CN107478840A - Monoclonal antibody matches detection method and its application, detection means - Google Patents
Monoclonal antibody matches detection method and its application, detection means Download PDFInfo
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- CN107478840A CN107478840A CN201710453589.4A CN201710453589A CN107478840A CN 107478840 A CN107478840 A CN 107478840A CN 201710453589 A CN201710453589 A CN 201710453589A CN 107478840 A CN107478840 A CN 107478840A
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The present invention discloses a kind of monoclonal antibody pairing detection method.Comprise the following steps：Balb/c mouse are immunized in comlete antigen with multiple antigenic determinants, different monoclonal antibodies are prepared；Monoclonal antibody and sheep anti-mouse igg are coated on NC films respectively, as detection line and nature controlling line；Every plant of monoclonal antibody is subjected to colloid gold label respectively；The antibody of colloid gold label is directly added dropwise in the sample pad of the colloid gold bar of coating monoclonal antibody in a manner of intersecting two-by-two, detected sample is also added dropwise in sample pad again, do blank control simultaneously, nature controlling line all outlets are chosen after reaction, positive detection line outlet, and the monoclonal antibody of blank control detection line not outlet is to as pairing monoclonal antibody.Monoclonal antibody provided by the invention matches detection method, and detection speed is fast, step is simplified, the time is short.The present invention also provides a kind of monoclonal antibody and matches the detection method application of helicobacter pylori and monoclonal antibody pairing detection means in human feces are detected.
Description
【Technical field】
The present invention relates to technical field of biological, and in particular to a kind of monoclonal antibody matches detection method and its should
With, detection means.
【Background technology】
1975, Kohler and Milstein established hybridoma technology, and they with predetermined antigens being immunized
Mouse boosting cell merged with the myeloma cell unrestrictedly grown in cultivating in vitro, formed B cell hybridoma, successfully
Establish monoclonal antibody technology of preparing.Hybridoma after fusion has double cytotropic features, both thin with myeloma
Born of the same parents unlimited fast breeding and the ability to live eternally in cultivating in vitro, have lymphocyte synthesis and secreting specificity antibody again
Ability.
It is conventional for same proteantigen but the matching method of two monoclonal antibodies of different epitopes in correlation technique
Using ELISE detection methods.ELISE detection methods have complicated enzyme mark process, wrapper sheet process, sample-adding process, and during reaction
Between it is longer, it is necessary to which professional and equipment could be carried out.
Therefore, it is necessary to providing a kind of new technology solves above-mentioned technical problem.
【The content of the invention】
The purpose of the present invention is to overcome above-mentioned technical problem, there is provided a kind of monoclonal antibody matches detection method, can not only
When quickly determining whether different monoclonal antibodies combines same site, while also simplify detecting step, shorten detection
Between.
The technical scheme is that：
A kind of monoclonal antibody matches detection method, comprises the following steps：
Step S1：Balb/c mouse are immunized in comlete antigen with multiple antigenic determinants, obtain more plants of stable cells
Strain, so as to which different positive monoclonal antibodies be prepared；
Step S2：Every plant of monoclonal antibody and sheep anti-mouse igg are coated on NC films respectively, as detection line and Quality Control
Sample pad and blotting paper are pasted in line, NC films both ends respectively, and making forms colloid gold bar；
Step S3：Every plant of monoclonal antibody is subjected to colloid gold label respectively；
Step S4：The antibody of colloid gold label is directly added dropwise in a manner of intersecting two-by-two and is being coated with the glue of monoclonal antibody
In the sample pad of body gold bar, then by detected sample dropwise addition in sample pad, while blank control is done, nature controlling line is chosen after reaction
Outlet, positive detection line outlet, and the monoclonal antibody of blank control detection line not outlet is to as pairing monoclonal antibody.
Preferably, in step S4, testing result judgement is carried out after reacting 10min.
Preferably, the colloid gold bar width is 4mm.
