CN104650237B - It a kind of cross-linking antibody and its is applied in immune detection - Google Patents
It a kind of cross-linking antibody and its is applied in immune detection Download PDFInfo
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Abstract
The present invention provides a kind of cross-linking antibody, the antibody includes being formed by the tetramer by the human antibody for being uninfected by TORCH pathogen of inhuman TORCH pathogen antigen and health, and the present invention also provides cross-linking antibodies as substituting application of the quality-control product of patient's positive blood in immunity detection reagent.The heterologous cross-linking agent production prepares simple, at low cost, while having extraordinary safety and stability, can effectively reduce detection error.
Description
The application is point application No. is the Chinese patent application on 28 days June 2013 201310267573.6, applying date
Case application.
Technical field
The present invention relates to a kind of heterologous cross-linking agents, specifically exactly a kind of to pass through cross-linking reaction by two kinds of heterologous antibody
And the heterologous cross-linking agent of the heterologous cross-linking antibody containing tetramer obtained, and its pathogen is anti-in patient's positive blood
Application in body substitute, belongs to immunological technique field.
Background technique
Nowadays, such as pathogen of virus, bacterium, fungi and helminth etc threatens increasing to mankind's bring.
Immunoassay method based on specificity between antigen and antibody and high sensitivity reaction, such as colloidal gold immunochromatographimethod detection, cream
The detection of glue immunochromatography, the detection of Placenta function (RIA), MBP enzyme linked immuno-adsorbent assay (ELISA), time-resolved fluoroimmunoassay
(TRFIA) or chemiluminescence immunoassay detects (CLIA), has been widely used among clinical immunization test.These immunoassays
Method is usually used to the pathogen antigen or antibody qualitatively or quantitatively detected in human blood, to quickly and correctly determine
Whether infection occurs and/or infects the locating stage.
Clinical immunization detection kit generally all includes negative quality-control product and positive quality control product.These quality-control products often from
The higher patient's positive blood of antibody titer is serially diluted product, or directly uses patient's positive blood of different antibodies potency
Liquid, including serum, blood plasma or whole blood.But had disadvantages that using patient's positive blood preparation positive quality control product: 1) being difficult to obtain
Obtain patient's positive blood simultaneously with high-titer and high specific；2) individual between antibody titer and specific difference compared with
Greatly, it is difficult to standardize；3) cost is relatively high, 4) some pathogenic infection cases are rare, are difficult to obtain in a short time enough
Positive blood；5) pathogen positive blood has potential infectiousness, even if by inactivation treatment, but still be difficult to ensure thoroughly
Inactivation, while when collecting and inactivating patient's positive blood, operator also has potential infected risk.For this purpose, people pass through
It is often used the quality-control product of substitution patient's positive blood.
0 330 902 A2 of European patent application EP is disclosed when detecting human blood specific antibody, using a kind of or
A variety of human monoclonal antibodies (such as people's rubella virus monoclonal antibody and people's varicella zoster virus monoclonal antibody) are made
It is compareed for test, but the human monoclonal antibodies constructed every time only include a kind of constant region of antibody, detect other Antibody types
It needs to rebuild, and due to not allowing ethically to carry out Antibody preparation using human body, thus has to utilize bacteriophage
The methods of display technique and transgenic animals manufacture human monoclonal antibodies, and that this allows for Antibody preparation is relatively difficult.The U.S. is special
Sharp US 6,015,662, which discloses human mouse chimeric antibody, can be used for the positive control of human blood antibody test, but building every time
People's mouse chimeric mAb only include the constant region of antibody a kind of, detect other Antibody types needs and rebuild, and
Technique is extremely complex, this is because also to carry out antibody base other than obtaining mouse monoclonal antibody using hybridoma technology
Because of the building and expression of clone and people's mouse mosaic gene.
At present in addition to the above method prepares antibody, has also appeared and prepare antibody using crosslinking agent.There are many kinds of utilize friendship
The method that connection agent prepares cross-linking antibody.Such as crosslinking agent succinimide pyridine dimercapto propyl ester [N-Succinimidyl 3- (2-
Pyridyldithio) propionate, SPDP] it can be used to for two different albumen (including antibody and enzyme) being crosslinked one
It rises and forms cross-linking agent (Carlsson J, Drevin H, and Ax é n R.Protein thiolation and
reversible protein-protein conjugation.N-Succinimidyl 3-(2-pyridyldithio)
propionate,a new heterobifunctional reagent.Biochem.J.,1978,173:723-737).Crosslinking
Agent phenylenedimaleimide (O-phenylenedi-maleimide, O-PDM) is also used to exist the segment crosslinking of two kinds of antibody
(Glennie M, McBride H, Worth A, et al..Preparation and performance of together
bispecific F(ab'gamma)2antibody containing thioether-linked Fab'gamma
fragments.J.Immunol.,1987,139:2367-2375).0 062 227A1 of European patent EP, which is disclosed, utilizes crosslinking
Agent glutaraldehyde or sodium metaperiodate by the antibody coupling in two kinds of different plant species sources together.In addition, United States Patent (USP) US 4,507,
234 disclose crosslinking agent dithio-bis-nitrobenzoic acid [5,5'-dithiobis- (2-nitrobenzoicacid), DTNB] can
It is used to prepare cross-linking antibody.United States Patent (USP) US 4,929,543 discloses crosslinking aid S-acetyl mercapto succinic anhydride (S-
Acetylmercaptosuccinic anhydride, AMSA) and maleimidocaproyl-N-hydroxy-succinamide ester
(Maleimidohexanoyl-N-hydroxysuccinimide ester, MHS) can be used to prepare cross-linking antibody.The U.S. is special
Sharp US 5,478,753 discloses crosslinking agent 4- maleimidobutyric acid-N- succinimide ester (N- γ-
Maleimidobutyryloxysuccinimide ester, GMBS) cross-linking antibody can be used to prepare.
Cross-linking antibody is also commonly used for the quality-control product of preparation substitution patient's positive blood.United States Patent (USP) US 4,929,543 takes off
Inhuman antibody or its Fab or F (ab') are shown2The heterologous cross-linking agent of segment and human immunoglobulin(HIg) or its Fc segment is used for people
Serum antibody diagnosis.United States Patent (USP) US 5,478,753 discloses the heterologous cross-linking agent of inhuman non-IgM and people IgM, is used for blood
Clear IgM diagnostic kit, the inhuman non-IgM in heterologous cross-linking agent specifically can identify and combine pathogen antigen, people IgM
It can be combined with labelled antibody.United States Patent (USP) US 5,491,218 discloses inhuman antibody or its segment and another antibody
Heterologous cross-linking agent, such as people IgM and anti-herpes simplex virus antibodies F (ab')2The heterologous cross-linking agent of segment, it is non-in heterologous cross-linking agent
A kind of specific pathogen is combined to the antibody specificity of people, combines a kind of specific antibody class to another antibody specificity
Type.Chinese patent ZL97109106.4 discloses inhuman Plasma donor or after processing with the piece of antibody activity
The heterologous cross-linking agent of section and human immunoglobulin(HIg), can be used as the substitute of antibody of AIDS virus in patient's positive blood.But
There is no the concrete compositions for disclosing the heterologous cross-linking agent prepared in the above patent, and it is different that this has resulted in heterologous cross-linking agent
Unstable quality is easy to produce detection error, alternative poor.
