CN103382224B - Heterologous cross-linking object containing heterologous cross-linking antibody in tetramer form and applications thereof - Google Patents
Heterologous cross-linking object containing heterologous cross-linking antibody in tetramer form and applications thereof Download PDFInfo
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Abstract
The invention provides a heterologous cross-linking object containing heterologous cross-linking antibody in a tetramer form, the heterologous cross-linking object is obtained from cross-linking reactions between two heterologous antibodies, the mass ratio of the heterologous cross-linking antibody in a tetramer form in the heterologous cross-linking object is preferably not less than 50%, the heterologous cross-linking object can be taken as a substituent of a pathogen antibody in the patients' positive blood, and can be applied to an immunity detection kit as a quality control object to substitute the patients' positive blood after being diluted by a diluting liquid. The heterologous cross-linking object has the advantages of simple production and preparation, low cost, and very good stability and safety. Furthermore, qualities of different batches of quality control objects are stable, so detection errors can be effectively reduced.
Description
Technical field
The present invention relates to a kind of allos cross-linking agent, it is exactly specifically a kind of allos cross-linking agent of the allos cross-linking antibody containing tetramer obtained by crosslinking reaction by the antibody of two kinds of allos, and the application in patient's positive blood on pathogen antigen surrogate, belong to immunological technique field.
Background technology
Nowadays, the threat that brings to the mankind of the pathogenic agent of such as virus, bacterium, fungi and parasite and so on is increasing.Based on the immune analysis method that specificity between antigen and antibody and high sensitivity are reacted, as colloidal gold immunochromatographimethod detection, the detection of latex immunochromatography, Placenta function (RIA), MBP enzyme linked immuno-adsorbent assay (ELISA), time-resolved fluoroimmunoassay detect (TRFIA) or chemiluminescence immunoassay detects (CLIA), has been widely used among clinical immunization test.These immune analysis methods are usually used to pathogen antigen that is qualitative or that detect quantitatively in human blood or antibody, thus determine the stage residing for whether occurring and/or infecting of infecting fast and exactly.
Clinical immunization detection kit generally all comprises negative quality control product and positive quality control product.These quality control products are often from the serial dilution product of the higher patient's positive blood of antibody titer, or the patient's positive blood directly using different antibodies to tire, and comprise serum, blood plasma or whole blood.But utilize patient's positive blood to prepare positive quality control product and have a lot of shortcoming: 1) be difficult to patient's positive blood that acquisition has high-titer and high specific simultaneously; 2) antibody titer between individuality and specificity differ greatly, and are difficult to stdn; 3) cost compare is high, 4) some pathogenic infection case is rare, is difficult at short notice obtain enough positive bloods; 5) pathogenic agent positive blood has potential infectivity, even if through inactivation treatment, but still is difficult to ensure thorough deactivation, and simultaneously when collection and deactivation patient positive blood, operator also have potential infected risk.For this reason, people often use the quality control product of alternative patient's positive blood.
European patent application EP 0 330 902 A2 discloses when detecting human blood specific antibody, use one or more human monoclonal antibodies (as people's rubella virus monoclonal antibody and people's varicella zoster virus monoclonal antibody) as test comparison, but each human monoclonal antibodies built only includes a kind of constant region of antibody, detect other Antibody types to need to rebuild, and owing to not allowing ethically to utilize human body to carry out antibody preparation, thus have to utilize the method such as display technique of bacteriophage and transgenic animal to carry out manufacturer's monoclonal antibody, it is more difficult that this just makes antibody prepare.US Patent No. 6,015,662 disclose the positive control that human mouse chimeric antibody may be used for human blood antibody test, but each people mouse chimeric mAb built only includes a kind of constant region of antibody, detect other Antibody types to need to rebuild, and technique is very complicated, this is because except utilizing hybridoma technology to obtain except mouse monoclonal antibody, structure and the expression of antibody cloning and people mouse mosaic gene also will be carried out.
At present except aforesaid method Dispersal risk, have also appeared and utilize linking agent Dispersal risk.The existing a variety of method utilizing linking agent to prepare cross-linking antibody.As linking agent succinimide pyridine dimercapto propyl ester ［ N-Succinimidyl 3-(2-pyridyldithio) propionate, SPDP ］ can be used to two kinds of different albumen (comprising antibody and enzyme) is crosslinked together and form 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).Linking agent phenylenedimaleimide (O-phenylenedi-maleimide, O-PDM) be also used to (Glennie M crosslinked together for the fragment of two kinds of antibody, McBride H, Worth A, et al.. Preparation and performance of bispecific F (ab'gamma)
2antibody containing thioether-linked Fab'gamma fragments.
j. Immunol., 1987,139:2367-2375).European patent EP 0 062 227 A1 disclose utilize linking agent glutaraldehyde or sodium periodate two kinds of different plant species to be originated antibody coupling together.In addition, US Patent No. 4,507,234 disclose linking agent dithio-bis-nitrobenzoic acid [5,5'-dithiobis-(2-nitrobenzoicacid), DTNB] can be used to prepare cross-linking antibody.US Patent No. 4; 929; 543 disclose crosslinking aid S-acetyl mercapto Succinic anhydried (S-acetylmercaptosuccinic anhydride; AMSA) and maleimidocaproyl-N-hydroxy-succinamide ester (Maleimidohexanoyl-N-hydroxysuccinimide ester, MHS) can be used to prepare cross-linking antibody.US Patent No. 5,478,753 disclose linking agent 4-maleimidobutyric acid-N-succinimide ester (N-γ-maleimidobutyryloxysuccinimide ester, GMBS) can be used to prepare cross-linking antibody.
Cross-linking antibody is also often used to the quality control product preparing alternative patient's positive blood.US Patent No. 4,929,543 disclose inhuman antibody or itself Fab or F (ab')
2the allos cross-linking agent of fragment and human normal immunoglobulin or its Fc fragment, diagnoses for Serum Antibodies.US Patent No. 5,478,753 disclose inhuman non-IgM and the allos cross-linking agent of people IgM, for serum IgM diagnostic kit, inhuman non-IgM in allos cross-linking agent can identify specifically and in conjunction with pathogen antigen, people IgM can combine with traget antibody.US Patent No. 5,491, the 218 allos cross-linking agents disclosing inhuman antibody or its fragment and another kind of antibody, as people IgM and anti-herpes simplex virus antibodies F (ab')
2the allos cross-linking agent of fragment, antibodies specific ground inhuman in allos cross-linking agent combines a kind of specific pathogenic agent, and another kind of antibodies specific ground combines a kind of specific Antibody types.Chinese patent ZL97109106.4 discloses inhuman Plasma donor or has the fragment of antibody activity and the allos cross-linking agent of human normal immunoglobulin after treatment, can be used as the surrogate of antibody of AIDS virus in patient's positive blood.But do not disclose the concrete composition of the allos cross-linking agent prepared in these patents above, it is unstable that this just causes quality between allos cross-linking agent different batches, easily produces metrical error, substituting poor.