Preferably, step S1 specifically comprises the following steps：
Balb/c mouse are immunized in comlete antigen with multiple antigenic determinants, and immune Balb/c mouse can be secreted
The splenocyte of specific antibody is merged with SP2/0 myeloma cell, filters out positive hybridoma cell strain；
Limited dilution cloning culture is carried out to the positive hybridoma cell strain, singling expands culture；
The positive hybridoma cell strain that singling expands after culture is injected into the Balb/c mouse peritoneals of sensitization respectively,
And gather ascites and purified, obtain the specific antibody secreted by different positive monoclonal antibody hybridoma cell strains.
The present invention, which also provides a kind of monoclonal antibody and matches detection method helicobacter pylori in human feces detects, to be answered
With.
The present invention also provides a kind of monoclonal antibody pairing detection means.The monoclonal antibody pairing detection means includes
PVC bottom plates, the NC films for being formed at the PVC bottom plates, it is formed on the NC films and spaced detection line and nature controlling line, solid
The sample pad and blotting paper at the NC films both ends are attached at due to the PVC board and respectively, the detection line is by monoclonal antibody
It is coated on the NC films and is formed, the nature controlling line is coated on the NC films by sheep anti-mouse igg and formed.
Compared with correlation technique, monoclonal antibody provided by the invention matches detection method, and beneficial effect is：
First, the present invention using the screening of double-antibody sandwich colloidal gold immunochromatographimethod detection method for same proteantigen but not
With two monoclonal antibodies of epitope, compared with conventional ELISE detection methods, eliminate the enzyme mark process of complexity, wrapper sheet process,
Sample-adding process, can be detected by an unaided eye within 10 minutes judged result after sample is added dropwise, detection time is quick, and without professional and
Professional equipment can complete detection operation.
2nd, the monoclonal antibody pairing detection method, is further improved to colloidal gold immunochromatographimethod technology, saves
The process of gold standard pad is prepared, and the step of cumbersome intersection pastes gold standard pad, the collaurum for having marked antibody is also direct
It is added dropwise in the sample pad of the colloid gold bar of coated antibody to be screened, it is easy to operate.
3rd, monoclonal antibody pairing detection method provided by the invention, suitable for the antigen with multiple antigenic determinants
Detection；The monoclonal antibody matches detection method, available for the pairing list prepared needed for collaurum double-antibody sandwich detection method
Clonal antibody；For preparing the antibody of the anti-HP for HP antigen different locis；It may be particularly used in quick detection human feces
Middle helicobacter pylori.
【Brief description of the drawings】
Fig. 1 is the structural representation that monoclonal antibody provided by the invention matches detection means；
Fig. 2 is that monoclonal antibody provided by the invention matches detection method testing result display figure.
【Embodiment】
Below in conjunction with drawings and embodiments embodiment, the invention will be further described.
Embodiment one
The present invention provides a kind of monoclonal antibody pairing detection means, and the device is double-antibody sandwich colloidal gold immunochromatographimethod
Detection card.Collaurum is by gold chloride (HAuCl4) reducing agent effect under, polymerization as particular size gold grain, and due to
Electrostatic interaction turns into a kind of colloidal state of stabilization.Colloidal gold immunochromatographimethod technology is to be used as tracer label thing application using collaurum
It is that immuno-gold labeling technology and antigen-antibody reaction are combined and formed in a kind of new immunolabelling technique of antigen-antibody
A kind of application form, have that fast and easy, cost are low, do not need special installation and reagent, stability is strong, result interpretation is directly perceived
The advantages that.
The monoclonal antibody pairing detection means 100 includes PVC bottom plates 11, is formed at the NC films of the PVC bottom plates 11
12nd, it is formed on the NC films 12 and spaced detection line 13 and nature controlling line 14, is fixed on the PVC board 11 and pastes respectively
Sample pad 15 and blotting paper 16 located at the both ends of NC films 12, the detection line 13 are coated in the NC films by monoclonal antibody
Formed on 12, the nature controlling line 14 is coated on the NC films 12 by sheep anti-mouse igg and formed.