Summary of the invention
The purpose of the present invention is to provide a kind of heterologous crosslinkings obtained by two kinds of heterologous antibody by cross-linking reaction
Object, the heterologous cross-linking agent include the heterologous cross-linking antibody of the tetramer formed by described two heterologous antibody.It is preferred that
Ground, described two heterologous antibody are the human antibody for being uninfected by pathogen of inhuman pathogen antigen and health；Preferably, institute
The heterologous cross-linking antibody of tetramer mass ratio shared in the heterologous cross-linking agent is stated not less than 50%.
It is an object of the invention to a kind of heterologous cross-linking agent in patient's positive blood answering on pathogen antigen substitute
With, the heterologous cross-linking agent is to be obtained by two kinds of heterologous antibody by cross-linking reaction, the heterologous cross-linking agent include by
The heterologous cross-linking antibody for the tetramer that described two heterologous antibody are formed.Preferably, described two heterologous antibody are
The human antibody for being uninfected by pathogen of inhuman pathogen antigen and health；Preferably, the heterologous crosslinking of the tetramer
Antibody mass ratio shared in the heterologous cross-linking agent is not less than 50%.
It is another object of the present invention to provide a kind of quality-control product for substituting patient's positive blood, the quality-control product includes
Dilution and a kind of heterologous cross-linking agent obtained by two kinds of heterologous antibody by cross-linking reaction, the heterologous cross-linking agent include
By the heterologous cross-linking antibody for the tetramer that described two heterologous antibody are formed, the dilution can be selected from calf serum or
Fetal calf serum, preferably calf serum, in the quality-control product, the heterologous cross-linking agent concentration is 5~25 μ g/ml, preferably 20 μ
g/ml.Preferably, described two heterologous antibody are the human antibody for being uninfected by pathogen of inhuman pathogen antigen and health；
Preferably, the heterologous cross-linking antibody of tetramer mass ratio shared in the heterologous cross-linking agent is not less than 50%.
Particularly, the present invention provides a kind of quality-control product for substituting patient TORCH pathogen antigen positive blood, and feature exists
Include dilution in, the quality-control product and a kind of TORCH pathogen is uninfected by by inhuman TORCH pathogen antigen and health
Human antibody by cross-linking reaction heterologous cross-linking agent obtained, the heterologous cross-linking agent includes by the inhuman TORCH disease
The heterologous cross-linking antibody for the tetramer that the human antibody for being uninfected by TORCH pathogen of mycoplasma antibody and the health is formed,
The dilution can be selected from calf serum or fetal calf serum, preferably calf serum, in the quality-control product, the heterologous crosslinking
Object concentration is 5~25 μ g/ml, preferably 20 μ g/ml.Preferably, the heterologous cross-linking antibody of the tetramer is described heterologous
Shared mass ratio is not less than 50% in cross-linking agent.
Compared with prior art, the present invention is obtained has the beneficial effect that
Firstly, heterologous cross-linking agent of the invention contains the heterologous cross-linking antibody of tetramer.The heterologous cross-linking agent is made
For the substitute of pathogen antigen in patient's positive blood, in the stability test after being saved one month at -20 DEG C and 4 DEG C,
The heterologous cross-linking agent all has extraordinary stability.
Secondly, the substitute of heterologous cross-linking agent of the invention as pathogen antigen in patient's positive blood, it can be fine
Ground substitutes the pathogen antigen in patient's positive blood.
Again, the people for being uninfected by pathogen of heterologous cross-linking agent of the invention from inhuman pathogen antigen and health
Antibody can be used as the quality-control product of substitution patient's positive blood, this quality-control product and patient's positive blood after diluted
Performance is suitable, and is avoided that the potentially infectious of patient's positive blood, while this heterologous cross-linking agent has production preparation simple
With feature at low cost.
Also, quality-control product difference between batch of the invention is smaller, can be effectively reduced detection error.
Specific embodiment
The present invention using such as SPDP, GMBS and the crosslinking agent of DTNB etc by two kinds it is heterologous it is antibody linked together with,
To form the heterologous cross-linking agent including the heterologous cross-linking antibody of tetramer.Present invention discover that working as the heterologous crosslinking
When object contains the heterologous cross-linking antibody of tetramer, the heterologous cross-linking agent can be used as pathogen antigen in patient's positive blood
Substitute, after diluted as substitution patient's positive blood quality-control product, be used for immune detection, such as colloid gold immune
Chromatography detection, the detection of latex immunochromatography, Placenta function, enzyme linked immunosorbent detection, time-resolved fluoroimmunoassay detection or change
Electrochemiluminescent immunoassay detection is learned, while there is extraordinary sensitivity, specificity and stability.The heterologous crosslinking of the tetramer
Antibody mass ratio shared in the heterologous cross-linking agent is preferably not less than 50%.
In the heterologous cross-linking antibody molecule of each tetramer, described two heterologous antibody molecule number ratios are
3:1 or 2:2 or 1:3.In structural formula 1, described two heterologous antibody molecule number ratios are 3:1.In structural formula 2 (a) or 2 (b)
In, described two heterologous antibody molecule number ratios are 2:2.In structural formula 3, described two heterologous antibody molecule number ratios are 1:
3.In these structural formulas, described two heterologous antibody are indicated with Ab1 and Ab2 respectively, and L is using crosslinking agent to described two
Heterologous antibody carries out the attachment of formation when cross-linking reaction.
Structural formula 1:
Structural formula 2 (a):
Structural formula 2 (b):
Structural formula 3:
In these structural formulas, Ab1 is preferably inhuman pathogen antigen, such as the anti-II herpes simplex virus type list of mouse
Clonal antibody, mouse anti-I type herpes simplex virus monoclonal antibody, mouse rubella virus monoclonal antibody, mouse anti human are huge
Cell virus monoclonal antibody, mouse monoclonal antibody against Toxoplasma gondii, mouse AIDS virus resisting monoclonal antibody, the anti-first of mouse
The anti-hepatitis A virus monoclonal antibody of Hepatitis virus monoclonal antibody, mouse, mouse anti-hepatitis B virus monoclonal are anti-
The anti-Hepatitis E disease of body, mouse anti-hepatitis c virus monoclonal antibody, mouse anti-hepatitis D virus monoclonal antibody, mouse
The anti-HGV RNA monoclonal antibody of malicious monoclonal antibody, mouse or the anti-other pathogens monoclonal antibody of mouse.Ab2 is preferred
For a kind of human antibody for being uninfected by pathogen of health, such as human IgG, IgA, IgD, IgE, IgM antibody monomer and its through handling
Afterwards with the antibody fragment or its mixture of intact Fc portion.L can be selected from structural formula 4 (a), and 4 (b) or 4 (c) and the neck
Other common structural formulas known to technical staff in domain.