Summary of the invention
The object of the present invention is to provide a kind of allos cross-linking agent obtained by crosslinking reaction by the antibody of two kinds of allos, described allos cross-linking agent comprises the allos cross-linking antibody of the tetramer formed by the antibody of described two kinds of allos.Preferably, the antibody of described two kinds of allos is people's antibody of inhuman pathogen antigen and healthy non-pathogen infection; Preferably, the mass ratio that the allos cross-linking antibody of described tetramer is shared in described allos cross-linking agent is not less than 50%.
The object of the invention is to the application of a kind of allos cross-linking agent in patient's positive blood on pathogen antigen surrogate, described allos cross-linking agent is obtained by crosslinking reaction by the antibody of two kinds of allos, and described allos cross-linking agent comprises the allos cross-linking antibody of the tetramer formed by the antibody of described two kinds of allos.Preferably, the antibody of described two kinds of allos is people's antibody of inhuman pathogen antigen and healthy non-pathogen infection; Preferably, the mass ratio that the allos cross-linking antibody of described tetramer is shared in described allos cross-linking agent is not less than 50%.
Another object of the present invention is the quality control product providing a kind of alternative patient's positive blood, described quality control product comprises diluent and a kind of allos cross-linking agent obtained by crosslinking reaction by the antibody of two kinds of allos, described allos cross-linking agent comprises the allos cross-linking antibody of the tetramer formed by the antibody of described two kinds of allos, described diluent is optional from calf serum or foetal calf serum, be preferably calf serum, in described quality control product, described allos cross-linking agent concentration is 5 ~ 25 μ g/ml, preferably 20 μ g/ml.Preferably, the antibody of described two kinds of allos is people's antibody of inhuman pathogen antigen and healthy non-pathogen infection; Preferably, the mass ratio that the allos cross-linking antibody of described tetramer is shared in described allos cross-linking agent is not less than 50%.
Especially, the invention provides a kind of quality control product of alternative patient TORCH pathogen antigen positive blood, it is characterized in that, described quality control product comprises diluent and a kind of allos cross-linking agent obtained by crosslinking reaction by inhuman TORCH pathogen antigen and the healthy people's antibody not infecting TORCH pathogenic agent, described allos cross-linking agent comprises the allos cross-linking antibody of the tetramer formed by the people's antibody not infecting TORCH pathogenic agent of described inhuman TORCH pathogen antigen and described health, described diluent is optional from calf serum or foetal calf serum, be preferably calf serum, in described quality control product, described allos cross-linking agent concentration is 5 ~ 25 μ g/ml, preferably 20 μ g/ml.Preferably, the mass ratio that the allos cross-linking antibody of described tetramer is shared in described allos cross-linking agent is not less than 50%.
Compared with prior art, the beneficial effect that the present invention obtains is:
First, allos cross-linking agent of the present invention contains the allos cross-linking antibody of tetramer.Described allos cross-linking agent is as the surrogate of pathogen antigen in patient's positive blood, and preserve at-20 DEG C and 4 DEG C in the stability test after month, described allos cross-linking agent all has extraordinary stability.
Secondly, allos cross-linking agent of the present invention, as the surrogate of pathogen antigen in patient's positive blood, can substitute the pathogen antigen in patient's positive blood well.
Again, allos cross-linking agent of the present invention derives from people's antibody of inhuman pathogen antigen and healthy non-pathogen infection, after diluted, can be used as the quality control product of alternative patient's positive blood, the performance of this quality control product and patient's positive blood is suitable, can avoid again the latent infection of patient's positive blood, this allos cross-linking agent has the advantages that manufacture is simple and cost is low simultaneously.
Further, quality control product difference between batch of the present invention is less, effectively can reduce metrical error.
Embodiment
The present invention utilize the linking agent of such as SPDP, GMBS and DTNB and so on by two kinds of allos antibody linked together with, thus form the allos cross-linking agent comprising the allos cross-linking antibody of tetramer.The present invention finds when described allos cross-linking agent contains the allos cross-linking antibody of tetramer, described allos cross-linking agent can be used as the surrogate of pathogen antigen in patient's positive blood, the quality control product of patient's positive blood as an alternative after diluted, for immunodetection, as colloidal gold immunochromatographimethod detects, latex immunochromatography detects, Placenta function, enzyme linked immunosorbent detection, time-resolved fluoroimmunoassay detect or chemiluminescence immunoassay detects, and have extraordinary sensitivity, specificity and stability simultaneously.The mass ratio that the allos cross-linking antibody of described tetramer is shared in described allos cross-linking agent is preferably not less than 50%.
In the allos cross-linking antibody molecule of each described tetramer, the antibody molecule number of described two kinds of allos is than being 3:1 or 2:2 or 1:3.In structural formula 1, the antibody molecule number of described two kinds of allos is than being 3:1.In structural formula 2 (a) or 2 (b), the antibody molecule number of described two kinds of allos is than being 2:2.In structural formula 3, the antibody molecule number of described two kinds of allos is than being 1:3.In these structural formulas, the antibody of described two kinds of allos represents with Ab1 and Ab2 respectively, and L is the connector of formation when utilizing the antibody of linking agent to described two kinds of allos to carry out crosslinking 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 little mouse-anti II herpes simplex virus type monoclonal antibody, mouse anti-I type hsv monoclonal antibody, mouse rubella virus monoclonal antibody, mouse anti human cytomegalovirus monoclonal antibody, mouse monoclonal antibody against Toxoplasma gondii, mouse AIDS virus resisting monoclonal antibody, little mouse-anti hepatitis A virus monoclonal antibody, little mouse-anti hepatitis A virus monoclonal antibody, mouse anti-hepatitis B virus monoclonal antibody, mouse anti-hepatitis c virus monoclonal antibody, mouse anti-hepatitis D virus monoclonal antibody, little mouse-anti E type hepatitis virus monoclonal antibody, little mouse-anti hepatitis G virus monoclonal antibody or other pathogenic agent monoclonal antibodies of little mouse-anti.Ab2 is preferably a kind of people's antibody of non-pathogen infection of health, such as human IgG, IgA, IgD, IgE, IgM antibody monomer and have antibody fragment or its mixture of intact Fc portion after treatment.The optional self-structure formula 4 (a) of L, 4 (b) or 4 (c), and other conventional structural formulas known to the skilled in this field.
