CN106680505A - Latex-enhanced immunoturbidimetry detection method based on multiple mutually-matchable monoclonal antibodies - Google Patents
Latex-enhanced immunoturbidimetry detection method based on multiple mutually-matchable monoclonal antibodies Download PDFInfo
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- CN106680505A CN106680505A CN201710004878.6A CN201710004878A CN106680505A CN 106680505 A CN106680505 A CN 106680505A CN 201710004878 A CN201710004878 A CN 201710004878A CN 106680505 A CN106680505 A CN 106680505A
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- latex
- monoclonal antibody
- monoclonal antibodies
- antibody
- many plants
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—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
Abstract
The invention relates to a latex-enhanced immunoturbidimetry detection method based on multiple mutually-matchable monoclonal antibodies; in the method, multiple monoclonal antibodies capable of matching pairwise to one another are used to modify latex microspheres, corresponding antigens are detected via latex-enhanced immunoturbidimetry to obtain antigen content information of a sample under high sensitivity and specificity. Compared with traditional polyclonal antibody labeled latex-enhanced immunoturbidimetry, the latex-enhanced immunoturbidimetry detection method based on multiple mutually-matchable monoclonal antibodies has the advantages that monoclonal antibodies have high specificity, more bonding sites are provided for antigens since the monoclonal antibodies which are matchable pairwise to one another are used, the antibodies never compete for bonding sites, and detection sensitivity can be improved accordingly.
Description
Technical field
The present invention relates to medical science detection and biological technical field, more particularly to Dan Ke is mutually paired based on many plants based on one kind
The latex enhancing immune of grand antibody is than turbid detection method.It is antigen detection means present invention can apply to the detection of hypersensitive antigen
A kind of alternative method is provided.
Background technology
Latex enhancing immune turbidimetry is that antigen-antibody combines dynamic assay method.Its general principle is when antigen and antibody
The reaction and when ratio is suitable in special dilution system, the coagulant of the soluble immune complex of formation in dilution system
In the presence of, separated out from liquid phase, forming particulate makes reaction solution turbidity occur, so as to calculate the content of antigen in measuring samples.
This method can detect that high degree of automation improves speed, sensitivity and the ease for operation of detection with instrument, be adapted to a large amount of
The clinical detection of sample.Traditional latex enhancing immune is with latex microsphere mark polyclonal antibody, but many grams than purifying method
Grand antibody can be combined with various epitopes, therefore polyclonal antibody is specific poor.Single monoclonal antibody is only recognized
A certain specific antigen epitope, therefore the specificity of monoclonal antibody is higher, but the specificity of this superelevation can drop sensitiveness
Low, so we devise the method detected using many plants of monoclonal antibodies, this method is to overcome polyclonal antibody
The low shortcoming of specificity, overcomes the low shortcoming of single monoclonal antibody sensitiveness, and utilize what is be mutually paired two-by-two again
Monoclonal antibody, the binding site for antigen is more, and the competition of binding site will not be formed between antibody and antibody, thus more
Plus the sensitivity that improve detection.
The content of the invention
The present invention devises the method that many plants of monoclonal antibodies are coated with microballoon simultaneously.The method and traditional polyclonal antibody
Mark latex enhancing immune is that the specificity of monoclonal antibody is higher than the advantage of purifying method.Single monoclonal antibody is only
Recognize a certain specific antigen epitope, the specificity of this superelevation can reduce sensitiveness, so we are devised using many plants of lists
The method that clonal antibody is detected, this method is to overcome the low shortcoming of polyclonal antibody specificity, is overcome again single
The low shortcoming of monoclonal antibody sensitiveness.
Many plants of monoclonal antibodies being mutually paired two-by-two are screened.Binding site for antigen is more, antibody and antibody
Between will not form the competition of binding site, thus detection sensitivity can be improved.
The many plants of monoclonal antibodies, are mainly characterized by：Including but not limited to three plants or more than three plants of monoclonal resists
Body.
It is described to determine that the experiment matched two-by-two be but not limited to ELISA and be related to double-antibody sandwich testing sieve select
Paired monoclonal antibody etc..
