CN1540351A - Colloidal Gold tag method for identifying virus single chained antibody of sprawn leuckoplakia yndrome - Google Patents
Colloidal Gold tag method for identifying virus single chained antibody of sprawn leuckoplakia yndrome Download PDFInfo
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- CN1540351A CN1540351A CNA2003101112932A CN200310111293A CN1540351A CN 1540351 A CN1540351 A CN 1540351A CN A2003101112932 A CNA2003101112932 A CN A2003101112932A CN 200310111293 A CN200310111293 A CN 200310111293A CN 1540351 A CN1540351 A CN 1540351A
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Abstract
In the method, single chained antibody A1 tagged by colloidal gold is obtained through following steps: purifying and dialyzing single chained antibody, and preparing pH value; determining optimal steady quantity of mixing solution of colloidal gold and single chained antibody, and mixing the said solution and antibody; centrifuging to remove supernatant solution so as to obtain single chained antibody A1 tagged by colloidal gold. The method simplifies operation step for detecting the virus, and decreases testing time and cost. The single chained antibody A1 tagged by colloidal gold is applicable for detecting virus, locating structure of virus, and tracking course of infection.
Description
Technical field:
The present invention relates to Protocols in Molecular Biology and be applied to culture fishery, more specifically relate to and use the colloid gold label single-chain antibody.
Background technology:
Since 1993, China's prawn disease burst is popular, causes the death of cultured prawn large tracts of land, the economic loss heaviness.Studies show that it mainly is by due to a kind of new dna double chain virus one prawn white spot syndrome virus (WSSV).This virus can infect the prawn kind of all artificial sea-farmings of China, and infectious strong, rapid onset, and the mortality ratio height brings great harm to shrimp culture industry; It is reported that also there is analogue other countries and regions.At present, prawn virus disease does not still have effective methods of treatment, avoid contacting with virus causing disease being considered to proper prophylactic methods, and this mainly depends on early stage quick diagnosis.At present reliable and fast diagnostic method mainly be PCR, Nucleic Acid Probe Technique and enzyme linked immunological adsorption technology.But PCR and Nucleic Acid Probe Technique can only depend on the laboratory, and technical conditions are had relatively high expectations, and vast shrimp farming is grasped this technology and had very big difficulty.Though being expected to develop into, the enzyme linked immunological adsorption technology can support the diagnostic techniques that use the shrimp field, but, limited application and the development of this technology aspect the diagnosis prawn virus disease greatly because required polyclonal antibody and the MONOCLONAL ANTIBODIES SPECIFIC FOR of this method bothers and the expense costliness very much.Because conventional immune marking method also needs two to resist, detection time and cost have inevitably been increased again.
" a kind of single-chain antibody A 1 of discerning white spot syndrome baculovirus and uses thereof ", number of patent application: 00131231.6, the applying date: on Dec 8th, 2000, applicant: Inst. of Hydrobiology, Chinese Academy of Sciences.This application describes dna sequence dna, the preparation method and its usage of the single-chain antibody A 1 of identification white spot syndrome baculovirus in detail.Owing to different names is arranged, the domestic now unified called after " white spot syndrome virus (WSSV) " of this name before this application described " white spot syndrome baculovirus ".
Colloidal gold-labeled method has been widely used in medical science and RESEARCH ON CELL-BIOLOGY field, is one of responsive method commonly used in the present immunohistochemistry technology.Collaurum is the hydrosol of gold chloride, has high electron density, and can combine with multiple biomacromolecule, is a kind of nonradioactive tracer commonly used in the immunolabelling technique.In recent years, the colloid gold label of IgG antibody is most widely used, and its technical conditions have versatility.And single-chain antibody only is the variable part of antibody, has only the molecular weight of complete antibody 1/6, so the mark of single-chain antibody can not must be groped condition again with reference to the condition of IgG fully.
Summary of the invention:
The colloid gold label method that the purpose of this invention is to provide a kind of Single-chain Antibody Recognizing Tache Blanche Virus Of Prawn A1, this method is purified with the white spot syndrome virus (WSSV) single-chain antibody, dialysis, allotment pH value, determine best stabilized amount that colloidal gold solution and single-chain antibody mix, mixed, centrifugal colloidal gold solution and single-chain antibody, remove supernatant, promptly obtain the single-chain antibody A 1 of colloid gold label.This method can be simplified the operation steps that detects white spot syndrome virus (WSSV), makes detection time and cost reduce greatly.