Structural formula 4 (a):
Structural formula 4 (b):
Structural formula 4 (c):
Wherein, the antibody of two kinds of allos is preferably people's antibody of the non-pathogen infection of a kind of inhuman pathogen antigen and a kind of health.Described pathogenic agent can be selected from hiv virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis G virus, hsv, rubella virus, human cytomegalic inclusion disease virus, human T lymphotropic virus, west Nile virus; Streptococcus aureus, intestinal bacteria, Pseudomonas aeruginosa, staphylococcus epidermidis, Klebsiella Pneumoniae, enterococcus faecalis, streptococcus pneumoniae; Toxoplasma gondii, schizotrypanum cruzi, treponema pallidum, plasmodium falciparum, Plasmodium vivax.Described inhuman pathogen antigen refers to from the monoclonal antibody removing the animals such as mankind mouse outside, rat, cavy, rabbit, chicken, pig, sheep, goat, horse, mule and camel, polyclonal antibody, genetic engineering antibody, the antibody utilizing described pathogen antigen immune animal to obtain and the fragment after treatment with antigen-binding activity thereof or its mixture.
Monoclonal antibody refers to only by immunocyte (as bone-marrow-derived lymphocyte) produced the immunoglobulin (Ig) be combined with a kind of epitope from the type in animal body.For the most frequently used mouse monoclonal antibody, with a kind of pathogen antigen immune mouse, after repeatedly immunity, isolate mouse spleen bone-marrow-derived lymphocyte, and merge with murine myeloma cell, hybridoma (K hle G, the Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. of the required monoclonal antibody of secretion just can be obtained by carrying out repeatedly subclone screening to produced hybridoma
nature, 1975,256:495-497; Albitar Maher. Monoclonal Antibodies:Methods in Molecular Biology, Vol. 378,2007).Then the hybridoma of the required monoclonal antibody of secretion is cultivated in vitro, collect nutrient solution, or hybridoma is expelled in the abdominal cavity of Syngenic mice, collect ascites, finally utilize caprylic acid-ammonium, saturated ammonium sulphate salt precipitation method or affinity column method just can isolate monoclonal antibody (Nakazawa M from nutrient solution or ascites, Mukumoto M, Miyatake K. Immunoelectron Microscopy:Methods in Molecular Biology, 2010,657:63-74; E. Lip river is breathed out, D. Lay grace. " antibody technique experiment guide ").
Polyclonal antibody refers to by from the produced immunoglobulin (Ig) that can be combined with plurality of antigens epi-position of the polytype immunocyte (as bone-marrow-derived lymphocyte) in animal body.For rabbit polyclonal antibody, with a kind of pathogen antigen repeatedly immune rabbit, then serum is collected, recycling caprylic acid-ammonium, saturated ammonium sulphate salt precipitation method or affinity column method or ion exchange chromatography isolate polyclonal antibody (Nakazawa M, Mukumoto M, Miyatake K. Immunoelectron Microscopy:Methods in Molecular Biology, 2010,657:63-74; E. Lip river is breathed out, D. Lay grace. " antibody technique experiment guide ").
Genetic engineering antibody refers to by the existing excellent non-human animal's species monoclonal antibody gene of genetic engineering techniques, reduce animal species origin components in antibody as far as possible, retain original antibodies specific simultaneously, thus create a kind of novel antibody, mainly comprise 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).
The antibody utilizing pathogen antigen immune animal to obtain refers to the monoclonal antibody or polyclonal antibody that utilize pathogen antigen immunity to obtain except mankind animal outside.
For inhuman pathogen antigen, the fragment after treatment with antigen-binding activity refers to the monoclonal antibody or polyclonal antibody that utilize pathogen antigen immunity to obtain except mankind animal outside and the genetic engineering antibody by genetic engineering technique, obtained monoclonal antibody being carried out to house of correction acquisition, through the antibody fragment still keeping antigen-binding matter that chemical means or collagenase treatment obtain, as monoclonal antibody after papain digestion the Fab fragment that produces, or the F produced after gastric pepsin digestion (ab')
2, wherein F (ab')
2also Fab' fragment can be obtained again after enzyme IdeS digests.
A kind of people's antibody of non-pathogen infection of health extracts from the human blood (comprising serum, blood plasma or whole blood) of the non-pathogen infection of health, optionally comprises IgG, IgA, IgD, IgE, IgM antibody monomer and has antibody fragment or its mixture of intact Fc portion after treatment.The method extracting antibody from blood can be selected from caprylic acid-ammonium, saturated ammonium sulphate salt precipitation method or affinity column method or ion exchange chromatography isolates monoclonal antibody (Nakazawa M, Mukumoto M, Miyatake K. Immunoelectron Microscopy:Methods in Molecular Biology, 2010,657:63-74; E. Lip river is breathed out, D. Lay grace. " antibody technique experiment guide ").Described antibody monomer refers to that the people's antibody for crosslinking reaction should be single aggressiveness, if not single aggressiveness, as the IgM antibody in human serum exists with pentamer form usually, then should in advance through process, them are allowed to become single aggressiveness (Ditzel H, Erb K, et 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).
For people's antibody of the non-pathogen infection of health, the antibody fragment after treatment with intact Fc portion refers to the intact Fc portion that IgG, IgA, IgD, IgE or IgM monomer produces after papoid or gastric pepsin digestion, or through other antibody fragments still with intact Fc portion that chemical means or collagenase treatment obtain.
By selecting suitable inhuman pathogen antigen, the invention provides the quality control product of a kind of alternative patient hiv virus, hepatitis virus or TORCH pathogen antigen positive blood, wherein hepatitis virus can be selected from hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis G virus, and TORCH pathogenic agent is selected from I herpes simplex virus type (HSV-I), II herpes simplex virus type (HSV-II), rubella virus (RV), Human cytomegalic inclusion disease virus (HCMV) and toxoplasma gondii (TOXO).
Especially, the invention provides a kind of quality control product of alternative patient TORCH pathogen antigen positive blood, described quality control product comprises diluent and a kind of allos cross-linking agent obtained by crosslinking reaction by inhuman TORCH pathogen antigen and the healthy people's antibody not infecting TORCH pathogenic agent, described allos cross-linking agent comprises the allos cross-linking antibody of the tetramer formed by the people's antibody not infecting TORCH pathogenic agent of described inhuman TORCH pathogen antigen and described health, described diluent is optional from calf serum or foetal calf serum, be preferably calf serum, in described quality control product, described allos cross-linking agent concentration is 5 ~ 25 μ g/ml, preferably 20 μ g/ml.Preferably, the mass ratio that the allos cross-linking antibody of described tetramer is shared in described allos cross-linking agent is not less than 50%.
Especially, the invention provides a kind of quality control product of alternative patient antibody of AIDS virus positive blood, described quality control product comprises diluent and a kind of allos cross-linking agent obtained by crosslinking reaction with people's antibody of healthy non-aids infection virus by inhuman antibody of AIDS virus, described allos cross-linking agent comprises the allos cross-linking antibody of the tetramer formed by people's antibody of the non-aids infection virus of described inhuman antibody of AIDS virus and described health, described diluent is optional from calf serum or foetal calf serum, be preferably calf serum, in described quality control product, described allos cross-linking agent concentration is 5 ~ 25 μ g/ml, preferably 20 μ g/ml.Preferably, the mass ratio that the allos cross-linking antibody of described tetramer is shared in described allos cross-linking agent is not less than 50%.