Structural formula 4 (a):
Structural formula 4 (b):
Structural formula 4 (c):
Wherein, two kinds of heterologous antibody are preferably that a kind of inhuman pathogen antigen and a kind of health are uninfected by pathogen
Human antibody.The pathogen can be selected from AIDS virus, hepatitis A virus, hepatitis type B virus, Hepatitis C Virus, fourth
Hepatitis virus, Hepatitis E virus, HGV RNA, herpes simplex virus, rubella virus, human cytomegalovirus, people T leaching
Bar cell virus, West Nile Virus；Staphylococcus aureus, Escherichia coli, pseudomonas aeruginosa, staphylococcus epidermis, lung
Scorching klebsiella, enterococcus faecalis, streptococcus pneumonia；Toxoplasma, schizotrypanum cruzi, microspironema pallidum, plasmodium falciparum, tertian fever are former
Worm.The inhuman pathogen antigen refer to from except mankind mouse outside, rat, cavy, rabbit, chicken, pig, sheep, goat,
It is the monoclonal antibodies of the animals such as horse, mule and camel, polyclonal antibody, genetic engineering antibody, immune using the pathogen antigen
Animal antibody obtained and its or mixtures thereof the segment after processing with antigen-binding activity.
Monoclonal antibody, which refers to, only to be produced by a type of immunocyte (such as bone-marrow-derived lymphocyte) in animal body
The only immunoglobulin in conjunction with a kind of epitope come.By taking most common mouse monoclonal antibody as an example, with a kind of cause of disease
Body mice immunized with antigen isolates mouse spleen bone-marrow-derived lymphocyte, and merge with murine myeloma cell after being repeatedly immunized,
By carrying out the hybridoma for being repeatedly subcloned monoclonal antibody needed for screening is achieved with secretion to generated hybridoma
Cell (G,Milstein C.Continuous cultures of fused cells secreting antibody
of predefined specificity.Nature,1975,256:495-497；Albitar Maher.Monoclonal
Antibodies:Methods in Molecular Biology,Vol.378,2007).Then in vitro needed for culture secretion
The hybridoma of monoclonal antibody collects culture solution, or hybridoma is injected into the abdominal cavity of Syngenic mice, collects
Ascites, finally can be from culture solution or abdomen using caprylic acid-ammonium, saturated ammonium sulfate salt precipitation method or affinity column method
Monoclonal antibody (Nakazawa M, Mukumoto M, Miyatake K.Immunoelectron is isolated in water
Microscopy:Methods in Molecular Biology,2010,657:63-74；E. Lip river, D. Lay grace " antibody skill are breathed out
Art experiment guide ").
Polyclonal antibody refers to be manufactured by a plurality of types of immunocytes (such as bone-marrow-derived lymphocyte) in animal body
Can be with the immunoglobulin in conjunction with a variety of epitopes.By taking rabbit polyclonal antibody as an example, with a kind of pathogen antigen multi-time no
Epidemic disease rabbit, then collects serum, recycle caprylic acid-ammonium, saturated ammonium sulfate salt precipitation method or affinity column method or
Ion exchange chromatography isolates polyclonal antibody (Nakazawa M, Mukumoto M, Miyatake
K.Immunoelectron Microscopy:Methods in Molecular Biology,2010,657:63-74；E. it breathes out
Lip river, D. Lay grace " antibody technique experiment guide ").
Genetic engineering antibody refers to through the existing excellent non-human animal's species monoclonal antibody of genetic engineering techniques
Gene reduces animal species origin components in antibody to the greatest extent, while retaining original antibody specificity, to create a kind of novel
Antibody mainly includes chimeric antibody, humanized antibody, single-chain antibody and single domain antibody (Chames, Patrick.Antibody
Engineering:Methods in Molecular Biology,Vol.907,2nd ed.2012；Proetzel G,
Ebersbach H.Antibody Methods and Protocols:Methods in Molecular Biology,
Vol.901,2012；Saerens D,Muyldermans S.Methods in Molecular Biology,Single
Domain Antibodies:Methods and Protocols,Vol.911,2012)。
Using pathogen antigen be immunized animal antibody obtained refer to using pathogen antigen it is immune except the mankind outside
Animal monoclonal antibody obtained or polyclonal antibody.
For inhuman pathogen antigen, the segment with antigen-binding activity refers to anti-using pathogen after processing
Original is immune to be removed the animal of the mankind outside monoclonal antibody obtained or polyclonal antibody and passes through technique for gene engineering to institute
The monoclonal antibody of acquisition is transformed genetic engineering antibody obtained, obtained by chemical means or collagenase treatment
The antibody fragment for still maintaining antigen binding property, as monoclonal antibody after papain digestion generated Fab segment,
Or the F (ab') generated after pepsin digestion2, wherein F (ab')2It also can get Fab' segment after enzyme IdeS digestion again.
A kind of human antibody for being uninfected by pathogen of health is human blood (including the blood for being uninfected by pathogen from health
Clearly, blood plasma or whole blood) in extract and go out, optionally include IgG, IgA, IgD, IgE, IgM antibody monomer and its through locating
With the antibody fragment or its mixture of intact Fc portion after reason.The method that antibody is extracted from blood can be selected from octanoic acid-sulphur
Sour ammonium method, saturated ammonium sulfate salt precipitation method or affinity column method or ion exchange chromatography isolate monoclonal antibody
(Nakazawa M,Mukumoto M,Miyatake K.Immunoelectron Microscopy:Methods in
Molecular Biology,2010,657:63-74；E. Lip river, D. Lay grace " antibody technique experiment guide " are breathed out).The antibody list
Body refers to that the human antibody for cross-linking reaction should be Dan Juti, if not single aggressiveness, as the IgM antibody in human serum usually with
Pentamer form exists, then them should be allowed to become single aggressiveness (Ditzel H, Erb K, et in advance by processing
al..Preparation of antigen-binding monomeric and half-monomeric fragments
from human monoclonal IgM antibodies against colorectal cancer-associated
antigens.Human Antibodies,1993,4(2):86-93)。
To health be uninfected by the human antibody of pathogen for, refer to after processing with the antibody fragment of intact Fc portion
IgG, IgA, IgD, IgE or IgM monomer generated intact Fc portion, Huo Zhejing after papain or pepsin digestion
It crosses chemical means or collagenase treatment is obtained still with other antibody fragments of intact Fc portion.
By selecting suitable inhuman pathogen antigen, the present invention provides a kind of alternative patient AIDS virus, liver
The quality-control product of scorching virus or TORCH pathogen antigen positive blood, wherein hepatitis virus can be selected from hepatitis A virus, B-mode liver
Scorching virus, Hepatitis C Virus, Hepatitis D virus, Hepatitis E virus, HGV RNA, TORCH pathogen are selected from I type
Herpes simplex virus (HSV-I), II herpes simplex virus type (HSV-II), rubella virus (RV), human cytomegalovirus (HCMV)
With toxoplasma (TOXO).
Particularly, the present invention provides a kind of quality-control product for substituting patient TORCH pathogen antigen positive blood, the Quality Control
Product include that dilution and a kind of human antibody for being uninfected by TORCH pathogen by inhuman TORCH pathogen antigen and health are led to
Cross cross-linking reaction heterologous cross-linking agent obtained, the heterologous cross-linking agent include by the inhuman TORCH pathogen antigen and
The heterologous cross-linking antibody for the tetramer that the human antibody for being uninfected by TORCH pathogen of the health is formed, the dilution
It can be selected from calf serum or fetal calf serum, preferably calf serum, in the quality-control product, the heterologous cross-linking agent concentration is 5
~25 μ g/ml, preferably 20 μ g/ml.Preferably, the heterologous cross-linking antibody of tetramer institute in the heterologous cross-linking agent
The mass ratio accounted for is not less than 50%.