In order to achieve the above object, the present invention adopts following technical scheme:
The colloid gold label method of a kind of Single-chain Antibody Recognizing Tache Blanche Virus Of Prawn A1 follows these steps to order and carries out:
1, (this single-chain antibody is application number: 00131231.6 to get Single-chain Antibody Recognizing Tache Blanche Virus Of Prawn A1, the applying date: on Dec 8th, 2000, applicant: Inst. of Hydrobiology, Chinese Academy of Sciences, denomination of invention is " single-chain antibody A 1 of identification white spot syndrome baculovirus " described in " a kind of single-chain antibody A 1 of discerning white spot syndrome baculovirus and uses thereof "), with affinity chromatography purification of single stranded antibody A 1, and in the solution of 0.005mol/L NaCl pH7.0, dialyse;
2, the colloidal gold solution for preparing 20-30nm with the sodium citrate reducing process is pH 5.8 with the isoelectric point of gel electrophoresis therapy determining single-chain antibody A 1, and the pH of colloidal gold solution is transferred to 6.2, makes the pH of colloidal gold solution be higher than single-chain antibody A 1 isoelectric point 0.5 pH unit;
3, mixed with 1 milliliter of colloidal gold solution with 0.1 milliliter of single-chain antibody A 1 that contains the different protein concentrations of 0.1-0.5 milligram, after 5 minutes, the NaCl solution that adds 0.1 milliliter 10%, placed 1-2 hour, observe the change color of solution, the best stabilized amount that is defined as antibody that protein concentration in the redness is minimum;
4,3 best stabilized amounts of determining set by step evenly join single-chain antibody A 1 in the colloidal gold solution stirring at room 30 minutes with 5 fens clock times;
5, with 12000 rev/mins centrifugal 60 minutes, remove supernatant, sediment is the single-chain antibody A 1 of colloid gold label.
6, sediment is dissolved in the preservation liquid of used colloidal gold solution 1/10 volume of step 4, in four degrees celsius, preserves;
Wherein: the prescription of preserving liquid is every liter and contains: sodium tetraborate 0.4 gram, bovine serum albumin(BSA) 1.0 grams, Sodium azide 0.1 restrain, the rest is ultrapure water, transfer pH to 7.4 with the hydrochloric acid of 6 equivalents.
The present invention compared with prior art has the following advantages and effect:
1, be used for the detection of white spot syndrome virus (WSSV): with testing sample 5 μ l points to the NC film, wrap by 5 minutes with 1%BSA/PBS, put into collaurum-A1 solution then took out in 5-10 minute, wash with water, observe spot colors, what the brownish red spot was arranged is to contain white spot syndrome virus (WSSV) in the sample.
2, be used for the colloid gold immune electron microscopy: after the sick shrimp organization embedding that white spot syndrome virus (WSSV) can be infected, the section, with collaurum-A1 insulation, flush away is the association colloid gold not, use electron microscopic observation then, can study the location of antigenic determinant on virus structure of single-chain antibody A 1, and the course of infection of spike virus.
3, the phage antibody display technique has not only been avoided in preparation polyclonal antibody process by tiring and narrow spectrum difference that individuality causes, also avoided numerous and diverse hybridoma technology in preparation monoclonal antibody process, and it is simple to operate, economical, the screening scope is wide, and making a large amount of preparation antibody or batch production produce antibody becomes possibility.Single-chain antibody A 1 can obtain the antibody of a large amount of anti-white spot syndrome virus (WSSV) by the cultivation of bacterium, makes the cost of Antibody Preparation reduce greatly.The success of single-chain antibody A 1 colloid gold label, the cost that makes white spot syndrome virus (WSSV) detect further reduces, and also can remove two from and resist, and makes trace routine obtain very big simplification.