Especially, the invention provides a kind of quality control product of alternative patient's hepatitis virus antibody positive blood, described quality control product comprises diluent and a kind of allos cross-linking agent obtained by crosslinking reaction by inhuman hepatitis virus antibody and the healthy people's antibody not infecting hepatitis virus, described allos cross-linking agent comprises the allos cross-linking antibody of the tetramer formed by the people's antibody not infecting hepatitis virus of described inhuman hepatitis virus antibody and described health, described diluent is optional from calf serum or foetal calf serum, be preferably calf serum, in described quality control product, described allos cross-linking agent concentration is 5 ~ 25 μ g/ml, preferably 20 μ g/ml.Preferably, the mass ratio that the allos cross-linking antibody of described tetramer is shared in described allos cross-linking agent is not less than 50%.
In addition, according to known to a person skilled in the art, by selecting suitable inhuman pathogen antigen, the invention provides a kind of quality control product of other pathogen antigen positive bloods of alternative patient.
When allos cross-linking agent of the present invention is used for immunodetection; usually the diluent adding a kind of diluting effect is also needed; utilize described diluent by the concentration dilution of described allos cross-linking agent most 5 ~ 25 μ g/ml; so just prepare the quality control product of alternative patient's positive blood; wherein said diluent is optional from calf serum or foetal calf serum, is preferably calf serum.
Preferably, allos cross-linking agent of the present invention is kept in a kind of frozen storing liquid at-20 DEG C, and described frozen storing liquid comprises stablizer, glycerine, sanitas.Wherein said stablizer is selected from BSA or gelatin, and preferred concentration is 1%; Described glycerine, is preferably 50% glycerine; Described sanitas is selected from sodium azide or Thiomersalate, and preferred concentration is 0.01%.In described frozen storing liquid, described allos cross-linking agent final concentration is 2 ~ 5mg/ml, is preferably 4mg/ml.
When allow the antibody generation crosslinking reaction of two kinds of allos time, a lot of linking agent can be selected, as SPDP, O-PDM, DTNB, AMSA, MHS or GMBS, and other linking agents known to those skilled in the art.The present invention utilizes crosslinking aid S PDP and reducing agent dithiothreitol (dithiothreitol, DTT), prepare little mouse-anti II herpes simplex virus type monoclonal antibody (anti-HSV-II McAb) and the healthy allos cross-linking agent not infecting the human IgG of HSV-II, mouse anti-I type hsv monoclonal antibody (anti-HSV-I McAb) and the healthy allos cross-linking agent not infecting the human IgG of HSV-I, mouse rubella virus monoclonal antibody (anti-RV McAb) and the healthy allos cross-linking agent not infecting the human IgG of RV, the allos cross-linking agent not infecting the human IgG of TOXO of the allos cross-linking agent of the human IgG of mouse anti human cytomegalovirus monoclonal antibody (anti-HCMV McAb) and healthy non-HCMV infection and mouse monoclonal antibody against Toxoplasma gondii (anti-TOXO McAb) and health.Similarly, SPDP linking agent and DTT is utilized also can to prepare allos cross-linking agent between people's antibody of other inhuman pathogen antigen and the non-pathogen infection of health.
Embodiment 1
SPDP linking agent is utilized (to buy from Thermo Scientific company, production code member 21857) prepare the allos cross-linking agent not infecting the human IgG of HSV-II of mouse anti-HSV-II McAb and health as HSV-II antibody surrogate thing in patient's positive blood, process is as follows:
(1) 5mg mouse anti-HSV-II McAb is dissolved in cross-linking buffer (0.1M potassiumphosphate, 0.1M NaCl, pH7.5), in, stir and add 50 μ l SPDP linking agents (3.2mg/ml is dissolved in dehydrated alcohol), react under room temperature after 2 hours, load dialysis tubing, with reduction damping fluid (0.1M sodium acetate, 0.1M NaCl, pH4.5) dialyse, change liquid four times;
(2) human IgG not infecting HSV-II of 5mg health is dissolved in cross-linking buffer, stirs and add 50 μ l SPDP linking agents, react after 2 hours under room temperature, load dialysis tubing, with cross-linking buffer dialysis, change liquid four times;
(3) add 30 μ l DTT(1M in (1), be dissolved in 0.01M sodium acetate solution, pH5.2), room temperature reaction, after 30 minutes, loads dialysis tubing, with cross-linking buffer dialysis, changes liquid four times;
(4) by the handled thing mixing in (2), (3), react 15 hours at 4 DEG C;
(5) in (4), add 0.4mg iodo-acid amide encapsulant, react 30 minutes to close unreacted active group, then load dialysis tubing, with cross-linking buffer dialysis, change liquid four times;
(6) product in (5) is loaded in HiLoad 16/60 Superdex 200 PG gel-filtration column (buying from GE Healthcare), wash-out is carried out with cross-linking buffer, and utilize HD-3 purple light analyser to detect the light absorption value of eluted product when 280nm wavelength in real time, draw chromatography collection of illustrative plates with the small-sized registering instrument of XWT-S simultaneously, collect eluted product by peak;
(7) the allos cross-linking agent of the allos cross-linking antibody containing tetramer is obtained, as the surrogate of HSV-II antibody in patient's positive blood.Wherein the allos cross-linking antibody molecule of each tetramer refers to and utilizes SPDP and DTT by 3 mouse anti-HSV-II McAb molecules and 1 healthy human IgG molecule not infecting HSV-II, or 2 mouse anti-HSV-II McAb molecules and 2 healthy human IgG molecules not infecting HSV-II, or 1 mouse anti-HSV-II McAb molecule with 3 healthy human IgGs not infecting HSV-II molecule crosslinked together with formed.In the allos cross-linking antibody molecule of formed each tetramer, to be mouse anti-HSV-II McAb, Ab2 the be healthy human IgG not infecting HSV-II of the Ab1 corresponding to structural formula 1,2 and 3, L for shown in structural formula 4 (a), namely
Embodiment 2 mouse anti-HSV-II McAb and the healthy allos cross-linking agent not infecting the human IgG of HSV-II is substituting, quality control product stability and frozen stability experiment
Using the mouse anti-HSV-II McAb for preparing in embodiment 1 and the healthy allos cross-linking agent not infecting the human IgG of HSV-II as the surrogate of HSV-II antibody in patient's positive blood, carry out experiment.
