsAg content in the solution of loading front and back, and the rate of recovery for calculating HBsAg is 83% or so.
From Fig. 1 (A) as can be seen that HBsAg is substantially all to flow through out chromatography media (retention time 14.08 minutes).It uses again
0.1M pH3.0 glycine-HCl is eluted, and is not had the appearance of the peak HBsAg after elution, is illustrated MabSelect SuRe affinity chromatography
Medium is very low to HBsAg non-specific adsorption.
(2) MabSelect SuRe affinity chromatography medium loading HBIG: will be with batch HBIG concentration dilution with physiological saline
To, loading volume 2mL identical as concentration in excessive antigenic compound is added, collection is all to flow through peak (retention time 28.70 minutes),
It is eluted again with 0.1M pH3.0 glycine-HCl, collects eluting peak.It is flowed through with ELISA method (dual-antigen sandwich method) detection
Anti-HBs in peak, result are feminine gender；Flowing through the human IgG in peak using ELISA method detection, (using anti-human igg, (Fc is special
Property) Peroxidase Antibody), result is feminine gender.
Result above proves that all IgG in HBIG are inhaled by MabSelectSuRe affinity chromatography medium with this condition
Attached, flowing through does not have anti-HBs in peak, nobody IgG yet.Fig. 1 (B) chromatographs a visible small peak in map, and analysis may be trace
IgM, IgA or human serum albumin.
(3) separation of MabSelect SuRe affinity chromatography medium plus excessive antigenic compound: will be added with physiological saline excessive
It is 120 μ g/mL, loading volume 2mL that antigenic compound, which is diluted to HBsAg protein concentration, and collection is all to flow through liquid, then uses 0.1M
PH3.0 glycine-HCl is eluted, and eluent is collected.The HBIG in peak and eluting peak, detection are flowed through with ELISA method detection
Flow through the HBsAg antigenic content in liquid.As a result prove to flow through there is no anti-HBs in peak, all anti-HBs are in eluent
In, thus preliminary proof adds excessive antigenic compound in eluent, without in flowing through peak.Add excessive antigenic compound solution
Flow through 1 position of peak (retention time 14.62 minutes) and simple HBsAg flows through peak position (retention time 14.08 minutes) unanimously,
Illustrate that flowing through peak 1 is the free HBsAg added in excessive antigenic compound；It detects this and flows through the HBsAg antigenic content in liquid, i.e.,
It can primarily determine the content to add the HBsAg that dissociates in excessive antigenic compound.Peak position is flowed through when flowing through peak 2 and independent HBIG chromatography
It sets consistent.Peak 3 is eluting peak.
Embodiment 5. " free HBsAg content " detection method repeatability verifying
Same batch of sample is in situation identical with Examples 1 and 2, and for three days on end, measurement 1 time, as a result as follows daily.
Table 1 is repeated (n=3)
Note: CV approving standard is≤30%
Embodiment 6. " free HBsAg content " detection method Intermediate precision verifying
Different personnel measure precision: three personnel detect same batch of sample according to the method for Examples 1 and 2,
As a result as follows.
2 same batches of samples of table, three personnel's testing results (n=3)
Note: CV approving standard is≤30%
Same personnel according to Examples 1 and 2 method to three crowdes of HBsAg in different time with identical preparing process system
Standby plus excessive antigenic compound carries out repeating to detect three times, as a result as follows.
3 Intermediate precision of table (n=3)
Note: CV approving standard is≤30%
Embodiment 7.CaptoTMHeparin chromatography
AKTApurifier protein chromatography system, XK16/20 chromatographic column, CaptoTMHeparin chromatography media 5-10mL；
Combination buffer (A): 10mM PB 20mM NaCl；Elution buffer (B): 10mM PB 2M NaCl；It is collected and is flowed according to A280
Wear peak and eluting peak, specific detection process is as follows: " adding excessive antigenic compound ", (HBsAg total amount was 120 μ g/mL, HBIG total amounts
For 37.14mg/mL) loading volume 0.5mL, chromatographic flow rates 2mL/ minutes, 6 minutes after loading started to be eluted with 5% B liquid
As wash conditions, collection flows through peak 120mL, is eluted completely (elution) after 61 minutes with 100%B liquid, collects elution
Peak 20mL (100%B liquid is eluted completely).HBIG control sample (HBIG normal saline solution, concentration 37.14mg/mL),
Method flows through peak 120mL (1-61 minutes) with " adding excessive antigenic compound ", collection, collects eluting peak 20mL.