Embodiment:
The colloid gold label method of a kind of Single-chain Antibody Recognizing Tache Blanche Virus Of Prawn A1 follows these steps to order and carries out:
1, (this single-chain antibody is application number: 00131231.6 to get Single-chain Antibody Recognizing Tache Blanche Virus Of Prawn A1, the applying date: on Dec 8th, 2000, applicant: Inst. of Hydrobiology, Chinese Academy of Sciences, denomination of invention is " single-chain antibody A 1 of identification white spot syndrome baculovirus " described in " a kind of single-chain antibody A 1 of discerning white spot syndrome baculovirus and uses thereof "), with affinity chromatography purification of single stranded antibody A 1, and in the solution of 0.005mol/L NaCl pH7.0, dialyse;
2, the colloidal gold solution for preparing 20-30nm with the sodium citrate reducing process is pH 5.8 with the isoelectric point of gel electrophoresis therapy determining single-chain antibody A 1, and the pH of colloidal gold solution is transferred to 6.2, makes the pH of colloidal gold solution be higher than single-chain antibody A 1 isoelectric point 0.5pH unit;
3, mixed with 1 milliliter of colloidal gold solution with 0.1 milliliter of single-chain antibody A 1 that contains the different protein concentrations of 0.1-0.5 milligram, after 5 minutes, the NaCl solution that adds 0.1 milliliter 10%, placed 1-2 hour, observe the change color of solution, the best stabilized amount that is defined as antibody that protein concentration in the redness is minimum;
4,3 best stabilized amounts of determining set by step evenly join single-chain antibody A 1 in the colloidal gold solution stirring at room 30 minutes with 5 fens clock times;
5, with 12000 rev/mins centrifugal 60 minutes, remove supernatant, sediment is the single-chain antibody A 1 of colloid gold label;
6, to other the colloid gold label method of single-chain antibody of identification white spot syndrome virus (WSSV), carry out according to the method for step 1-5 equally.
7, sediment is dissolved in the preservation liquid of used colloidal gold solution 1/10 volume of step 4, in four degrees celsius, preserves;
Wherein: the prescription of preserving liquid is every liter and contains: sodium tetraborate 0.4 gram, bovine serum albumin(BSA) 1.0 grams, Sodium azide 0.1 restrain, the rest is redistilled water, transfer pH to 7.4 with the hydrochloric acid of 6 equivalents.
Claims (2)
1, the colloid gold label method of a kind of Single-chain Antibody Recognizing Tache Blanche Virus Of Prawn A1 is characterized in that, this method follows these steps to order and carries out:
A, get Single-chain Antibody Recognizing Tache Blanche Virus Of Prawn A1,, and in the solution of 0.005mol/L NaCl pH7.0, dialyse with affinity chromatography purification of single stranded antibody A 1;
B, preparing the colloidal gold solution of 20-30nm with the sodium citrate reducing process, is pH5.8 with the isoelectric point of gel electrophoresis therapy determining single-chain antibody A 1, and the pH of colloidal gold solution is transferred to 6.2, makes the pH of colloidal gold solution be higher than single-chain antibody A 1 isoelectric point 0.5pH unit;
C, mixed with 0.1 milliliter of single-chain antibody A 1 that contains the different protein concentrations of 0.1-0.5 milligram with 1 milliliter of colloidal gold solution, after 5 minutes, the NaCl solution that adds 0.1 milliliter 10%, placed 1-2 hour, observe the change color of solution, the best stabilized amount that is defined as antibody that protein concentration in the redness is minimum;
D, the best stabilized amount determined of c set by step evenly join single-chain antibody A 1 in the colloidal gold solution stirring at room 30 minutes with 5 fens clock times;
E, with 12000 rev/mins centrifugal 60 minutes, remove supernatant, sediment is the single-chain antibody A 1 of colloid gold label.