(1) quality control product is prepared.The allos cross-linking agent of the mass ratio shared in prepared allos cross-linking agent of the allos cross-linking antibody making tetramer successively 50%, 70%, 90% and 100% time as patient's positive blood in the surrogate of HSV-II antibody.When the mass ratio that the allos cross-linking antibody of tetramer is shared in prepared allos cross-linking agent is 50%, 70% or 90%, the allos cross-linking agent of residue 50%, 30% or 10% comprises the allos cross-linking antibody of the dimeric forms formed by mouse anti-HSV-II McAb and human IgG and/or the allos cross-linking antibody of trimeric form, but does not comprise the mouse anti-HSV-II McAb and the healthy human IgG not infecting HSV-II that are cross-linked.Utilize the cross-linking buffer in embodiment 1 to dilute, make the starting point concentration of these allos cross-linking agents be 0.5mg/ml, and utilize calf serum to carry out 20 times to be diluted to 25 μ g/ml, just to prepare the quality control product of alternative patient HSV-II antibody positive blood.
(2) utilize indirect elisa method to detect, detailed process is as follows:
(1) bag quilt: be buffered liquid (1.59g sodium carbonate, 2.93g sodium bicarbonate are dissolved in 1000ml distilled water, pH are adjusted to 9.6 simultaneously, be configured to bag and be buffered liquid) with bag and dilute II herpes simplex virus type antigen, make antigen concentration be 1 μ g/ml; Add the antigenic solution after 100 μ l dilutions in each hole of enzyme plate, and cover enzyme plate with plastics film, spend the night at 4 DEG C, next day, discard solution in hole; Add 200 μ l washing solns (0.5ml Tween 20 being joined 1000ml bag to be buffered in liquid) detersive enzyme target, repeat 3 times;
(2) close: add 200 μ l lock solution (1g BSA is dissolved in 100ml washing soln) in enzyme plate hole, and cover enzyme plate with plastics film, after hatching 2 hours at 37 DEG C, discard solution in hole; Add 200 μ l washing soln detersive enzyme targets, repeat 3 times;
(3) quality control product is added: in holes different on enzyme plate successively, add the quality control product 100 μ l and patient's positive blood standard substance 100 μ l that prepare in (one), wherein every special quality control product should repeat to add once, namely be added in two adjacent holes, every hole is 100 μ l, and cover enzyme plate with plastics film, after hatching 30 minutes at 37 DEG C, discard solution in hole, add 200 μ l washing soln detersive enzyme targets, repeat 5 times;
(4) ELIAS secondary antibody is added: add ELIAS secondary antibody (goat anti-human igg of horseradish peroxidase-labeled) working fluid 100 μ l in each hole of enzyme plate, and cover enzyme plate with plastics film, after hatching 30 minutes at 37 DEG C, discard solution in hole, add 200 μ l washing soln detersive enzyme targets, repeat 5 times;
(5) detect: add nitrite ion A(containing citric acid 35.8g/L, Na
2hPO
412H
20 9.34g/L, 30% H
2o
2660 μ l) and nitrite ion B(containing 3,3,5,5-tetramethyl benzidine 0.2g/L, dimethyl sulfoxide (DMSO) 5ml/L, 6N HCl 1ml/L) each 50 μ l, and cover enzyme plate with plastics film, after hatching 10 minutes at 37 DEG C, add stop bath (2N H
2sO
4) 50 μ l, with the absorbance (A450/A630) during microplate reader reading 450nm and 630nm wavelength, and correct, namely A450 value deducts A630 value (OD
450-630).Experimental result is as shown in table 1.
Patient's positive blood standard substance refer to the patient's positive serum collected through multiplex screening, the serum containing interference material is rejected, thus avoids occurring false positive, be mainly used in the validity of evaluation experimental result, stability and comparability.
Patient HSV-II antibody positive blood is substituted, proposed standard in the industry: utilize the indirect elisa method in the present embodiment to record the OD of patient's positive blood standard substance and prepared quality control product under the same conditions to enable prepared quality control product
450-630> 0.8, the best is 2.0.As shown in Table 1, when the mass ratio that the allos cross-linking antibody of tetramer is shared in prepared allos cross-linking agent is respectively 50%, 70%, 90% and 100%, prepared quality control product OD
450-630all much larger than 0.8, meet proposed standard in the industry, compared with patient's positive blood standard substance, performance is quite even better, therefore alternative patient's positive blood standard substance.When the mass ratio that the allos cross-linking antibody of tetramer is shared in prepared allos cross-linking agent is higher, OD
450-630higher, this quality control product substituting better, and as prepared quality control product OD
450-630during > 2.0, this just shows that prepared quality control product also should dilute further, thus the consumption of quality control product when each detection prepared by reducing.In addition, every special quality control product utilize calf serum to carry out diluting producing by prepared allos cross-linking agent, this just shows that the allos cross-linking agent of the allos cross-linking antibody comprising tetramer is as HSV-II antibody surrogate thing in patient's positive blood, can substitute the HSV-II antibody in patient's positive blood well.
The substituting experiment of allos cross-linking agent of table 1. mouse anti-HSV-II McAb and human IgG
In the allos cross-linking agent selecting the allos cross-linking antibody of tetramer prepared in embodiment 1, shared mass ratio is that the allos cross-linking agent of 90% is as HSV-II antibody surrogate thing in patient's positive blood, described allos cross-linking agent dilutes 20 times through calf serum and prepares quality control product to 25 μ g/ml, the quality control product of three different batches is therefrom selected to carry out experiment as patient's positive blood surrogate, to assess the difference between different batches quality control product, still select indirect ELISA method to carry out experiment, result is as shown in table 2.
Table 2. quality control product difference between batch evaluation form
CV=1.09% between being criticized by the data calculating in table 2, much smaller than 5%, difference between the quality control product of alternative patient's positive blood of known different batches is very little, illustrate that quality control product of the present invention has stability and consistence between extraordinary batch, therefore, it is possible to effectively reduce the metrical error caused because of differences between batches.
Allos cross-linking agent when shared mass ratio is 50%, 70%, 90% and 100% in the allos cross-linking agent that the allos cross-linking antibody of tetramer is prepared in embodiment 1 all utilizes the cross-linking buffer in embodiment 1 to be diluted to 0.5mg/ml, get the allos cross-linking agent solution after 0.5ml dilution, be put in 1ml centrifuge tube, preserve at-20 DEG C and 4 DEG C respectively after one month, dilute 100 times with cross-linking buffer, still utilize indirect elisa method to detect the stability of these allos cross-linking agents.Experimental result shows that the allos cross-linking agent of the allos cross-linking antibody containing tetramer prepared by the present invention is as HSV-II antibody surrogate thing in patient's positive blood, has extraordinary stability.