The NC films 12 represent nitrocellulose filter, and supporting body is used as in colloid gold test paper, while are also immune response
Point.
The width of the NC films 12 is 4mm, and the spacing between the detection line 13 and the nature controlling line 14 is 5mm, and institute
The line width for stating detection line 13 and the nature controlling line 14 is 1mm.
Compared with colloidal gold immunochromatographimethod detection card in correlation technique, gold standard pad structure is eliminated, it is simplified and makes work
Skill.
Embodiment two
It is row with helicobacter pylori in human feces (HP), elaborates monoclonal antibody pairing detection provided by the invention
Method.
A kind of monoclonal antibody matches detection method, comprises the following steps：
Step S1：Balb/c mouse are immunized in comlete antigen with multiple antigenic determinants, obtain more plants of stable cells
Strain, so as to which different positive monoclonal antibodies be prepared；
Specifically, including：
Step S11, the preparation of anti-human HP monoclonal antibody hybridoma cells
Appropriate HP antigens are taken to be mixed and made into emulsion in equal volume with Freund's complete adjuvant, 6-8 week old is immunized in subcutaneous multi-point injection
Balb/c female mices；Emulsion, subcutaneous multiple spot are mixed and made into incomplete Freund's adjuvant and appropriate HP antigens in equal volume after three weeks
Injecting immune mouse, the potency of blood sampling Elisa indirect Determination antibody after one week；Through people's HP antigen booster immunizations are injected intraperitoneally
Mouse, after three days, mouse is put to death after taking a blood, take out spleen and isolate splenocyte, splenocyte and SP2/0 cells are passed through
PEG mediated fusions；Then selective culture is carried out in HAT culture mediums, only merging successful cell could select to train in HAT
Support and survived in base.
Step S12, the screening of anti-human HP monoclonal antibody hybridoma cells
Take be dissolved in sodium carbonate buffer concentration be 500ng/ml people's HP antigenic solutions 100ul be added to the small of 96 hole elisa Plates
Kong Zhong, 4 DEG C overnight coating, next day by be coated with people's HP antigens 96 hole elisa Plates with washing lotion (PBST buffer solutions) board-washing once, so
Afterwards plus 37 DEG C of confining liquid closes two hours；Confining liquid is outwelled and patted dry, the Hybridoma Cell Culture for then taking step S11 to obtain
Supernatant 100ul is added in 96 hole elisa Plates holes, is incubated at room temperature 1 hour, and board-washing three times, then adds the sheep of 100ul HRP marks
Dynamics, be incubated at room temperature 1 hour, board-washing three times, add 100ul chromogenic substrates (TMB), room temperature lucifuge colour developing 10min,
OD is surveyed at 450nm with ELIASA, clone of the picking OD values more than 0.8 is cooked further subclone experiment, obtain 20 plants of positives
Hybridoma.
Step S13, the colonized culture of hybridoma
Monoclonal hybridoma can be obtained by limiting dilution assay culture.Specific method is as follows, the sun that step S12 is obtained
Property hybridoma count, prepare the single cell suspension that concentration is 1 cell/200ul by a series of dilution；According to
Single cell suspension is assigned in the aperture of 96 porocyte culture plates by the amount in 200ul/ holes, is placed in 37 DEG C, 5% carbon dioxide cell
Cultivated in incubator；About 7 days or so observation number of cell clones, detect antibody, such as positive, then clone, general to clone 3 times.Choosing
The single clone that antibody positive is strong, cell growth is good is selected, culture is enlarged and preserves.
Step S14, the production of anti-human HP monoclonal antibodies
Step S13 is expanded into the positive hybridoma cell strain after culture to be injected into 1x106/ml density respectively
The Balb/c mouse peritoneals of sensitization, belly is substantially expanded, skin has the tight mouse for collapsing sense when touching, and can use No. 12 syringe needles
Ascites is gathered, is further purified with Protein G affinity columns, is purified into by different positive monoclonal antibody hybridomas
The specific antibody of strain secretion, for matching the screening of antibody.
