CN103910795A - Monoclonal antibody resistant to Bohle iridescent virus and application of monoclonal antibody - Google Patents
Monoclonal antibody resistant to Bohle iridescent virus and application of monoclonal antibody Download PDFInfo
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- CN103910795A CN103910795A CN201410169309.3A CN201410169309A CN103910795A CN 103910795 A CN103910795 A CN 103910795A CN 201410169309 A CN201410169309 A CN 201410169309A CN 103910795 A CN103910795 A CN 103910795A
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- glass
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Abstract
The invention discloses a monoclonal antibody resistant to Bohle iridescent virus and an application of the monoclonal antibody. The monoclonal antibody resistant to Bohle iridescent virus is secreted by a mouse hybridoma cell strain with the collection number CGMCC No. 8555. The monoclonal antibody resistant to Bohle iridescent virus can be used for detecting Bohle iridescent virus in cell culture. The invention further discloses an ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting Bohle iridescent virus.
Description
Technical field
The invention belongs to field of immunology, be specifically related to the monoclonal antibody that a kind of anti-glass is strangled irido virus, hybridoma cell strain, and application.
Background technology
Glass is strangled definite member that irido virus (Bohle virus, BIV) belongs to Iridoviridae Ranavirus.Ranavirus is take frog virus 3 type (FV3) as representative species, determine that member strangles irido virus (Bohle virus except glass, BIV) outer Ou Channel-catfish virus (European catfish virus in addition, ECV), Europe catfish virus (European sheatfish virus, ESV) and Sang Dikupa frog virus (Santee-Cooper ranavirus) etc.The virion of frog virus is large (150～180nm), is icosahedron three-dimensional symmetrical.Genome is the double-stranded DNA of 150～170kb, in karyon and kytoplasm, all can copy.
This viral host range is very extensive, comprises fish, batrachians, reptiles and some susceptible other animal species.In order to prevent that in time in water surrounding glass from strangling the expansion of irido virus hazardness, need further to understand its host's scope, this just requires to develop as early as possible a kind of effectively detection technique.
At present, virus separation and PCR method are to be applied to glass to strangle the common method that irido virus is diagnosed.But isolation of virus is consuming time longer, be difficult for as quick diagnosis; PCR method, although the used time is shorter, cost is higher, is difficult for as large-scale inquiry.
Summary of the invention
Technical problem to be solved by this invention is to provide the ELISA test kit that a kind of anti-glass is strangled the monoclonal antibody of irido virus, the detection glass of applying this monoclonal antibody is strangled irido virus, and vitro detection glass is strangled the method for irido virus.
For solving the problems of the technologies described above, the present invention adopts following technical proposals:
Anti-glass provided by the present invention is strangled the monoclonal antibody of irido virus, is to be CGMCCNo.8555 by deposit number hybridoma cell strain secretion produces.This mouse hybridoma cell strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 4th, 2013 and (is called for short CGMCC, address is No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute), its deposit number is CGMCC No.8555, and Classification And Nomenclature is the hybridoma cell strain that glass is strangled irido virus.
Deposit number is that the mouse hybridoma cell strain of CGMCC No.8555 also belongs to protection scope of the present invention.
The present invention also provides a kind of ELISA test kit that detects glass and strangle irido virus, and this test kit comprises the solid phase carrier of coated monoclonal antibody of the present invention.Preferably, also comprise that goat-anti glass strangles the polyclonal antibody of irido virus, and the antibody of the anti-sheep of enzyme mark.Described enzyme is horseradish peroxidase.
The present invention adopts the antibody of the anti-sheep of enzyme mark of commercialization purchase, is because on the one hand, if the monoclonal antibody of explaining in mark the present invention or polyclonal antibody, process more complicated.Even if success all may affect tiring or other aspects of antibody of antibody, make whole test kit research and development more loaded down with trivial details, consumption power, consuming time.And there is not this problem in the antibody of the anti-sheep of application commercialization enzyme mark; On the other hand, if the monoclonal antibody in mark the present invention or polyclonal antibody, the cost of whole test kit is just very high, and it is infeasible applying for test kit.The antibody (sigma) of the anti-sheep of application commercialization enzyme mark, every 1ml expense is 1000 yuan of left and right, has reduced the cost of test kit.