In addition, frozen stability test is carried out during shared in the allos cross-linking agent prepared in embodiment 1 for the allos cross-linking antibody of tetramer mass ratio 90%.The described allos cross-linking agent of three different batches is joined in appropriate frozen storing liquid and be 5mg/ml to final concentration, wherein this frozen storing liquid comprises 50% glycerine, the BSA of 1% and the Thiomersalate of 0.01%, then the frozen storing liquid that 0.1ml adds described allos cross-linking agent is got respectively, load in the centrifuge tube of three different 0.5ml, these three centrifuge tubes are placed in the refrigerator of-20 DEG C and preserve 4 months, this three centrifuge tubes are taken out after 4 months, after freeze thawing, solution cross-linking buffer in each centrifuge tube is diluted to 25 μ g/ml, still indirect elisa method is utilized to detect, result is as shown in table 3:
Frozen stability experiment at the allos cross-linking agent-20 DEG C of table 3. mouse anti-HSV-II McAb and human IgG
From the data in table 3, with obtain under same testing conditions frozen before data (see table 2) compared with, the detection of the allos cross-linking agent of these three different batches is tired and to be preserved at-20 DEG C after 4 months all without remarkable reduction in frozen storing liquid, therefore it is as HSV-II antibody surrogate thing in patient's positive blood, has extraordinary frozen stability.
Embodiment 3 mouse anti-HSV-I McAb and the healthy substituting experiment of allos cross-linking agent not infecting the human IgG of HSV-I
SPDP linking agent is utilized to prepare the allos cross-linking agent not infecting the human IgG of HSV-I of mouse anti-HSV-I McAb and health as HSV-I antibody surrogate thing in patient's positive blood, except replacing except mouse anti-HSV-II McAb with mouse anti-HSV-I McAb, the same with embodiment 1 of detailed process.
The mouse anti-HSV-I McAb utilizing embodiment 3 to prepare and the healthy allos cross-linking agent not infecting the human IgG of HSV-I carry out experiment as the surrogate of HSV-I antibody in patient's positive blood.Make the allos cross-linking agent of mass ratio 50%, 70%, 90% and 100% time that the allos cross-linking antibody of tetramer is shared in prepared allos cross-linking agent successively.When the mass ratio that the allos cross-linking antibody of tetramer is shared in prepared allos cross-linking agent is 50%, 70% or 90%, the allos cross-linking agent of residue 50%, 30% or 10% comprises the allos cross-linking antibody of dimeric forms and/or the allos cross-linking antibody of trimeric form that are formed by the human IgG not infecting HSV-I of mouse anti-HSV-I McAb and health, but does not comprise the human IgG not infecting HSV-I of mouse anti-HSV-I McAb and the health be cross-linked.The cross-linking buffer in embodiment 1 is utilized to dilute, the starting point concentration of these allos cross-linking agents is made to be 0.5mg/ml, and utilize calf serum to carry out 100 times to be diluted to 5 μ g/ml and to prepare quality control product, then utilize indirect elisa method to detect and carry out detecting (concrete steps are with reference to embodiment 2).Experimental result is as shown in table 4.
Patient HSV-I antibody positive blood standards product are substituted, proposed standard in the industry: utilize the indirect elisa method in embodiment 2 to record the OD of patient's positive blood standard substance and prepared quality control product under the same conditions to enable prepared quality control product
450-630> 1.5, the best is 2.0 ~ 2.5.As shown in Table 4, when the mass ratio that the allos cross-linking antibody of tetramer is shared in prepared allos cross-linking agent is respectively 50%, 70%, 90% and 100%, prepared quality control product OD
450-630all much larger than 1.5, meet proposed standard in the industry, compared with patient's positive blood standard substance, performance is quite even better, therefore alternative patient's positive blood standard substance.When the mass ratio that the allos cross-linking antibody of tetramer is shared in prepared allos cross-linking agent is higher, OD
450-630higher, this quality control product substituting better, and as prepared quality control product OD
450-630during > 2.5, this just shows that prepared quality control product also should dilute further, thus the consumption of quality control product when each detection prepared by reducing.In addition, every special quality control product utilize calf serum to carry out diluting producing by prepared allos cross-linking agent, this just shows that the allos cross-linking agent of the allos cross-linking antibody comprising tetramer is as HSV-I antibody surrogate thing in patient's positive blood, can substitute the HSV-I antibody in patient's positive blood well.
The substituting experiment of allos cross-linking agent of table 4. mouse anti-HSV-I McAb and human IgG
Allos cross-linking agent when shared mass ratio is 50%, 70%, 90% and 100% in the allos cross-linking agent that the allos cross-linking antibody of tetramer is prepared in embodiment 3 all utilizes the cross-linking buffer in embodiment 1 to be diluted to 0.5mg/ml, get the allos cross-linking agent solution after 0.5ml dilution, be put in 1ml centrifuge tube, preserve at-20 DEG C and 4 DEG C respectively after one month, dilute 100 times with cross-linking buffer, still utilize indirect elisa method to detect the stability of these allos cross-linking agents.Experimental result shows that the allos cross-linking agent of the allos cross-linking antibody containing tetramer prepared by the present invention is as HSV-I antibody surrogate thing in patient's positive blood, has extraordinary stability.
Embodiment 4 mouse anti-RV McAb and the healthy substituting experiment of allos cross-linking agent not infecting the human IgG of RV
SPDP linking agent is utilized to prepare the allos cross-linking agent not infecting the human IgG of RV of mouse anti-RV McAb and health as RV antibody surrogate thing in patient's positive blood, except replacing except mouse anti-HSV-II McAb with mouse anti-RV McAb, the same with embodiment 1 of detailed process.
The mouse anti-RV McAb utilizing embodiment 4 to prepare and the healthy allos cross-linking agent not infecting the human IgG of RV carry out experiment as the surrogate of RV antibody in patient's positive blood.Make the allos cross-linking agent of mass ratio 50%, 70%, 90% and 100% time that the allos cross-linking antibody of tetramer is shared in prepared allos cross-linking agent successively.When the mass ratio that the allos cross-linking antibody of tetramer is shared in prepared allos cross-linking agent is 50%, 70% or 90%, the allos cross-linking agent of residue 50%, 30% or 10% comprises the allos cross-linking antibody of dimeric forms and/or the allos cross-linking antibody of trimeric form that are formed by the human IgG not infecting RV of mouse anti-RV McAb and health, but does not comprise the human IgG not infecting RV of mouse anti-RV McAb and the health be cross-linked.The cross-linking buffer in embodiment 1 is utilized to dilute, the starting point concentration of these allos cross-linking agents is made to be 0.5mg/ml, and utilize calf serum to carry out 20 times to be diluted to 25 μ g/ml and to prepare quality control product, then utilize indirect elisa method to carry out detecting (concrete steps are with reference to embodiment 2).Experimental result is as shown in table 5.