Particularly, the present invention provides a kind of quality-control product for substituting patient antibody of AIDS virus positive blood, the Quality Control
Product include that dilution and a kind of human antibody for being uninfected by AIDS virus by inhuman antibody of AIDS virus and health pass through
Cross-linking reaction heterologous cross-linking agent obtained, the heterologous cross-linking agent include by the inhuman antibody of AIDS virus and described
The heterologous cross-linking antibody for the tetramer that the human antibody for being uninfected by AIDS virus of health is formed, the dilution can be selected from
Calf serum or fetal calf serum, preferably calf serum, in the quality-control product, the heterologous cross-linking agent concentration is 5~25 μ g/
Ml, preferably 20 μ g/ml.Preferably, the heterologous cross-linking antibody of tetramer matter shared in the heterologous cross-linking agent
Amount is than being not less than 50%.
Particularly, the present invention provides a kind of quality-control product for substituting patient's hepatitis virus antibody positive blood, the quality-control product
It is anti-by being crosslinked including dilution and a kind of human antibody for being uninfected by hepatitis virus by inhuman hepatitis virus antibody and health
Answer heterologous cross-linking agent obtained, the heterologous cross-linking agent include by the inhuman hepatitis virus antibody and the health not
Infect hepatitis virus human antibody formed tetramer heterologous cross-linking antibody, the dilution can be selected from calf serum or
Fetal calf serum, preferably calf serum, in the quality-control product, the heterologous cross-linking agent concentration is 5~25 μ g/ml, preferably 20 μ
g/ml.Preferably, the heterologous cross-linking antibody of tetramer mass ratio shared in the heterologous cross-linking agent is not less than
50%.
In addition, by selecting suitable inhuman pathogen antigen, the present invention mentions according to known to a person skilled in the art
For a kind of quality-control product for substituting patient's other pathogens antibody positive blood.
When heterologous cross-linking agent of the invention is used for immune detection, the dilution that a kind of diluting effect is added usually also is needed
Liquid thus prepares substitution disease using the dilution by the concentration dilution of the heterologous cross-linking agent most 5~25 μ g/ml
The quality-control product of people's positive blood, wherein the dilution can be selected from calf serum or fetal calf serum, preferably calf serum.
Preferably, heterologous cross-linking agent of the present invention is stored in a kind of frozen stock solution at -20 DEG C, and the frozen stock solution includes
Stabilizer, glycerol, preservative.Wherein the stabilizer is selected from BSA or gelatin, preferred concentration 1%；The glycerol, preferably
50% glycerol；The preservative is selected from Sodium azide or thimerosal, preferred concentration 0.01%.It is described different in the frozen stock solution
Source cross-linking agent final concentration of 2~5mg/ml, preferably 4mg/ml.
When allowing two kinds of heterologous antibody to crosslink reaction, many crosslinking agents may be selected, as SPDP, O-PDM, DTNB,
AMSA, MHS or GMBS and other crosslinking agents known to those skilled in the art.The present invention utilizes crosslinking aid S PDP and reduction
Agent dithiothreitol (DTT) (dithiothreitol, DTT) prepares the anti-II herpes simplex virus type monoclonal antibody (anti-of mouse
HSV-II McAb) and health the human IgG for being uninfected by HSV-II heterologous cross-linking agent, mouse anti-I type herpes simplex virus Dan Ke
Heterologous cross-linking agent, the mouse rubella virus of the human IgG for being uninfected by HSV-I of grand antibody (anti-HSV-I McAb) and health
The heterologous cross-linking agent of the human IgG for being uninfected by RV of monoclonal antibody (anti-RV McAb) and health, mouse anti human giant cell disease
The heterologous cross-linking agent and the anti-bow of mouse of the human IgG for being uninfected by HCMV of malicious monoclonal antibody (anti-HCMV McAb) and health
The heterologous cross-linking agent of the human IgG for being uninfected by TOXO of shape worm monoclonal antibody (anti-TOXO McAb) and health.Similarly, sharp
With SPDP crosslinking agent and DTT can also prepare other inhuman pathogen antigens and health the human antibody for being uninfected by pathogen it
Between heterologous cross-linking agent.
Embodiment 1
Preparation mouse (is bought from Thermo Scientific company, product number 21857) using SPDP crosslinking agent
The heterologous cross-linking agent of anti-HSV-II McAb and the human IgG for being uninfected by HSV-II of health is as HSV- in patient's positive blood
II antibody substitute, process are as follows:
(1) by 5mg mouse anti-HSV-II McAb be dissolved in cross-linking buffer (0.1M potassium phosphate, 0.1M NaCl,
PH7.5 in), 50 μ l SPDP crosslinking agents (3.2mg/ml is dissolved in dehydrated alcohol) are added in stirring, react 2 hours at room temperature
Afterwards, it is packed into bag filter, with reduction buffer (0.1M sodium acetate, 0.1M NaCl, pH4.5) dialysis, is changed liquid four times；
(2) human IgG for being uninfected by HSV-II of 5mg health is dissolved in cross-linking buffer, 50 μ l SPDP are added in stirring
Crosslinking agent after reacting 2 hours at room temperature, is packed into bag filter, is dialysed, changed liquid four times with cross-linking buffer；
(3) 30 μ l DTT (1M is dissolved in 0.01M sodium acetate solution, pH5.2) is added in (1), reacts at room temperature 30 minutes
Afterwards, it is packed into bag filter, is dialysed, is changed liquid four times with cross-linking buffer；
(4) processed material in (2), (3) is mixed, 15 hours is reacted at 4 DEG C；
(5) 0.4mg iodoacetamide sealer is added into (4), reacts 30 minutes to close unreacted active group, so
After be packed into bag filter, dialysed, changed liquid four times with cross-linking buffer；
(6) product in (5) HiLoad 16/60Superdex 200PG solvent resistant column is loaded into (to buy from GE
Healthcare it in), is eluted with cross-linking buffer, and is existed using HD-3 purple light analyzer real-time detection eluted product
Light absorption value when 280nm wavelength, while chromatography map is drawn with the small-sized recorder of XWT-S, eluted product is collected by peak；
(7) the heterologous cross-linking agent for obtaining the heterologous cross-linking antibody containing tetramer, as in patient's positive blood
The substitute of HSV-II antibody.Wherein the heterologous cross-linking antibody molecule of each tetramer refers to 3 using SPDP and DTT
Mouse anti-HSV-II McAb molecule and 1 healthy the human IgG molecule for being uninfected by HSV-II or 2 mouse anti-
HSV-II McAb molecule and 2 healthy human IgG molecules for being uninfected by HSV-II or 1 mouse anti-HSV-II McAb
Molecule is formed by together with 3 healthy human IgG molecule cross-links for being uninfected by HSV-II.It is being formed by each tetramer
In the heterologous cross-linking antibody molecule of form, the Ab1 corresponding to structural formula 1,2 and 3 is that mouse anti-HSV-II McAb, Ab2 are
Shown in structural formula 4 (a), i.e., the human IgG for being uninfected by HSV-II of health, L are
The heterologous cross-linking agent of 2 mouse anti-HSV-II McAb of embodiment and the human IgG for being uninfected by HSV-II of health replaces
Dai Xing, quality-control product stability and freeze stability experiment
By the mouse anti-HSV-II McAb prepared in embodiment 1 and the human IgG for being uninfected by HSV-II of health
Substitute of the heterologous cross-linking agent as HSV-II antibody in patient's positive blood carries out experiment.