Compared with prior art, the invention has the characteristics that：
Traditional latex enhancing immune is with latex microsphere mark polyclonal antibody, but Anti-TNF-α physical efficiency than purifying method
Combined with various epitopes, therefore polyclonal antibody is specific poor.Single monoclonal antibody only recognizes a certain specific
Epitope, therefore the specificity of monoclonal antibody is higher, but the specificity of this superelevation can reduce sensitiveness, so I
Devise the method detected using many plants of monoclonal antibodies, this method is that to overcome polyclonal antibody specificity low
Shortcoming, overcomes the low shortcoming of single monoclonal antibody sensitiveness again, and we are using the list that mutually can be matched two-by-two
Clonal antibody more increased the sensitiveness of detection.
Brief description of the drawings
Below in conjunction with drawings and Examples, the invention will be further described.
Fig. 1 is a kind of to be illustrated than turbid detection method flow based on many plants of latex enhancing immunes for being mutually paired monoclonal antibody
Figure.
Fig. 2 is that latex microsphere is coated with three plants of monoclonal antibodies, latex microsphere coating tri- kinds of methods of polyclonal antibody and ELISA
The ROC curve of GP73 contents in detection serum.
Specific embodiment
Below in conjunction with example, the invention will be further described：
The latex enhancing immune of monoclonal antibody is mutually matched two-by-two than turbid detection based on many plants the invention discloses a kind of
Method, latex microsphere is modified with many plants of monoclonal antibodies that mutually can be matched two-by-two, is carried out latex enhancing immune and is compared purifying method
Detection correspondence antigen, you can obtain antigenic content information in sample.In one of the invention preferably example, with carboxylated
Latex microsphere is coated with three plants of monoclonal antibodies matched two-by-two, and the detection of signal is carried out on automatic clinical chemistry analyzer.
Embodiment：Detected by object of hepatic carcinoma mark GP73 albumen.
1. monoclonal antibody is coated with latex microsphere.By the latex microsphere (100 μ L with 5%wt/vol) of 107nm
It is scattered in the HEPES solution (pH 7.0) of 50mM, adds three plants of monoclonal antibodies (0.5mg/mL) to be incubated on shaking table
30min.Add the NHS (N-hydroxysulfosuccinimide) and EDC (1-ethyl-3- [3- of 0.5mg/mL
Dimethylaminopropyl carbodiimide hydrochloride) be incubated 30min after with 1% confining liquid BSA
(Bovine Serum Albumin) closes 30min.The coated antibody of latex microsphere is dispersed in the Tris-HCl containing 1%BSA
In (1.0Mm, pH 6.8), ultrasound to solution printing opacity.Reagent be stored in 4 DEG C it is stand-by.
2. pattern detection.Pattern detection is carried out in automatic biochemistry analyzer, the reagent 1 of the sample of 10 μ L and 240 μ L
(10mM Tris-HCl (pH 7.0), 150mM NaCl, 0.01%Tween-20and PEG-6000) is added in reaction tray,
After being incubated 5min, reagent 2 (the coated monoclonal antibody of latex microsphere) is added to start immunoturbidimetry reaction.Recorded after reaction 5min
At 570nm wavelength at 5min and 10min absorbance change.
3. immunoturbidimetry testing result is as shown in Figure 2.Latex microsphere is coated with three plants of reaction system detections of monoclonal antibody
The Sensitivity and Specificity (96.7% and 93.3%) of GP73 is above latex microsphere coating Anti-TNF-α in liver cancer serum sample
Body (94.6% and 72.4%) and ELISA (70.0% and 83.3%).
The above, is only presently preferred embodiments of the present invention, and any formal limitation is not made to the present invention, any ripe
Professional and technical personnel is known, it is without departing from the scope of the present invention, real to more than according to technical spirit of the invention
Apply any simple modification, equivalent that example made and improve etc., still fall within technical solution of the present invention protection domain it
It is interior.
Claims (4)
- It is 1. a kind of to be mutually paired the latex enhancing immune of monoclonal antibody than turbid detection method based on many plants, it is characterised in that：With Monoclonal antibody modifies latex microsphere, latex enhancing immune is carried out than purifying method detection correspondence antigen, so as to resist in obtaining sample Former content information；Monoclonal antibody employed in it is many plants of monoclonal antibodies, and many plants of monoclonal antibodies mutual two Two pairings.