Patient RV antibody positive blood standards product are substituted, proposed standard in the industry: utilize the indirect elisa method in embodiment 2 to record the OD of patient's positive blood standard substance and prepared quality control product under the same conditions to enable prepared quality control product
450-630> 2.0, the best is 2.75.As shown in Table 5, when the mass ratio that the allos cross-linking antibody of tetramer is shared in prepared allos cross-linking agent is respectively 50%, 70%, 90% and 100%, prepared quality control product OD
450-630all much larger than 2.0, meet proposed standard in the industry, compared with patient's positive blood standard substance, performance is quite even better, therefore alternative patient's positive blood standard substance.When the mass ratio that the allos cross-linking antibody of tetramer is shared in prepared allos cross-linking agent is higher, OD
450-630higher, this quality control product substituting better, and as prepared quality control product OD
450-630during > 2.75, this just shows that prepared quality control product also should dilute further, thus the consumption of quality control product when each detection prepared by reducing.In addition, every special quality control product utilize calf serum to carry out diluting producing by prepared allos cross-linking agent, this just shows that the allos cross-linking agent of the allos cross-linking antibody comprising tetramer is as RV antibody surrogate thing in patient's positive blood, can substitute the RV antibody in patient's positive blood well.
The substituting experiment of allos cross-linking agent of table 5. mouse anti-RV McAb and human IgG
Allos cross-linking agent when shared mass ratio is 50%, 70%, 90% and 100% in the allos cross-linking agent that the allos cross-linking antibody of tetramer is prepared in example 4 all utilizes the cross-linking buffer in embodiment 1 to be diluted to 0.5mg/ml, get the allos cross-linking agent solution after 0.5ml dilution, be put in 1ml centrifuge tube, preserve at-20 DEG C and 4 DEG C respectively after one month, dilute 100 times with cross-linking buffer, still utilize indirect elisa method to detect the stability of these allos cross-linking agents.Experimental result shows that the allos cross-linking agent of the allos cross-linking antibody containing tetramer prepared by the present invention is as RV antibody surrogate thing in patient's positive blood, has extraordinary stability.
The substituting experiment of allos cross-linking agent of the human IgG of embodiment 5 mouse anti-HCMV McAb and healthy non-HCMV infection
SPDP linking agent is utilized to prepare the allos cross-linking agent of the human IgG of mouse anti-HCMV McAb and healthy non-HCMV infection as HCMV antibody surrogate thing in patient's positive blood, except replacing except mouse anti-HSV-II McAb with mouse anti-HCMV McAb, the same with embodiment 1 of detailed process.
The allos cross-linking agent of the human IgG of mouse anti-HCMV McAb and the healthy non-HCMV infection prepared is utilized to carry out experiment as the surrogate of HCMV antibody in patient's positive blood.Make the allos cross-linking agent of mass ratio 50%, 70%, 90% and 100% time that the allos cross-linking antibody of tetramer is shared in prepared allos cross-linking agent successively.When the mass ratio that the allos cross-linking antibody of tetramer is shared in prepared allos cross-linking agent is 50%, 70% or 90%, the allos cross-linking agent of residue 50%, 30% or 10% comprises the allos cross-linking antibody of dimeric forms and/or the allos cross-linking antibody of trimeric form that are formed by the human IgG of the non-HCMV infection of mouse anti-HCMV McAb and health, but does not comprise the human IgG of the non-HCMV infection of mouse anti-HCMV McAb and the health be cross-linked.The cross-linking buffer in embodiment 1 is utilized to dilute, the starting point concentration of these allos cross-linking agents is made to be 0.5mg/ml, and utilize calf serum to carry out 100 times to be diluted to 5 μ g/ml and to prepare quality control product, then utilize indirect elisa method to carry out detecting (concrete steps are with reference to embodiment 2).Experimental result is as shown in table 6.
Patient HCMV antibody positive blood is substituted, proposed standard in the industry: utilize the indirect elisa method in embodiment 2 to record the OD of patient's positive blood standard substance and prepared quality control product under the same conditions to enable prepared quality control product
450-630> 1.5, the best is 2.6.As shown in Table 6, when the mass ratio that the allos cross-linking antibody of tetramer is shared in prepared allos cross-linking agent is respectively 50%, 70%, 90% and 100%, prepared quality control product OD
450-630all much larger than 1.5, meet proposed standard in the industry, compared with patient's positive blood standard substance, performance is quite even better, therefore alternative patient's positive blood standard substance.When the mass ratio that the allos cross-linking antibody of tetramer is shared in prepared allos cross-linking agent is higher, OD
450-630higher, this quality control product substituting better, and as prepared quality control product OD
450-630during > 2.6, this just shows that prepared quality control product also should dilute further, thus the consumption of quality control product when each detection prepared by reducing.In addition, every special quality control product utilize calf serum to carry out diluting producing by prepared allos cross-linking agent, this just shows that the allos cross-linking agent of the allos cross-linking antibody comprising tetramer is as HCMV antibody surrogate thing in patient's positive blood, can substitute the HCMV antibody in patient's positive blood well.
The substituting experiment of allos cross-linking agent of table 6. mouse anti-HCMV McAb and human IgG
Allos cross-linking agent when shared mass ratio is 50%, 70%, 90% and 100% in the allos cross-linking agent that the allos cross-linking antibody of tetramer is prepared in embodiment 5 all utilizes the cross-linking buffer in embodiment 1 to be diluted to 0.5mg/ml, get the allos cross-linking agent solution after 0.5ml dilution, be put in 1ml centrifuge tube, preserve at-20 DEG C and 4 DEG C respectively after one month, dilute 100 times with cross-linking buffer, still utilize indirect elisa method to detect the stability of these allos cross-linking agents.Experimental result shows that the allos cross-linking agent of the allos cross-linking antibody containing tetramer prepared by the present invention is as HCMV antibody surrogate thing in patient's positive blood, has extraordinary stability.
Embodiment 6 mouse anti-TOXO McAb and the healthy substituting experiment of allos cross-linking agent not infecting the human IgG of TOXO
SPDP linking agent is utilized to prepare the allos cross-linking agent not infecting the human IgG of TOXO of mouse anti-TOXO McAb and health as TOXO antibody surrogate thing in patient's positive blood, except replacing except mouse anti-HSV-II McAb with mouse anti-TOXO McAb, the same with embodiment 1 of detailed process.
The allos cross-linking agent not infecting the human IgG of TOXO of mouse anti-TOXO McAb and the health prepared is utilized to carry out experiment as the surrogate of TOXO antibody in patient's positive blood.Make the allos cross-linking agent of mass ratio 50%, 70%, 90% and 100% time that the allos cross-linking antibody of tetramer is shared in prepared allos cross-linking agent successively.When the mass ratio that the allos cross-linking antibody of tetramer is shared in prepared allos cross-linking agent is 50%, 70% or 90%, the allos cross-linking agent of residue 50%, 30% or 10% comprises the allos cross-linking antibody of dimeric forms and/or the allos cross-linking antibody of trimeric form that are formed by the human IgG not infecting TOXO of mouse anti-TOXO McAb and health, but does not comprise the human IgG not infecting TOXO of mouse anti-TOXO McAb and the health be cross-linked.The cross-linking buffer in embodiment 1 is utilized to dilute, the starting point concentration of these allos cross-linking agents is made to be 0.4mg/ml, and utilize calf serum to carry out 20 times to be diluted to 20 μ g/ml and to prepare quality control product, then utilize indirect elisa method to carry out detecting (concrete steps are with reference to embodiment 2).Experimental result is as shown in table 7.