Step S2：Every plant of monoclonal antibody and sheep anti-mouse igg are coated on NC films respectively, as detection line and Quality Control
Sample pad and blotting paper are pasted in line, NC films both ends respectively, are cut into the wide bars of 4mm, and making forms colloid gold bar；
Step S3：Every plant of monoclonal antibody is subjected to colloid gold label respectively；
Step S4：The antibody of colloid gold label is directly added dropwise in a manner of intersecting two-by-two and is being coated with the glue of monoclonal antibody
In the sample pad of body gold bar, then detection sample is also added dropwise in sample pad, while does blank control, nature controlling line is chosen after reaction
All outlets, positive detection line outlet, and the monoclonal antibody of blank control detection line not outlet is to as pairing monoclonal antibody；
Specifically, take the good μ l of Mab17 antibody 2 of colloid gold label that the colloid gold bar in the coated antibody cut is directly added dropwise
Sample pad on, then the μ l of detected sample 50 are also added dropwise in sample pad, while do blank control, react timing 10min, choosing
Take nature controlling line all outlets, positive detection line outlet and more obvious, and the antibody of blank control detection line not outlet is to making
To match monoclonal antibody.
Referring to Fig. 2, match detection method testing result display figure for monoclonal antibody provided by the invention.And combine table
1：
Table 1：Mab17 gold labeling antibodies match result with 20 plants of coated antibodies
Can be drawn with reference to the result that Fig. 1 and table 1 are shown, after Mab17 antibody labelings with Mab1, Mab4, Mab8, Mab10,
Seven kinds of monoclonal antibodies of Mab13, Mab16, Mab20 are successfully matched.
Other two monoclonal antibodies for being directed to same proteantigen but different epitopes are with reference to above detection method.
Compared with correlation technique, monoclonal antibody provided by the invention matches detection method, and beneficial effect is：
First, the present invention using the screening of double-antibody sandwich colloidal gold immunochromatographimethod detection method for same proteantigen but not
With two monoclonal antibodies of epitope, compared with conventional ELISE detection methods, eliminate the enzyme mark process of complexity, wrapper sheet process,
Sample-adding process, can be detected by an unaided eye within 10 minutes judged result after sample is added dropwise, detection time is quick, and without professional and
Professional equipment can complete detection operation.
2nd, the monoclonal antibody pairing detection method, is further improved to colloidal gold immunochromatographimethod technology, saves
The process of gold standard pad is prepared, and the step of cumbersome intersection pastes gold standard pad, the collaurum for having marked antibody is also direct
It is added dropwise in the sample pad of the colloid gold bar of coated antibody to be screened, it is easy to operate.
3rd, monoclonal antibody pairing detection method provided by the invention, suitable for the antigen with multiple antigenic determinants
Detection；The monoclonal antibody matches detection method, available for the pairing list prepared needed for collaurum double-antibody sandwich detection method
Clonal antibody；For preparing the antibody of the anti-HP for HP antigen different locis；It may be particularly used in quick detection human feces
Middle helicobacter pylori.
Above-described is only embodiments of the present invention, it should be noted here that for one of ordinary skill in the art
For, without departing from the concept of the premise of the invention, improvement can also be made, but these belong to the protection model of the present invention
Enclose.
Claims (6)
1. a kind of monoclonal antibody matches detection method, it is characterised in that comprises the following steps：
Step S1：Balb/c mouse are immunized in comlete antigen with multiple antigenic determinants, obtain more plants of stable cell lines, from
And different positive monoclonal antibodies is prepared；
Step S2：Every plant of monoclonal antibody and sheep anti-mouse igg are coated on NC films respectively, as detection line and nature controlling line, NC
Sample pad and blotting paper are pasted in film both ends respectively, and making forms colloid gold bar；
Step S3：Every plant of monoclonal antibody is subjected to colloid gold label respectively；
Step S4：The antibody of colloid gold label is directly added dropwise in a manner of intersecting two-by-two and is being coated with the collaurum of monoclonal antibody
In the sample pad of bar, then by detected sample dropwise addition in sample pad, while blank control is done, nature controlling line is chosen after reaction and is gone out
Line, positive detection line outlet, and the monoclonal antibody of blank control detection line not outlet is to as pairing monoclonal antibody.
