CN108490171A - High-sensitivity saliva antibody detection kit - Google Patents
High-sensitivity saliva antibody detection kit Download PDFInfo
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- CN108490171A CN108490171A CN201810174292.9A CN201810174292A CN108490171A CN 108490171 A CN108490171 A CN 108490171A CN 201810174292 A CN201810174292 A CN 201810174292A CN 108490171 A CN108490171 A CN 108490171A
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- Prior art keywords
- antibody
- detection kit
- highly sensitive
- antibody detection
- salivary
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- 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
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- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
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- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
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- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a high-sensitivity saliva antibody detection kit and a preparation method thereof. Used for detecting a trace amount of virus-specific antigen contained in saliva. The colloidal gold compound connects the recognition antibody and the colloidal gold particles through bridging molecules, so that the labeling efficiency is improved, and the steric hindrance of the colloidal gold is reduced. The invention has high coincidence rate, improves the sensitivity of the saliva antibody detection colloidal gold test paper, has more obvious color development and more accurate detection result.
Description
Technical field
The present invention relates to field of biological detection more particularly to a kind of highly sensitive salivary antibody detection kits.
Technical background
Antibody in traditional human antibodies detection mainly detection blood, is tried as anti-HBs detect
Agent box, human immune defect virus antibody detection kit etc..But in application process, the blood sample of acquisition may carry
Virus is the potential source of infection.Exist to medical testing staff and environment potential dangerous.In addition, lancing test can bring wound
Mouthful, increase the probability of infection.So on the basis of traditional antibody assay kit, antibodies in saliva detection reagent is developed
Box.
The content of antibody is about the 1/50 to 1/800 of Serum Antibody content in saliva.Relative to serum, antibody content pole
Low, if directly directly detecting saliva with the antigen detection kit of detection blood, sensitivity is too low, easily causes false negative.
This just needs to improve detection reagent sensitivity.Present invention employs a kind of method of new colloidal gold labeled monoclonal antibody, antibody with
Between colloid gold particle, bridging molecules are added, prepares gold mark particle composites, the efficiency of colloid gold label is made to significantly improve, from
And enhance colour developing degree, signal amplification is carried out, the sensitivity of Test paper is improved.In clinical detection, the antibody of body generation
As the index for judging whether virus infection, so the use of mouse anti-human igg monoclonal antibody being gold marked reagent, mouse anti-human igg energy
The ends FC of enough specific recognitions and binding domain-immunoglobulin IgG, form three-layer sandwich shape structure, ensure that the accuracy of detection.
Invention content
Present invention the problem of background puts forward in view of the above technology, provide one kind can under the premise of keeping accuracy,
The method for improving detection sensitivity.
A kind of highly sensitive salivary antibody detection kit, including gold mark particle composites are marked in sample pad, in nitric acid
Recombinant antigen is marked in cellulose chromatographic film detection line.For detecting specific antibody in corresponding saliva.
A kind of preparation of the quick anti-liquid detection kit of high clever body saliva, the main preparation for including colloidal gold composite, nitric acid are fine
The plain film coating of dimension.Processes, the specific methods such as sample pad preparation are shown in specific embodiment.
As a further solution of the present invention, the raw material for preparing of gold mark particle includes：The colloidal gold of a diameter of 20-40nm
Grain, Avidin mouse anti-human IgG antibodies, bridge joint immune molecule, bridge joint immune molecule antibody etc., under certain reaction condition, shape
At " Avidin mouse anti-human igg-bridging molecules-gold particle " compound, will be expanded on further in a particular embodiment.
The invention has the advantages that being：By the present invention in that with new colloidal gold-labeled method, make mouse anti-human igg
A certain distance is generated between colloid gold particle, is reduced due to steric hindrance caused by colloidal gold diameter, is made determined antigen
Reaction efficiency between mouse anti-human igg improves；Since there is the biotin of tetravalence to know at the ends FC of the mouse anti-human igg of Avidin
Other site.By the way that the connection function of the bridging molecules of biotin is marked, 2 colloid gold particles theoretically can be at least carried,
The labeling effciency that colloid gold particle and gold marked reagent can be improved makes detection line colour developing deepen, to improve the sensitivity of detection.
Specific implementation mode
In order to make the present invention advantage and technical solution illustrate be more clear, with reference to embodiments, to the present invention into
Row is further elucidated above.
Embodiment 1.
The present embodiment is with hepatitis B virus antibody in saliva（HCV）To detect object.