The detection of 8. protein concentration of embodiment and calculating
Detect the eluting peak and stream for adding excessive antigenic compound respectively using Lowry method low protein content assay kit
The protein content at peak and the eluting peak of HBIG control sample and the protein content for flowing through peak are worn, is examined according to kit specification
It surveys.Standard items are serially diluted, and standard concentration gradient is 20 μ g/mL, 15 μ g/mL, 8 μ g/mL, 4 μ g/mL, 2 μ g/mL, 0 μ
G/mL is detected according to kit specification.
As a result it calculates:
HBsAg/anti-HBs complex content=(excessive antigenic compound solution eluting peak protein content-HBIG is added to elute
Peak protein content)-dissociate HBsAg content=(262.6 μ g/mL-80.56 μ g/mL) -32.89 μ g/mL=149.15 μ g/mL
Free IgG content=plus excessive antigenic compound solution flows through peak protein content × flows through peak volume/1mL/1000
=283 μ g/mL × 120mL/1mL/1000=33.96mg/mL
The verifying of " HBsAg/anti-HBs compound " the detection method specificity of embodiment 9.
HBsAg control sample, HBIG control sample plus excessive antigenic compound carry out Capto respectivelyTMHeparin layers
Analysis.
(1) individually HBsAg solution chromatographs
HBsAg control sample carries out CaptoTMHeparin chromatography, collection flow through sample, 5%B liquid washing sample and
100%B liquid elution samples carry out HBsAg qualitative detection；(changed using hepatitis b virus s antigen immue quantitative detection reagent box
Learn luminescence method) respectively to sample carries out the detection of HBsAg antigenic content before loading and after elution, and calculate the HBsAg rate of recovery.
By Fig. 2 (A) as it can be seen that individually HBsAg carries out CaptoTMHeparin chromatography, after flowing through peak or the elution of 5%B liquid,
It can't detect HBsAg；After the elution of 100%B liquid, it is seen that obvious protein peak.Using chemoluminescence method respectively to before loading and eluting
Sample carries out the detection of HBsAg antigenic content afterwards, and calculates the rate of recovery.The amount 73.65 μ g, HBsAg after elution of HBsAg before loading
The rate of recovery that amount is 70.51 μ g, HBsAg is 95.74%.Illustrate that most HBsAg are adsorbed in CaptoTMHeparin chromatography
Medium needs stronger elution requirement that can just be eluted.
(2) individually HBIG solution chromatographs
HBIG control sample carries out CaptoTMHeparin chromatography, collection flow through peak and 5%B liquid eluting peak, sample are closed
And it is denoted as " flowing through peak "；100%B liquid eluting peak, is denoted as " eluting peak ".Using Lowry method respectively to before loading, flow through peak and
Eluting peak sample carries out protein concentration detection, and calculates protein recovery.
By Fig. 2 (B) as it can be seen that individually HBIG carries out CaptoTMHeparin chromatography, it is seen that flow through peak；After the elution of 5%B liquid,
It can be seen that protein peak；After the elution of 100%B liquid, it is seen that compared with little albumen peak.Peak 1,2 sample of peak merge, and are denoted as " flowing through peak "；Peak 3 is denoted as
" eluting peak ".Using Lowry method respectively to before loading, flow through peak and eluting peak sample and carry out protein concentration detection, and calculate egg
Bai Liang.
Protein content is 24330 μ g before loading, and flowing through peak protein content is 24758.4 μ g, and eluting peak protein content is 14.52 μ g.
The protein recovery of HBIG is 101.82%.Illustrate that overwhelming majority IgG is directly flowed through or eluted through 5%B liquid in HBIG,
Only extremely least a portion of IgG is by CaptoTMThe absorption of Heparin chromatography media, needs 100%B liquid to elute.
(3) plus excessive antigenic compound chromatographs
Excessive antigenic compound is added to carry out CaptoTMHeparin chromatography, collection flows through peak and 5%B liquid eluting peak, by sample
Product merge, and are denoted as " flowing through peak "；100%B liquid eluting peak, is denoted as " eluting peak ".The qualitative inspection of HBsAg is carried out respectively to peak is flowed through
It surveys, anti-HBs qualitative detection；Using Lowry method respectively to before adding excessive antigenic compound loading, flow through peak and eluting peak sample
Product carry out protein concentration detection, and calculate protein recovery.