2. monoclonal antibody according to claim 1 matches detection method, it is characterised in that in step S4, reacts 10min
Testing result judgement is carried out afterwards.
3. monoclonal antibody according to claim 1 matches detection method, it is characterised in that the colloid gold bar width is
4mm。
4. monoclonal antibody according to claim 1 matches detection method, it is characterised in that step S1 specifically includes as follows
Step：
Balb/c mouse are immunized in comlete antigen with multiple antigenic determinants, and immune Balb/c mouse can be secreted specifically
The splenocyte of property antibody is merged with SP2/0 myeloma cell, filters out positive hybridoma cell strain；
Limited dilution cloning culture is carried out to the positive hybridoma cell strain, singling expands culture；
The positive hybridoma cell strain that singling expands after culture is injected into the Balb/c mouse peritoneals of sensitization respectively, and adopted
Collection ascites is purified, and obtains the specific antibody secreted by different positive monoclonal antibody hybridoma cell strains.
5. a kind of monoclonal antibody as any one of claim 1-4 is matched detection method and imprisoned in human feces are detected
The application of Helicobacter pylori.
6. a kind of monoclonal antibody matches detection means, it is characterised in that including PVC bottom plates, is formed at the NC of the PVC bottom plates
Film, it is formed on the NC films and spaced detection line and nature controlling line, is fixed on the PVC board and is attached at respectively described
The sample pad and blotting paper at NC films both ends, the detection line are coated on the NC films by monoclonal antibody and formed, the Quality Control
Line is coated on the NC films by sheep anti-mouse igg and formed.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113219178A (en) * | 2021-05-12 | 2021-08-06 | 上海理工大学 | Trace antibody rapid pairing immune test paper and pairing method thereof |
| CN114113615A (en) * | 2021-12-03 | 2022-03-01 | 河南省农业科学院 | Immunochromatographic test strip for screening universal monoclonal antibody and detection method |
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| CN104991069A (en) * | 2015-07-17 | 2015-10-21 | 上海理工大学 | Fruit blotch immune colloidal gold detection test strip, and monoclonal antibody and hybridoma cell strain thereof |
| CN105785000A (en) * | 2016-04-21 | 2016-07-20 | 卢连伟 | Kit for rapidly detecting EHFV-IgM antibody |
| CN106632619A (en) * | 2016-12-02 | 2017-05-10 | 杭州贤至生物科技有限公司 | Influenza A virus recombinant protein and preparation of monoclonal antibody thereof |
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2017
- 2017-06-15 CN CN201710453589.4A patent/CN107478840A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104991069A (en) * | 2015-07-17 | 2015-10-21 | 上海理工大学 | Fruit blotch immune colloidal gold detection test strip, and monoclonal antibody and hybridoma cell strain thereof |
| CN105785000A (en) * | 2016-04-21 | 2016-07-20 | 卢连伟 | Kit for rapidly detecting EHFV-IgM antibody |
| CN106632619A (en) * | 2016-12-02 | 2017-05-10 | 杭州贤至生物科技有限公司 | Influenza A virus recombinant protein and preparation of monoclonal antibody thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113219178A (en) * | 2021-05-12 | 2021-08-06 | 上海理工大学 | Trace antibody rapid pairing immune test paper and pairing method thereof |
| CN114113615A (en) * | 2021-12-03 | 2022-03-01 | 河南省农业科学院 | Immunochromatographic test strip for screening universal monoclonal antibody and detection method |
| CN114113615B (en) * | 2021-12-03 | 2024-03-05 | 河南省农业科学院 | Immunochromatography detection test strip for screening universal monoclonal antibodies and detection method |
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