Patient TOXO antibody positive blood is substituted, proposed standard in the industry: utilize the indirect elisa method in embodiment 2 to record the OD of patient's positive blood standard substance and prepared quality control product under the same conditions to enable prepared quality control product
450-630> 1.5, the best is 2.4.As shown in Table 7, when the mass ratio that the allos cross-linking antibody of tetramer is shared in prepared allos cross-linking agent is respectively 50%, 70%, 90% and 100%, prepared quality control product OD
450-630all much larger than 1.5, meet proposed standard in the industry, compared with patient's positive blood standard substance, performance is quite even better, therefore alternative patient's positive blood standard substance.When the mass ratio that the allos cross-linking antibody of tetramer is shared in prepared allos cross-linking agent is higher, OD
450-630higher, this quality control product substituting better, and as prepared quality control product OD
450-630during > 2.4, this just shows that prepared quality control product also should dilute further, thus the consumption of quality control product when each detection prepared by reducing.In addition, every special quality control product utilize calf serum to carry out diluting producing by prepared allos cross-linking agent, this just shows that the allos cross-linking agent of the allos cross-linking antibody comprising tetramer is as TOXO antibody surrogate thing in patient's positive blood, can substitute the TOXO antibody in patient's positive blood well.
The substituting experiment of allos cross-linking agent of table 7. mouse anti-TOXO McAb and human IgG
Allos cross-linking agent when shared mass ratio is 50%, 70%, 90% and 100% in the allos cross-linking agent that the allos cross-linking antibody of tetramer is prepared in embodiment 6 all utilizes the cross-linking buffer in embodiment 1 to be diluted to 0.5mg/ml, get the allos cross-linking agent solution after 0.5ml dilution, be put in 1ml centrifuge tube, preserve at-20 DEG C and 4 DEG C respectively after one month, dilute 100 times with cross-linking buffer, still utilize indirect elisa method to detect the stability of these allos cross-linking agents.Experimental result shows that the allos cross-linking agent of the allos cross-linking antibody containing tetramer prepared by the present invention is as TOXO antibody surrogate thing in patient's positive blood, has extraordinary stability.
Claims (4)
1. the allos cross-linking agent obtained by crosslinking reaction by the antibody of two kinds of allos, it is characterized in that, described allos cross-linking agent comprises the allos cross-linking antibody of the tetramer formed by mouse anti-HSV-II McAb and the healthy human IgG not infecting HSV-II; Described allos cross-linking agent is prepared by following methods:
(1) 5mg mouse anti-HSV-II McAb is dissolved in cross-linking buffer, stirs and add 50 μ l SPDP linking agents, react after 2 hours under room temperature, load dialysis tubing, with the dialysis of reduction damping fluid, change liquid four times; Wherein said cross-linking buffer comprises 0.1M potassiumphosphate, 0.1M NaCl, pH7.5; Described crosslinker concentration is 3.2mg/ml, and linking agent is dissolved in dehydrated alcohol; Described reduction damping fluid comprises 0.1M sodium acetate, 0.1M NaCl, pH4.5;
(2) human IgG not infecting HSV-II of 5mg health is dissolved in cross-linking buffer, stirs and add 50 μ l SPDP linking agents, react after 2 hours under room temperature, load dialysis tubing, with cross-linking buffer dialysis, change liquid four times;
(3) add 30 μ l DTT in (1), room temperature reaction, after 30 minutes, loads dialysis tubing, with cross-linking buffer dialysis, changes liquid four times; Described DTT concentration is 1M, is that DTT to be dissolved in 0.01M sodium acetate solution to final concentration be 1M, pH5.2;
(4) by the handled thing mixing in (2), (3), react 15 hours at 4 DEG C;
(5) in (4), add 0.4mg iodo-acid amide encapsulant, react 30 minutes to close unreacted active group, then load dialysis tubing, with cross-linking buffer dialysis, change liquid four times;
(6) product in (5) is loaded in HiLoad 16/60 Superdex 200 PG gel-filtration column, wash-out is carried out with cross-linking buffer, and utilize HD-3 purple light analyser to detect the light absorption value of eluted product when 280nm wavelength in real time, draw chromatography collection of illustrative plates with the small-sized registering instrument of XWT-S simultaneously, collect eluted product by peak;
(7) the allos cross-linking agent of the allos cross-linking antibody containing tetramer is obtained.
2. allos cross-linking agent as claimed in claim 1, it is characterized in that, described allos cross-linking agent is kept in a kind of frozen storing liquid, and preserves at-20 DEG C, and described frozen storing liquid comprises stablizer, glycerine and sanitas.
3. allos cross-linking agent as claimed in claim 2, it is characterized in that, described stablizer can be selected from BSA or gelatin, and described sanitas can be selected from sodium azide or Thiomersalate.
4. the allos cross-linking agent as described in one of claims 1 to 3 is preparing the application in immunity detection reagent.
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| CN201510016461.2A CN104650238B (en) | 2013-06-28 | 2013-06-28 | Human cytomegalovirus cross-linking antibody and its application in immunity detection reagent |
| CN201510017003.0A CN104650239B (en) | 2013-06-28 | 2013-06-28 | A kind of antibody and its application in Toxoplasma immune detection |
| CN201510015885.7A CN104650236B (en) | 2013-06-28 | 2013-06-28 | A kind of heterologous cross-linking agent and its application in rubella virus detects |
| CN201510016141.7A CN104650237B (en) | 2013-06-28 | 2013-06-28 | It a kind of cross-linking antibody and its is applied in immune detection |
| CN201310267573.6A CN103382224B (en) | 2013-06-28 | 2013-06-28 | Heterologous cross-linking object containing heterologous cross-linking antibody in tetramer form and applications thereof |
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| CN201510016141.7A Division CN104650237B (en) | 2013-06-28 | 2013-06-28 | It a kind of cross-linking antibody and its is applied in immune detection |
| CN201510016461.2A Division CN104650238B (en) | 2013-06-28 | 2013-06-28 | Human cytomegalovirus cross-linking antibody and its application in immunity detection reagent |
| CN201510017003.0A Division CN104650239B (en) | 2013-06-28 | 2013-06-28 | A kind of antibody and its application in Toxoplasma immune detection |
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