By Fig. 2 (C) as it can be seen that plus excessive antigenic compound progress CaptoTMHeparin chromatography, it is seen that flow through peak；5%B liquid
After elution, it is seen that protein peak；After the elution of 100%B liquid, it is seen that compared with little albumen peak.Peak 1,2 sample of peak merge, and are denoted as " flowing through peak "；
Peak 3 is denoted as " eluting peak ".Using Lowry method respectively to before loading, flow through peak and eluting peak sample and carry out protein concentration detection, and
Calculate protein content.Protein content is 25048 μ g before loading, and flowing through peak protein content is 26464.8 μ g, and eluting peak protein content is 95.76 μ
g.The protein recovery for adding excessive antigenic compound is 106%.Analyzed in conjunction with Fig. 2A, 2B, 2C: peak 1, peak 2 are free
The peak IgG, peak 3 are mainly in addition to this HBsAg/anti-HBs compound and free HBsAg should also contain minimal amount of
The IgG of non-specific adsorption.Therefore when carrying out the calculating of HBsAg/anti-HBs complex content, it should with the total albumen in peak 3
The IgG of amount (being measured by MabSelect SuRe affinity chromatography method) and non-specific adsorption that amount subtracts free HBsAg (leads to
Cross the parallel Capto for carrying out HBIGTMHeparin chromatography, detection 3 protein content of peak measure).
4 Lowry method of table detects solution protein concentration
The verifying of " HBsAg/anti-HBs compound " the detection method repeatability of embodiment 10.
Same batch of sample under identical circumstances, according to the method for embodiment 7 and 8, for three days on end, tie by measurement 1 time daily
Fruit is as follows.
The repeated result of table 5
Note: CV approving standard is≤30%
The verifying of " HBsAg/anti-HBs compound " the detection method precision of embodiment 11.
Different personnel measure precision: three personnel detect same batch of sample according to the method for embodiment 7 and 8,
As a result as follows.
6 same batches of samples of table, three personnel's testing results (n=3)
Note: CV approving standard is≤30%.
Three personnel's testing results of same batch of sample (HBsAg/anti-HBs complex content: μ g/mL).
7 same batches of samples of table, three personnel's testing results (n=3)
Note: CV approving standard is≤30%.
Same personnel (" add excessive anti-three batches of samples using three crowdes of HBsAg what different time was prepared with identical technique
Former compound ") it carries out repeating to detect three times, it is as a result as follows.
8 Intermediate precision of table (free IgG content: mg/mL, n=3)
Note: CV approving standard is≤15%.
9 Intermediate precision of table (HBsAg/anti-HBs complex content: μ g/mL, n=3)
Note: CV approving standard is≤30%
Embodiment 12. " HBsAg/anti-HBs compound " detection method Accuracy Verification
Without " adding excessive antigenic compound standard substance " (i.e. vaccinogen liquid standard items), it is immune to choose a batch
Complex hepatitis B vaccine stoste (" adds excessive antigenic compound "), and the HBsAg that high, medium and low three kinds of concentration are added is molten
Liquid carries out Capto according to embodiment 7 to acquired solutionTMHeparin affinity chromatography passes through the inspection to the HBsAg rate of recovery is added
It surveys, to investigate the accuracy of this method.
Specifically, to be divided into volume 6 parts identical for the solution that " will add excessive antigenic compound ", wherein 3 parts of additions it is high, in,
Low different amounts of HBsAg sample is made HBsAg and 3 different recycling samples of concentration is added；" add excessive antigen compound at another 3 parts
3 basic samples are made in the corresponding physiological saline that same volume is added in object " solution.3 parts of recycling samples and corresponding basis
Sample uses CaptoTMHeparin medium carries out affinity chromatography, collects eluting peak (respectively detecting 1, detection 2, detection 3).Together
When set HBIG control, equally operated.Protein concentration detection is carried out to eluting peak solution and HBsAg stoste using Lowry method.
Basic concentration of specimens is basic sample through CaptoTMThe protein concentration of eluting peak after Heparin chromatography.
Recycling concentration of specimens is recycling sample through CaptoTMAfter Heparin chromatography, the protein concentration of eluting peak.
Recycle HBsAg/anti-HBs complex content and HBsAg protein content=recycling concentration of specimens × elution in sample
Peak volume/loading volume-HBIG elutes Cmax/loading volume；
HBsAg/anti-HBs complex content and HBsAg protein content=basis concentration of specimens × elution in basic sample
Peak volume/loading volume-HBIG elutes Cmax/loading volume；
Sample and basic sample are recycled the difference is that the former joined part HBsAg, so the difference between the two recycles again
HBsAg concentration, that is, the actually detected amount arrived of the HBsAg being added.
The concentration that HBsAg sample of the HBsAg concentration to be added in " adding excessive antigenic compound " is added (is added
HBsAg concentration theoretical value).
Rate of recovery %=recycles HBsAg concentration/addition HBsAg concentration × 100%
The various concentration HBsAg rate of recovery is added in table 10
The result shows that: utilize CaptoTMHeparin affinity chromatography method, high, medium and low various concentration HBsAg addition " add
The rate of recovery of HBsAg is respectively 94.68%, 95.49%, 81.37% in excessive antigenic compound " solution, illustrates this method
Accuracy is good.