(1) quality-control product is prepared.The heterologous cross-linking antibody of tetramer is successively made in prepared heterologous crosslinking
Heterologous cross-linking agent of the shared mass ratio at 50%, 70%, 90% and 100% is as HSV-II in patient's positive blood in object
The substitute of antibody.When the heterologous cross-linking antibody of tetramer mass ratio shared in prepared heterologous cross-linking agent is
50%, 70% or 90% when, the heterologous cross-linking agent of residue 50%, 30% or 10% include by mouse anti-HSV-II McAb and
The heterologous cross-linking antibody for the dimeric forms that human IgG is formed and/or the heterologous cross-linking antibody of trimeric form, but do not include not sending out
The mouse anti-HSV-II McAb of raw crosslinking and the human IgG for being uninfected by HSV-II of health.It is slow using the crosslinking in embodiment 1
Fliud flushing is diluted, and making the initial concentration of these heterologous cross-linking agents is 0.5mg/ml, and dilute using 20 times of calf serum progress
Release the quality-control product that substitution patient HSV-II antibody positive blood is just prepared to 25 μ g/ml.
(2) it is detected using indirect elisa method, detailed process is as follows:
(1) it is coated with: (1.59g sodium carbonate, 2.93g sodium bicarbonate being dissolved in 1000ml distilled water, together with coating buffer
When pH is adjusted to 9.6, be configured to coating buffer) dilution II herpes simplex virus type antigen so that antigen concentration is 1 μ g/ml；
Antigenic solution after 100 μ l dilution is added covers ELISA Plate with plastic film into each hole of ELISA Plate, overnight at 4 DEG C, secondary
Day, discard solution in hole；200 μ l washing solution (0.5ml Tween 20 is added in 1000ml coating buffer) is added to wash
ELISA Plate is washed, is repeated 3 times；
(2) it closes: 200 μ l lock solutions (1g BSA is dissolved in 100ml washing solution) is added into ELISA Plate hole, and
ELISA Plate is covered with plastic film, after 37 DEG C are incubated for 2 hours, discards solution in hole；200 μ l washing solution detersive enzyme mark is added
Plate is repeated 3 times；
(3) quality-control product is added: be successively added in hole different on ELISA Plate in (one) the 100 μ l of quality-control product for preparing with
And 100 μ l of patient's positive blood standard items is added in two adjacent holes wherein every special quality control product should repeat to be added once,
Every hole is 100 μ l, and covers ELISA Plate with plastic film, after 37 DEG C are incubated for 30 minutes, discards solution in hole, 200 μ are added
L washs solution and washs ELISA Plate, is repeated 5 times；
(4) ELIAS secondary antibody is added: 100 μ of ELIAS secondary antibody (goat anti-human igg of horseradish peroxidase-labeled) working solution is added
L covers ELISA Plate with plastic film into each hole of ELISA Plate, after 37 DEG C are incubated for 30 minutes, discards solution in hole, is added
200 μ l wash solution and wash ELISA Plate, are repeated 5 times；
(5) it detects: developing solution A (35.8g/L containing citric acid, Na is added2HPO4·12H20 9.34g/L, 30%H2O2 660
μ l) and developing solution B (containing 3,3,5,5- tetramethyl benzidine 0.2g/L, dimethyl sulfoxide 5ml/L, 6N HCl 1ml/L) each 50 μ
L, and ELISA Plate is covered with plastic film, it is incubated at 37 DEG C after ten minutes, stop bath (2N H is added2SO4) 50 μ l, with enzyme mark
Instrument reads the absorbance value (A450/A630) when 450nm and 630nm wavelength, and is corrected, i.e., A450 value subtracts A630 value
(OD450-630).Experimental result is as shown in table 1.
Patient's positive blood standard items refer to patient's positive serum of collection by multiplex screening, containing interference object
The serum of matter is rejected, to avoid the occurrence of false positive, is mainly used for the validity, stability and comparativity of evaluation experimental result.
To enable prepared quality-control product to substitute patient's HSV-II antibody positive blood, proposed standard in the industry:
Patient's positive blood standard items and prepared quality-control product are measured using the indirect elisa method in the present embodiment under the same terms
OD450-630> 0.8, most preferably 2.0.As shown in Table 1, when the heterologous cross-linking antibody of tetramer is prepared heterologous
When shared mass ratio is respectively 50%, 70%, 90% and 100% in cross-linking agent, prepared quality-control product OD450-630It is all remote
Greater than 0.8, meet proposed standard in the industry, compared with patient's positive blood standard items, performance is quite even better, therefore alternative
Patient's positive blood standard items.When the heterologous cross-linking antibody of tetramer matter shared in prepared heterologous cross-linking agent
Amount than it is higher when, OD450-630It is higher, this quality-control product it is alternative better, and work as prepared quality-control product OD450-630>
When 2.0, this indicates that prepared quality-control product should also further dilute, and is examining every time to reduce prepared quality-control product
Dosage when survey.In addition, every special quality control product are to be diluted using calf serum by prepared heterologous cross-linking agent and generated
, this indicates that the heterologous cross-linking agent of the heterologous cross-linking antibody comprising tetramer is anti-as HSV-II in patient's positive blood
Body substitute can substitute the HSV-II antibody in patient's positive blood well.
The alternative experiment of heterologous cross-linking agent of 1. mouse anti-HSV-II McAb of table and human IgG
Select the heterologous cross-linking antibody of tetramer matter shared in prepared heterologous cross-linking agent in embodiment 1
Amount is than the heterologous cross-linking agent for 90% as HSV-II antibody substitute in patient's positive blood, and the heterologous cross-linking agent is through calf
Serum dilutes 20 times and prepares quality-control product to 25 μ g/ml, therefrom selects the quality-control product of three different batches as patient's positive
Blood surrogate carries out experiment, and to assess the difference between different batches quality-control product, still selection indirect ELISA method is carried out real
It tests, the results are shown in Table 2.
2. quality-control product difference between batch of table assesses table
CV=1.09% between criticizing is calculated by the data in table 2, is much smaller than 5%, it is known that the substitution patient of different batches is positive
Difference between the quality-control product of blood is very small, illustrate quality-control product of the invention have extraordinary batch between stability with it is consistent
Property, therefore can be effectively reduced because of detection error caused by differences between batches.
By shared mass ratio in the heterologous cross-linking antibody of tetramer in embodiment 1 prepared heterologous cross-linking agent
Heterologous cross-linking agent when being 50%, 70%, 90% and 100% is all diluted to 0.5mg/ using the cross-linking buffer in embodiment 1
Ml, the heterologous cross-linking agent solution after taking 0.5ml to dilute, is put in 1ml centrifuge tube, saves one month at -20 DEG C and 4 DEG C respectively
Afterwards, 100 times are diluted with cross-linking buffer, the stability of these heterologous cross-linking agents is still detected using indirect elisa method.Experiment knot
Fruit shows the heterologous cross-linking agent for containing the heterologous cross-linking antibody of tetramer prepared by the present invention as patient's positive blood
HSV-II antibody substitute in liquid has extraordinary stability.