Step 1：Colloid gold particle is prepared, using citric acid reduction method, the colloid gold particle for preparing 20-40nm diameters is standby
With.
Step 2:Prepare bridging molecules：As an implementation, select oligo DNA chain as bridging molecules, length is about
20-30bp uses biotin labeling, 5 ' ends to use digoxin at 3 ' ends（Bridging molecules 1）And FAM（Bridging molecules 2）It marks respectively
Note.Bridging molecules can be bought by business DNA Synesis Company, and the bridging molecules 1 and bridging molecules 2 in the present embodiment are bought in Shanghai
Raw work.
Bridging molecules 1：bio-TTTTTTTTTTTTTTTTTTTT-DIG.
Bridging molecules 2：Bio-TTTTTTTTTTTTTTTTTTTT-FAM.
Step 3：Colloid gold particle is coated with：Gold mark particle 1 is that DigiTAb is marked, and it is anti-that gold mark particle 2 has been coated with FAM
Body, monoclonal antibody are purchased in biological agent company.Labeling method is：Under stirring, PH is reconciled using carbonic acid buffer
To between 6.5-7.0, addition 3-4ul DigiTAbs or FAM antibody continue stirring 30 minutes, and detection absorbance is OD525-
530nm=1.7-2.0. carries out centrifugal treating.It goes after supernatant, precipitation is answered using the buffer solution that PH is 6.5-7.0
Melt.
Step 4：Above-mentioned coated colloid gold particle 1 and 2 is mixed in equal volume, mixed system is made.Again by above-mentioned mixing
Liquid and bridging molecules 1, bridging molecules 2, Avidin mouse anti-human igg with 4：2：2:1 is stirred mixing, and reaction temperature is normal
Temperature, NaCl concentration is 0.85%, PH 6-9 in reaction system.During the reaction, lasting stirring makes solution keep clarification shape
State.After reacting 30min, digoxin and FAM molecules is added, neutralizes recognition site, terminates reaction.Then carry out cryogenic freezing centrifugation
It is purified on machine, time 50-60min, revolution 1-8000-12000rpm, 4-8 DEG C of temperature.Discard supernatant, sediment is answered melt to
30-40OD。
Step 5:Protected protein and surfactant is added in coated colloidal gold composite, is sprayed into glass fiber sample
On pad.Drying for standby.
HCV haptens is coated in detection line by step 6, and Quality Control antibody protein is marked on control line.It is dry.
Experiment grouping：100 parts of the HCV saliva samples of all ages and classes, different gradient of infection are chosen, while to sample supplier
Blood specimen collection is carried out, is detected by fluorescence quantitative PCR method, positive sample is 61 parts, and wherein weakly positive sample is 12
Part.
Above-mentioned sample is detected.Using two kinds of test strips.One is the highly sensitive examinations of above-described embodiment description
Paper, another kind are directly to carry out contrast test using test paper made of mouse anti-human igg label gold particle.
The actually detected result of the saliva HCV detection kits prepared by above-described embodiment is it is found that the label for passing through innovation
Method can reduce the detection limit of product, and to improve the sensitivity of detection, and it is uniform to develop the color, and is easy to interpretation.
The above, preferable specific embodiment only of the invention, but protection scope of the present invention are not limited to above-mentioned
Embodiment, any one skilled in the art is within the scope of the present invention, the optimization that can readily occur in or
It replaces, should all be included within the scope of the present invention.
Claims (6)
1. a kind of highly sensitive salivary antibody detection kit, it is characterised in that：It is compound including spraying colloidal gold on the glass fibers
Object is coated with recombinant antigen on cellulose nitrate film, for detecting the special viral antibody in saliva.
2. a kind of highly sensitive salivary antibody detection kit according to claim 1, it is characterised in that：Colloidal gold composite
Structure be：The biotin and avidin identification of bridging molecules one end combines, the digoxin specific binding of other end label
The FAM specific bindings of the DigiTAb of label on the gold particle, label mark FAM antibody on the gold particle, form glue
Body Au composite.
3. a kind of highly sensitive salivary antibody detection kit according to claim 2, it is characterised in that：The bridge joint point
Son is the DNA fragmentation of one section of 20-30bp, and marks biotin molecule at one end, and the other end marks digoxin or FAM deciles
Son.
4. a kind of highly sensitive salivary antibody detection kit according to claim 2, it is characterised in that：The bridge joint point
Sub- specific antibody is digoxin monoclonal antibody or FAM antibody.