- 2. it is according to claim 1 to be mutually paired the latex enhancing immune of monoclonal antibody than turbid detection side based on many plants Method, it is characterised in that：The many plants of monoclonal antibodies include three plants or more than three plants of monoclonal antibody.
- 3. it is according to claim 1 to be mutually paired the latex enhancing immune of monoclonal antibody than turbid detection side based on many plants Method, it is characterised in that：The monoclonal antibody matched two-by-two is the paired monoclonal antibody by filtering out.
- 4. it is according to claim 3 to be mutually paired the latex enhancing immune of monoclonal antibody than turbid detection side based on many plants Method, it is characterised in that：The monoclonal antibody matched two-by-two is the paired list selected by using double-antibody sandwich testing sieve Clonal antibody.
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| CN201710004878.6A CN106680505A (en) | 2017-01-04 | 2017-01-04 | Latex-enhanced immunoturbidimetry detection method based on multiple mutually-matchable monoclonal antibodies |
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| CN201710004878.6A CN106680505A (en) | 2017-01-04 | 2017-01-04 | Latex-enhanced immunoturbidimetry detection method based on multiple mutually-matchable monoclonal antibodies |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108008130A (en) * | 2017-11-24 | 2018-05-08 | 海格德生物科技（深圳）有限公司 | Kit based on latex enhancing immune turbidimetry for Determination Lp-PLA2 and preparation method thereof |
| CN110865192A (en) * | 2019-11-21 | 2020-03-06 | 安徽大千生物工程有限公司 | Kit for determining GP73 based on latex enhanced immunoturbidimetry and preparation and use methods thereof |
Citations (5)
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| JPH10123137A (en) * | 1996-10-16 | 1998-05-15 | Sekisui Chem Co Ltd | Highly sensitive immunoassay method |
| EP2042870A1 (en) * | 2007-09-28 | 2009-04-01 | Fujifilm Corporation | Method of high sensitive immunoassay |
| CN102944679A (en) * | 2012-11-15 | 2013-02-27 | 北京康美天鸿生物科技有限公司 | Kit for performing retinol binding protein detection by using latex turbidimetry |
| CN104407145A (en) * | 2014-10-28 | 2015-03-11 | 绍兴市疾病预防控制中心 | Enterovirus 71 type latex agglutination detection kit, preparation and application |
| CN105548557A (en) * | 2015-12-08 | 2016-05-04 | 苏州德沃生物技术有限公司 | Hypersensitive latex-reinforced immunological turbidimetry detection agent and its preparation method and use |
-
2017
- 2017-01-04 CN CN201710004878.6A patent/CN106680505A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10123137A (en) * | 1996-10-16 | 1998-05-15 | Sekisui Chem Co Ltd | Highly sensitive immunoassay method |
| EP2042870A1 (en) * | 2007-09-28 | 2009-04-01 | Fujifilm Corporation | Method of high sensitive immunoassay |
| CN102944679A (en) * | 2012-11-15 | 2013-02-27 | 北京康美天鸿生物科技有限公司 | Kit for performing retinol binding protein detection by using latex turbidimetry |
| CN104407145A (en) * | 2014-10-28 | 2015-03-11 | 绍兴市疾病预防控制中心 | Enterovirus 71 type latex agglutination detection kit, preparation and application |
| CN105548557A (en) * | 2015-12-08 | 2016-05-04 | 苏州德沃生物技术有限公司 | Hypersensitive latex-reinforced immunological turbidimetry detection agent and its preparation method and use |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108008130A (en) * | 2017-11-24 | 2018-05-08 | 海格德生物科技（深圳）有限公司 | Kit based on latex enhancing immune turbidimetry for Determination Lp-PLA2 and preparation method thereof |
| CN110865192A (en) * | 2019-11-21 | 2020-03-06 | 安徽大千生物工程有限公司 | Kit for determining GP73 based on latex enhanced immunoturbidimetry and preparation and use methods thereof |
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Application publication date: 20170517 |