In addition, shared in the heterologous cross-linking agent prepared in embodiment 1 with the heterologous cross-linking antibody of tetramer
It carries out freezing stability test for when mass ratio 90%.The heterologous cross-linking agent of three different batches is added to appropriate jelly
To final concentration be 5mg/ml in liquid storage, wherein this frozen stock solution include 50% glycerol, 1% BSA and 0.01% thimerosal,
Then it takes 0.1ml that the frozen stock solution of the heterologous cross-linking agent is added respectively, is fitted into the centrifuge tube of three different 0.5ml, by this
Three centrifuge tubes are placed on save 4 months, 4 months in -20 DEG C of refrigerator after take out these three centrifuge tubes, will be every after freeze thawing
Solution in a centrifuge tube is diluted to 25 μ g/ml with cross-linking buffer, is still detected using indirect elisa method, as a result such as 3 institute of table
Show:
Stability experiment is frozen at -20 DEG C of heterologous cross-linking agent of 3. mouse anti-HSV-II McAb of table and human IgG
By the data in table 3 it is found that data (referring to table 2) phase before being frozen with what is obtained under same testing conditions
Than the detection potency of the heterologous cross-linking agent of these three different batches is dropped without significant after saving 4 months at -20 DEG C in frozen stock solution
It is low, therefore it is as HSV-II antibody substitute in patient's positive blood, freezes stability with extraordinary.
The heterologous cross-linking agent of 3 mouse anti-HSV-I McAb of embodiment and the human IgG for being uninfected by HSV-I of health substitution
Property experiment
The heterologous of the human IgG for being uninfected by HSV-I of mouse anti-HSV-I McAb and health is prepared using SPDP crosslinking agent
Cross-linking agent is as HSV-I antibody substitute in patient's positive blood, in addition to replacing mouse with mouse anti-HSV-I McAb
Except anti-HSV-II McAb, detailed process is as in embodiment 1.
The mouse anti-HSV-I McAb prepared using embodiment 3 is different with the human IgG for being uninfected by HSV-I of health
Source cross-linking agent carries out experiment as the substitute of HSV-I antibody in patient's positive blood.Successively make the different of tetramer
Cross-linking antibody mass ratio shared in prepared heterologous cross-linking agent in source is heterologous at 50%, 70%, 90% and 100%
Cross-linking agent.When the heterologous cross-linking antibody of tetramer mass ratio shared in prepared heterologous cross-linking agent is 50%,
When 70% or 90%, the heterologous cross-linking agent of residue 50%, 30% or 10% includes by mouse anti-HSV-I McAb and health
The heterologous crosslinking for being uninfected by the heterologous cross-linking antibody and/or trimeric form of the dimeric forms of the human IgG formation of HSV-I is anti-
Body, but do not include the human IgG for being uninfected by HSV-I of the mouse anti-HSV-I McAb not crosslinked and health.Utilize implementation
In example 1 cross-linking buffer dilution, making the initial concentration of these heterologous cross-linking agents is 0.5mg/ml, and using calf serum into
Row 100 is diluted to 5 μ g/ml again and prepares quality-control product, is then detected (specific steps ginseng using indirect elisa method
According to embodiment 2).Experimental result is as shown in table 4.
To enable prepared quality-control product to substitute patient's HSV-I antibody positive blood standards product, recommend mark in the industry
It is quasi-: to measure patient's positive blood standard items and prepared matter using the indirect elisa method in embodiment 2 under the same conditions
The OD of control product450-630> 1.5, most preferably 2.0~2.5.As shown in Table 4, when the heterologous cross-linking antibody of tetramer is made
When shared mass ratio is respectively 50%, 70%, 90% and 100% in standby heterologous cross-linking agent out, prepared quality-control product
OD450-6301.5 are all much larger than, meets proposed standard in the industry, compared with patient's positive blood standard items, performance is quite even more
It is good, therefore alternative patient's positive blood standard items.When the heterologous cross-linking antibody of tetramer is in prepared heterologous friendship
When mass ratio shared by joining in object is higher, OD450-630It is higher, this quality-control product it is alternative better, and when prepared
Quality-control product OD450-630When > 2.5, this indicates that prepared quality-control product should also further dilute, thus prepared by reducing
Quality-control product dosage at each test.In addition, every special quality control product are to utilize small ox blood by prepared heterologous cross-linking agent
It is diluted and generates clearly, this indicates that the heterologous cross-linking agent of the heterologous cross-linking antibody comprising tetramer as patient's sun
HSV-I antibody substitute in property blood, can substitute the HSV-I antibody in patient's positive blood well.
The alternative experiment of heterologous cross-linking agent of 4. mouse anti-HSV-I McAb of table and human IgG
By shared mass ratio in the heterologous cross-linking antibody of tetramer in embodiment 3 prepared heterologous cross-linking agent
Heterologous cross-linking agent when being 50%, 70%, 90% and 100% is all diluted to 0.5mg/ using the cross-linking buffer in embodiment 1
Ml, the heterologous cross-linking agent solution after taking 0.5ml to dilute, is put in 1ml centrifuge tube, saves one month at -20 DEG C and 4 DEG C respectively
Afterwards, 100 times are diluted with cross-linking buffer, the stability of these heterologous cross-linking agents is still detected using indirect elisa method.Experiment knot
Fruit shows the heterologous cross-linking agent for containing the heterologous cross-linking antibody of tetramer prepared by the present invention as patient's positive blood
HSV-I antibody substitute in liquid has extraordinary stability.
The alternative experiment of heterologous cross-linking agent of 4 mouse anti-RV McAb of embodiment and the human IgG for being uninfected by RV of health
Utilize the heterologous cross-linking agent of SPDP crosslinking agent preparation mouse anti-RV McAb and the human IgG for being uninfected by RV of health
As RV antibody substitute in patient's positive blood, in addition to replacing mouse anti-HSV-II McAb with mouse anti-RV McAb
Except, detailed process is as in embodiment 1.
The heterologous crosslinking of the human IgG for being uninfected by RV of the mouse anti-RV McAb and health that are prepared using embodiment 4
Object carries out experiment as the substitute of RV antibody in patient's positive blood.Successively make the heterologous cross-linking antibody of tetramer
Heterologous cross-linking agent of the shared mass ratio at 50%, 70%, 90% and 100% in prepared heterologous cross-linking agent.When
The heterologous cross-linking antibody of tetramer mass ratio shared in prepared heterologous cross-linking agent is 50%, 70% or 90%
When, the heterologous cross-linking agent of residue 50%, 30% or 10% includes by the people for being uninfected by RV of mouse anti-RV McAb and health
The heterologous cross-linking antibody for the dimeric forms that IgG is formed and/or the heterologous cross-linking antibody of trimeric form, but do not include not occurring
The mouse anti-RV McAb of crosslinking and the human IgG for being uninfected by RV of health.It is carried out using the cross-linking buffer in embodiment 1 dilute
It releases, making the initial concentration of these heterologous cross-linking agents is 0.5mg/ml, and carries out 20 times using calf serum and be diluted to 25 μ g/ml
And quality-control product is prepared, then (specific steps are referring to embodiment 2) is detected using indirect elisa method.Experimental result such as table 5
It is shown.