5. a kind of highly sensitive salivary antibody detection kit according to claim 2, it is characterised in that：Colloid gold particle is straight
Diameter is 20-40nm.
6. a kind of highly sensitive salivary antibody detection kit according to claim 3, it is characterised in that：Two kinds of bridges
The phenomenon that connecing between molecule without base complementrity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810174292.9A CN108490171A (en) | 2018-03-02 | 2018-03-02 | High-sensitivity saliva antibody detection kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810174292.9A CN108490171A (en) | 2018-03-02 | 2018-03-02 | High-sensitivity saliva antibody detection kit |
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| CN201810174292.9A Pending CN108490171A (en) | 2018-03-02 | 2018-03-02 | High-sensitivity saliva antibody detection kit |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111856003A (en) * | 2020-02-04 | 2020-10-30 | 潍坊市康华生物技术有限公司 | Novel coronavirus (2019-nCoV) IgM and IgG antibody detection test strip, kit and method |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5989813A (en) * | 1995-07-13 | 1999-11-23 | Molecular Innovations, Inc. | Detection of amplified nucleic acid sequences using bifunctional haptenization and dyed microparticles |
| CN1847852A (en) * | 2006-05-10 | 2006-10-18 | 杭州优思达生物技术有限公司 | Fast mononucleotide polymorphism detecting test paper strip and its detection method |
| CN101943699A (en) * | 2010-08-31 | 2011-01-12 | 广州万孚生物技术有限公司 | Test strip for detecting HIV antibodies in spittle and preparation method thereof |
| CN102539755A (en) * | 2011-12-20 | 2012-07-04 | 广州万孚生物技术有限公司 | Test strip for detecting influenza A virus antigen in secretion and preparation method thereof |
| CN104105965A (en) * | 2011-11-21 | 2014-10-15 | 爱贝斯股份有限公司 | Signal amplification in lateral flow and related immunoassays |
| CN104726606A (en) * | 2015-04-09 | 2015-06-24 | 南京爱佩捷生物科技有限公司 | Method for detecting pathogenic microorganisms by using PCR (polymerase chain reaction) enzyme-linked double-cross method |
| CN105154576A (en) * | 2015-10-26 | 2015-12-16 | 西南大学 | Primer pair for rapidly detecting silkworm microsporidia and reagent kit and detection method thereof |
| CN106248974A (en) * | 2016-07-11 | 2016-12-21 | 武汉百美生物科技有限公司 | A kind of hypersensitivity immunity chromatography detection test paper |
-
2018
- 2018-03-02 CN CN201810174292.9A patent/CN108490171A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5989813A (en) * | 1995-07-13 | 1999-11-23 | Molecular Innovations, Inc. | Detection of amplified nucleic acid sequences using bifunctional haptenization and dyed microparticles |
| CN1847852A (en) * | 2006-05-10 | 2006-10-18 | 杭州优思达生物技术有限公司 | Fast mononucleotide polymorphism detecting test paper strip and its detection method |
| CN101943699A (en) * | 2010-08-31 | 2011-01-12 | 广州万孚生物技术有限公司 | Test strip for detecting HIV antibodies in spittle and preparation method thereof |
| CN104105965A (en) * | 2011-11-21 | 2014-10-15 | 爱贝斯股份有限公司 | Signal amplification in lateral flow and related immunoassays |
| CN102539755A (en) * | 2011-12-20 | 2012-07-04 | 广州万孚生物技术有限公司 | Test strip for detecting influenza A virus antigen in secretion and preparation method thereof |
| CN104726606A (en) * | 2015-04-09 | 2015-06-24 | 南京爱佩捷生物科技有限公司 | Method for detecting pathogenic microorganisms by using PCR (polymerase chain reaction) enzyme-linked double-cross method |
| CN105154576A (en) * | 2015-10-26 | 2015-12-16 | 西南大学 | Primer pair for rapidly detecting silkworm microsporidia and reagent kit and detection method thereof |
| CN106248974A (en) * | 2016-07-11 | 2016-12-21 | 武汉百美生物科技有限公司 | A kind of hypersensitivity immunity chromatography detection test paper |
Non-Patent Citations (1)
| Title |
|---|
| 候巧华: "基于LCR、PCR核酸试纸条检测食源性致病菌方法的研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111856003A (en) * | 2020-02-04 | 2020-10-30 | 潍坊市康华生物技术有限公司 | Novel coronavirus (2019-nCoV) IgM and IgG antibody detection test strip, kit and method |
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