Claims (13)
1. a kind of method of the amount of effective component in detection HBsAg/anti-HBs complex systems, comprising:
1) HBsAg/anti-HBs complex systems are provided, which includes HBsAg/anti-HBs compound, free IgG and trip
From HBsAg；The effective component is the free HBsAg and the HBsAg/anti-HBs compound；
2) affinity chromatography is carried out to the HBsAg/anti-HBs complex systems, wherein the medium of the affinity chromatography can be inhaled
Attached HBsAg/anti-HBs compound and free IgG；It collects and flows through peak a containing free HBsAg；
3) it provides referring to HBsAg solution, it is described to contain and the HBsAg/anti-HBs complex systems referring to HBsAg solution
The identical HBsAg of HBsAg total amount；The affinity chromatography is carried out referring to HBsAg solution to described, collects flowing through containing HBsAg
Peak b；
4) specifically detection flows through the amount a of free HBsAg in peak a, and flows through the amount b of the HBsAg in peak b, dosage a,
The HBsAg total amount in b and the HBsAg/anti-HBs complex systems is measured, it is compound to calculate the HBsAg/anti-HBs
The amount of free HBsAg in objects system；
5) affinity chromatography is carried out to the HBsAg/anti-HBs complex systems, wherein the medium of the affinity chromatography can be inhaled
Attached HBsAg/anti-HBs compound and free HBsAg；By the HBsAg/anti-HBs compound being adsorbed on chromatography media and
Free HBsAg is eluted, and collects the eluting peak a containing the HBsAg/anti-HBs compound and the free HBsAg；
6) protein content in the eluting peak a, the HBsAg/anti-HBs composite body obtained by the protein content and step 4) are detected
The amount of free HBsAg in system calculates the HBsAg/anti-HBs compound in the HBsAg/anti-HBs complex systems
Amount.
2. according to the method described in claim 1, wherein the HBsAg/anti-HBs complex systems are immune complex type
Hepatitis B vaccine stoste.
3. according to the method described in claim 1, wherein step 2) and 3) described in affinity chromatography be protein A affinity chromatography.
4. according to the method described in claim 1, wherein affinity chromatography described in step 5) is heparin affinity chromatography.
5. according to the method described in claim 1, specifically detecting the amount using immunological method wherein in step 4)
The a and amount b.
6. according to the method described in claim 1, wherein in step 4), the ratio of calculation amount a proportion b, and calculate the ratio
With the product of the HBsAg total amount in the HBsAg/anti-HBs complex systems, the HBsAg/anti-HBs is thus obtained
The amount of free HBsAg in complex systems.
7. according to the method described in claim 1, subtracting step with the protein content in the eluting peak a wherein in step 6)
4) amount of the free HBsAg in gained HBsAg/anti-HBs complex systems, it is multiple thus to obtain the HBsAg/anti-HBs
Close the amount of the HBsAg/anti-HBs compound in objects system.
8. according to the method described in claim 1, wherein elution described in step 5) is using phosphate buffer as eluent.
9. according to the method described in claim 1, wherein the method also includes i) providing referring to HBIG solution, reference HBIG
Solution contains IgG identical with the IgG total amount of the HBsAg/anti-HBs complex systems；Reference HBIG solution is carried out
Affinity chromatography described in step 5) will be washed referring to non-specific adsorption in HBIG solution in the IgG on the medium of the affinity chromatography
It takes off, collects the eluting peak b containing the IgG, detect the protein content in eluting peak b；And
Wherein in step 6), with the protein content in the eluting peak b, the protein content in the eluting peak a and step 4) institute
The amount for obtaining the free HBsAg in HBsAg/anti-HBs complex systems, calculates the HBsAg/anti-HBs complex systems
In HBsAg/anti-HBs compound amount.
10. according to the method described in claim 9, wherein being subtracted with the protein content in the eluting peak a: in the eluting peak b
Protein content and the step 4) obtained by free HBsAg in HBsAg/anti-HBs complex systems amount, thus obtain
The amount of HBsAg/anti-HBs compound in the HBsAg/anti-HBs complex systems.
11. according to the method described in claim 9, being wherein eluted in described in step i) identical with the elution described in step 5)
Under the conditions of carry out.
12. it further include collecting to flow through peak c containing free IgG according to the method described in claim 1, wherein in step 5),
It detects this and flows through the protein content in peak c, to obtain the amount of the free IgG in the HBsAg/anti-HBs complex systems.
13. method described in any one of -12 is controlled in the quality of immune complex type hepatitis B vaccine according to claim 1
In application.
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