To enable prepared quality-control product to substitute patient's RV antibody positive blood standards product, proposed standard in the industry:
Patient's positive blood standard items and prepared Quality Control are measured using the indirect elisa method in embodiment 2 under the same conditions
The OD of product450-630> 2.0, most preferably 2.75.As shown in Table 5, when the heterologous cross-linking antibody of tetramer is prepared
When shared mass ratio is respectively 50%, 70%, 90% and 100% in heterologous cross-linking agent, prepared quality-control product OD450-630
2.0 are all much larger than, meets proposed standard in the industry, compared with patient's positive blood standard items, performance is quite even better, therefore can
Substitute patient's positive blood standard items.When the heterologous cross-linking antibody of tetramer is shared in prepared heterologous cross-linking agent
Mass ratio it is higher when, OD450-630It is higher, this quality-control product it is alternative better, and work as prepared quality-control product
OD450-630When > 2.75, this indicates that prepared quality-control product should also further dilute, to reduce prepared Quality Control
The dosage of product at each test.In addition, every special quality control product are to be carried out by prepared heterologous cross-linking agent using calf serum
Dilution and generate, this indicates that the heterologous cross-linking agent of the heterologous cross-linking antibody comprising tetramer as patient's positive blood
Middle RV antibody substitute can substitute the RV antibody in patient's positive blood well.
The alternative experiment of heterologous cross-linking agent of 5. mouse anti-RV McAb of table and human IgG
By shared mass ratio in the heterologous cross-linking antibody of tetramer in example 4 prepared heterologous cross-linking agent
Heterologous cross-linking agent when being 50%, 70%, 90% and 100% is all diluted to 0.5mg/ using the cross-linking buffer in embodiment 1
Ml, the heterologous cross-linking agent solution after taking 0.5ml to dilute, is put in 1ml centrifuge tube, saves one month at -20 DEG C and 4 DEG C respectively
Afterwards, 100 times are diluted with cross-linking buffer, the stability of these heterologous cross-linking agents is still detected using indirect elisa method.Experiment knot
Fruit shows the heterologous cross-linking agent for containing the heterologous cross-linking antibody of tetramer prepared by the present invention as patient's positive blood
RV antibody substitute in liquid has extraordinary stability.
The heterologous cross-linking agent of 5 mouse anti-HCMV McAb of embodiment and the human IgG for being uninfected by HCMV of health is alternative
Experiment
Utilize the heterologous friendship of SPDP crosslinking agent preparation mouse anti-HCMV McAb and the human IgG for being uninfected by HCMV of health
Join object as HCMV antibody substitute in patient's positive blood, in addition to replacing mouse anti-with mouse anti-HCMV McAb
Except HSV-II McAb, detailed process is as in embodiment 1.
Heterologous cross-linking agent using the human IgG for being uninfected by HCMV of the mouse anti-HCMV McAb and health that prepare is made
Carry out experiment for the substitute of HCMV antibody in patient's positive blood.The heterologous cross-linking antibody for successively making tetramer exists
Heterologous cross-linking agent of the shared mass ratio at 50%, 70%, 90% and 100% in prepared heterologous cross-linking agent.When four
The heterologous cross-linking antibody of dimer form mass ratio shared in prepared heterologous cross-linking agent is 50%, 70% or 90%
When, residue 50%, 30% or 10% heterologous cross-linking agent include by mouse anti-HCMV McAb and health be uninfected by HCMV
The heterologous cross-linking antibody of the heterologous cross-linking antibodies of dimeric forms and/or trimeric form that is formed of human IgG, but do not include not
The human IgG for being uninfected by HCMV of the mouse anti-HCMV McAb and health that crosslink.It is buffered using the crosslinking in embodiment 1
Liquid is diluted, and making the initial concentration of these heterologous cross-linking agents is 0.5mg/ml, and carries out 100 times of dilutions using calf serum
Quality-control product is prepared to 5 μ g/ml, is then detected (specific steps are referring to embodiment 2) using indirect elisa method.Experiment
The results are shown in Table 6.
To enable prepared quality-control product to substitute patient's HCMV antibody positive blood, proposed standard in the industry: in phase
Patient's positive blood standard items and prepared quality-control product are measured using the indirect elisa method in embodiment 2 under the conditions of
OD450-630> 1.5, most preferably 2.6.As shown in Table 6, when the heterologous cross-linking antibody of tetramer is in prepared heterologous friendship
When shared mass ratio is respectively 50%, 70%, 90% and 100% in connection object, prepared quality-control product OD450-630It is all long-range
In 1.5, meet proposed standard in the industry, compared with patient's positive blood standard items, performance is quite even better, therefore alternative disease
People's positive blood standard items.When the heterologous cross-linking antibody of tetramer quality shared in prepared heterologous cross-linking agent
Than it is higher when, OD450-630It is higher, this quality-control product it is alternative better, and work as prepared quality-control product OD450-630> 2.6
When, this indicates that prepared quality-control product should also further dilute, and is detecting every time to reduce prepared quality-control product
When dosage.In addition, every special quality control product are to be diluted using calf serum by prepared heterologous cross-linking agent and generated,
This indicates that the heterologous cross-linking agent of the heterologous cross-linking antibody comprising tetramer is replaced as HCMV antibody in patient's positive blood
For object, the HCMV antibody in patient's positive blood can be substituted well.
The alternative experiment of heterologous cross-linking agent of 6. mouse anti-HCMV McAb of table and human IgG
By shared mass ratio in the heterologous cross-linking antibody of tetramer heterologous cross-linking agent prepared in embodiment 5
Heterologous cross-linking agent when being 50%, 70%, 90% and 100% is all diluted to 0.5mg/ using the cross-linking buffer in embodiment 1
Ml, the heterologous cross-linking agent solution after taking 0.5ml to dilute, is put in 1ml centrifuge tube, saves one month at -20 DEG C and 4 DEG C respectively
Afterwards, 100 times are diluted with cross-linking buffer, the stability of these heterologous cross-linking agents is still detected using indirect elisa method.Experiment knot
Fruit shows the heterologous cross-linking agent for containing the heterologous cross-linking antibody of tetramer prepared by the present invention as patient's positive blood
HCMV antibody substitute in liquid has extraordinary stability.
The heterologous cross-linking agent of 6 mouse anti-TOXO McAb of embodiment and the human IgG for being uninfected by TOXO of health is alternative
Experiment
Utilize the heterologous friendship of SPDP crosslinking agent preparation mouse anti-TOXO McAb and the human IgG for being uninfected by TOXO of health
Join object as TOXO antibody substitute in patient's positive blood, in addition to replacing mouse anti-with mouse anti-TOXO McAb
Except HSV-II McAb, detailed process is as in embodiment 1.
Heterologous cross-linking agent using the human IgG for being uninfected by TOXO of the mouse anti-TOXO McAb and health that prepare is made
Carry out experiment for the substitute of TOXO antibody in patient's positive blood.The heterologous cross-linking antibody for successively making tetramer exists
Heterologous cross-linking agent of the shared mass ratio at 50%, 70%, 90% and 100% in prepared heterologous cross-linking agent.When four
The heterologous cross-linking antibody of dimer form mass ratio shared in prepared heterologous cross-linking agent is 50%, 70% or 90%
When, residue 50%, 30% or 10% heterologous cross-linking agent include by mouse anti-TOXO McAb and health be uninfected by TOXO
The heterologous cross-linking antibody of the heterologous cross-linking antibodies of dimeric forms and/or trimeric form that is formed of human IgG, but do not include not
The human IgG for being uninfected by TOXO of the mouse anti-TOXO McAb and health that crosslink.It is buffered using the crosslinking in embodiment 1
Liquid is diluted, and making the initial concentration of these heterologous cross-linking agents is 0.4mg/ml, and carries out 20 times of dilutions using calf serum
Quality-control product is prepared to 20 μ g/ml, is then detected (specific steps are referring to embodiment 2) using indirect elisa method.Experiment
The results are shown in Table 7.