2, the colloid gold label method of a kind of Single-chain Antibody Recognizing Tache Blanche Virus Of Prawn according to claim 1, it is characterized in that, the single-chain antibody A 1 of colloid gold label is dissolved in the preservation liquid of used colloidal gold solution 1/10 volume in claim 1 step, in four degrees celsius, preserves;
Wherein: the prescription of preserving liquid is every liter and contains: sodium tetraborate 0.4 gram, bovine serum albumin(BSA) 1.0 grams, Sodium azide 0.1 restrain, the rest is redistilled water, transfer pH to 7.4 with the hydrochloric acid of 6 equivalents.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101112932A CN1325916C (en) | 2003-10-30 | 2003-10-30 | Colloidal Gold tag method for identifying virus single chained antibody of sprawn leuckoplakia yndrome |
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| CNB2003101112932A CN1325916C (en) | 2003-10-30 | 2003-10-30 | Colloidal Gold tag method for identifying virus single chained antibody of sprawn leuckoplakia yndrome |
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| CN1540351A true CN1540351A (en) | 2004-10-27 |
| CN1325916C CN1325916C (en) | 2007-07-11 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1322009C (en) * | 2004-12-17 | 2007-06-20 | 中国海洋大学 | Monoclonal antibody of virus receptor of anti leukoplakia disease and preparation method |
| CN101915834A (en) * | 2010-06-02 | 2010-12-15 | 南京农业大学 | Colloidal gold colloidal gold detection test paper strip of shrimp TSV (Taura Syndrome Virus) |
| CN1932520B (en) * | 2006-09-29 | 2012-03-21 | 武汉大学 | Test paper bar for detecting praw white spot syndrome virus and producing process thereof |
| CN102776237A (en) * | 2012-06-12 | 2012-11-14 | 西安交通大学 | Cavitation-bubble-mediated laser cell transfection method |
| CN103995121A (en) * | 2014-02-21 | 2014-08-20 | 大连大学 | Preparation method for colloidal-gold test paper based on single-chain antibody |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002040027A (en) * | 2000-07-27 | 2002-02-06 | Yakult Honsha Co Ltd | Immunological measuring method |
| CN1184329C (en) * | 2000-08-30 | 2005-01-12 | 中国水产科学研究院黄海水产研究所 | Reagent kit and method for testing epidemic disease pathogen of prawn |
| CN1118704C (en) * | 2000-12-08 | 2003-08-20 | 中国科学院水生生物研究所 | Reagent kit for diagnosing white-spot baculovirus of prawn and its test method |
-
2003
- 2003-10-30 CN CNB2003101112932A patent/CN1325916C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1322009C (en) * | 2004-12-17 | 2007-06-20 | 中国海洋大学 | Monoclonal antibody of virus receptor of anti leukoplakia disease and preparation method |
| CN1932520B (en) * | 2006-09-29 | 2012-03-21 | 武汉大学 | Test paper bar for detecting praw white spot syndrome virus and producing process thereof |
| CN101915834A (en) * | 2010-06-02 | 2010-12-15 | 南京农业大学 | Colloidal gold colloidal gold detection test paper strip of shrimp TSV (Taura Syndrome Virus) |
| CN101915834B (en) * | 2010-06-02 | 2013-05-01 | 南京农业大学 | Colloidal gold colloidal gold detection test paper strip of shrimp TSV (Taura Syndrome Virus) |
| CN102776237A (en) * | 2012-06-12 | 2012-11-14 | 西安交通大学 | Cavitation-bubble-mediated laser cell transfection method |
| CN103995121A (en) * | 2014-02-21 | 2014-08-20 | 大连大学 | Preparation method for colloidal-gold test paper based on single-chain antibody |
| CN103995121B (en) * | 2014-02-21 | 2015-11-18 | 大连大学 | Based on the preparation method of the gold test strip of single-chain antibody |
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| CN1325916C (en) | 2007-07-11 |
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Assignee: Suzhou Haid Feeds Co., Ltd. Assignor: Institute of Hydrobiology, Chinese Academy of Sciences Contract fulfillment period: 2007.9.1 to 2012.8.31 contract change Contract record no.: 2008420000010 Denomination of invention: Colloidal Gold tag method for identifying virus single chained antibody of sprawn leuckoplakia yndrome Granted publication date: 20070711 License type: Exclusive license Record date: 2008.7.7 |
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Free format text: EXCLUSIVE LICENCE; TIME LIMIT OF IMPLEMENTING CONTACT: 2007.9.1 TO 2012.8.31 Name of requester: SUZHOU SEA BIG FEED CO., LTD. Effective date: 20080707 |
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