To enable prepared quality-control product to substitute patient's TOXO antibody positive blood, proposed standard in the industry: in phase
Patient's positive blood standard items and prepared quality-control product are measured using the indirect elisa method in embodiment 2 under the conditions of
OD450-630> 1.5, most preferably 2.4.As shown in Table 7, when the heterologous cross-linking antibody of tetramer is in prepared heterologous friendship
When shared mass ratio is respectively 50%, 70%, 90% and 100% in connection object, prepared quality-control product OD450-630It is all long-range
In 1.5, meet proposed standard in the industry, compared with patient's positive blood standard items, performance is quite even better, therefore alternative disease
People's positive blood standard items.When the heterologous cross-linking antibody of tetramer quality shared in prepared heterologous cross-linking agent
Than it is higher when, OD450-630It is higher, this quality-control product it is alternative better, and work as prepared quality-control product OD450-630> 2.4
When, this indicates that prepared quality-control product should also further dilute, and is detecting every time to reduce prepared quality-control product
When dosage.In addition, every special quality control product are to be diluted using calf serum by prepared heterologous cross-linking agent and generated,
This indicates that the heterologous cross-linking agent including the heterologous cross-linking antibody comprising tetramer is anti-as TOXO in patient's positive blood
Body substitute can substitute the TOXO antibody in patient's positive blood well.
The alternative experiment of heterologous cross-linking agent of 7. mouse anti-TOXO McAb of table and human IgG
By shared mass ratio in the heterologous cross-linking antibody of tetramer heterologous cross-linking agent prepared in embodiment 6
Heterologous cross-linking agent when being 50%, 70%, 90% and 100% is all diluted to 0.5mg/ using the cross-linking buffer in embodiment 1
Ml, the heterologous cross-linking agent solution after taking 0.5ml to dilute, is put in 1ml centrifuge tube, saves one month at -20 DEG C and 4 DEG C respectively
Afterwards, 100 times are diluted with cross-linking buffer, the stability of these heterologous cross-linking agents is still detected using indirect elisa method.Experiment knot
Fruit shows the heterologous cross-linking agent for containing the heterologous cross-linking antibody of tetramer prepared by the present invention as patient's positive blood
TOXO antibody substitute in liquid has extraordinary stability.
Claims (4)
1. a kind of cross-linking antibody as patient's positive blood pathogen antigen substitute, which is characterized in that the crosslinking is anti-
Body is formed by tetramer shape by cross-linking reaction by the human IgG for being uninfected by HSV-I of mouse anti-HSV-I McAb and health
The heterologous cross-linking antibody of formula forms, and the attachment structural formula L that the cross-linking reaction is formed is selected from 4 (a), 4 (a):
,
The preparation method of the cross-linking antibody is as follows: (1) 5mg mouse anti-HSV-I McAb being dissolved in crosslinking buffering
It in liquid, stirs and 50 μ l SPDP crosslinking agents is added, after reacting 2 hours at room temperature, be packed into bag filter, dialysed with reduction buffer,
It changes liquid four times, wherein the cross-linking buffer is 0.1M potassium phosphate, 0.1M NaCl, pH7.5, the reduction buffer is 0.1M
Sodium acetate, 0.1M NaCl, pH4.5, the SPDP crosslinking agent are dissolved in dehydrated alcohol, concentration 3.2mg/ml；
(2) human IgG for being uninfected by HSV-I of 5mg health is dissolved in cross-linking buffer, 50 μ l SPDP crosslinking is added in stirring
Agent after reacting 2 hours at room temperature, is packed into bag filter, is dialysed, changed liquid four times with cross-linking buffer；
(3) 30 μ lDTT are added in (1) to be packed into bag filter after room temperature reaction 30 minutes, dialysed with cross-linking buffer, change liquid four
It is secondary, wherein the DTT is dissolved in 0.01M sodium acetate solution, concentration 1M, pH5.2；
(4) processed material in (2), (3) is mixed, 15 hours is reacted at 4 DEG C；
(5) 0.4mg iodoacetamide sealer is added into (4), reacts 30 minutes to close unreacted active group, then fills
Enter bag filter, dialysed with cross-linking buffer, is changed liquid four times；
(6) product in (5) is loaded into 16/60 Superdex of HiLoad, 200 PG solvent resistant column, to be crosslinked buffering
Liquid is eluted, and the light absorption value using HD-3 purple light analyzer real-time detection eluted product in 280nm wavelength, is used simultaneously
The small-sized recorder of XWT-S draws chromatography map, collects eluted product by peak；
(7) the heterologous cross-linking agent of the heterologous cross-linking antibody containing tetramer is obtained.
2. cross-linking antibody as described in claim 1, which is characterized in that the cross-linking antibody is stored in a kind of frozen stock solution, and
It is saved at -20 DEG C, the frozen stock solution includes stabilizer, glycerol and preservative.
3. cross-linking antibody as claimed in claim 2, which is characterized in that the stabilizer is selected from BSA or gelatin, the preservative
Selected from Sodium azide or thimerosal.
4. the cross-linking antibody as described in one of claims 1 to 3 is preparing the application in HSV-I immunity detection reagent.
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| CN1167920A (en) * | 1997-05-14 | 1997-12-17 | 卫生部武汉生物制品研究所 | Safety human immunodeficiency virus antibody positive serum substitute |
| CN1488944A (en) * | 2002-10-09 | 2004-04-14 | 上海市刑事科学技术研究所 | HIV positive serum surrogate |
| CN1924579A (en) * | 2005-07-04 | 2007-03-07 | 上海富纯中南生物技术有限公司 | Cross-linked composite used as standard diagnosing reagent replacing positive serum and method for use as standard reagent |
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| CN101676726A (en) * | 2008-09-18 | 2010-03-24 | 卫生部北京医院 | Preparation method of E hepatitis rabbit-human chimeric antibody quality control substance |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1167920A (en) * | 1997-05-14 | 1997-12-17 | 卫生部武汉生物制品研究所 | Safety human immunodeficiency virus antibody positive serum substitute |
| CN1488944A (en) * | 2002-10-09 | 2004-04-14 | 上海市刑事科学技术研究所 | HIV positive serum surrogate |
| CN101484182A (en) * | 2005-04-06 | 2009-07-15 | Ibc药品公司 | Methods for generating stably linked complexes composed of homodimers, homotetramers or dimers of dimers and uses |
| CN1924579A (en) * | 2005-07-04 | 2007-03-07 | 上海富纯中南生物技术有限公司 | Cross-linked composite used as standard diagnosing reagent replacing positive serum and method for use as standard reagent |
| CN101676726A (en) * | 2008-09-18 | 2010-03-24 | 卫生部北京医院 | Preparation method of E hepatitis rabbit-human chimeric antibody quality control substance |
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