Further, for the ease of detecting, test kit of the present invention also comprises positive control and negative control, and ELISA reacts required enzyme linked immunosorbent detection reagent.Wherein, described positive control is that glass is strangled irido virus solution, and negative control is fish cell suspension or healthy tissues homogenate.Described enzyme linked immunosorbent detection reagent, is conventional enzyme linked immunosorbent detection reagent, includes but not limited to substrate reactions liquid, washings and the reaction terminating liquid of enzyme.
The preparation process of polyclonal antibody that goat-anti glass of the present invention is strangled irido virus is as follows: strangle irido virus as antigen with glass, multi-point injection immune goat, be respectively immunity in every 1 week once, altogether immunity 4 times, get serum, be goat-anti glass and strangle the polyclonal antibody of irido virus.
The present invention also provides a kind of method that detects glass and strangle irido virus, it is characterized in that, the method comprises the following steps:
(1) by testing sample application of sample in the solid phase carrier that is coated with monoclonal antibody claimed in claim 1;
(2) the polyclonal antibody application of sample of goat-anti glass being strangled to irido virus is in the solid phase carrier of step (1) acquisition;
(3) solid phase carrier antibody application of sample of the anti-sheep of enzyme mark being obtained in step (2);
(4) detect enzyme labelling thing, whether determine existence that glass in testing sample strangles irido virus or the amount existing, thereby determine that existence that glass strangles irido virus whether.
Beneficial effect of the present invention is as follows:
1. after the hybridoma cell strain propagation of secretion monoclonal antibody of the present invention, can prepare a large amount of required specific antibodies.After hybridoma injection mouse, the ascites MAb mediated ELISA of generation is tired as 1:1 × 10
6.Hybridoma cell strain activity is high, in liquid nitrogen after frozen 8-10 month, and still can rapid fluid resuscitation and keep excellent activity.In the preparation of described monoclonal antibody, cell confluency is 97.6%, and positive rate is 98.0%.
2. the antigen of immune mouse is the principle according to immunological tolerance, using the thick concentrated viral suspension of purifying of simple differential centrifugation method as immunogen, and immune mouse.This immunization method has confirmed that through many experiments data its main advantage is to have guaranteed the positive effect after cytogamy, has simplified again the preparation method of antigen.
3. in screening process, apply indirect ELISA method, in this method, embedding plank a large amount of elisa plate of having taked disposable embedding, guarantee the unity of whole experiment, and then have guaranteed to obtain the anti-glass of preparation and strangle the accuracy of the monoclonal antibody of irido virus.
4. the detection glass of setting up is strangled the sandwich ELISA method of irido virus, and whether the virus that can detect accurately in cell suspension exists.This detection method, strangles aspect irido virus favourable condition is provided at inspection and quarantine glass for importing and exporting inspection and quarantine mechanism.
Embodiment
In order to be illustrated more clearly in the present invention, below in conjunction with preferred embodiment, the present invention is described further.It will be appreciated by those skilled in the art that specifically described content is illustrative and nonrestrictive below, should not limit the scope of the invention with this.
The anti-glass of embodiment 1 is strangled the preparation of the monoclonal antibody of irido virus
1. the preparation of antigen
The thick glass of purifying of method with differential centrifugation is strangled irido virus (Bohle virus, BIV) (J Pallister, A Gould, D Harrison, A Hyatt, J Jancovich and H Heine1.Development of real-time PCR assays for the detection and differentiation of Australian and European ranaviruses, Journal of Fish Diseases2007, 30, 427-438) suspension, be first 8000r/min, after centrifugal 35min, 24000r/min, centrifugal 2hr, collecting precipitation, resuspended with the PBS of the 0.01mol/L of 0.5ML,-80 ℃ of preservations.
2. immune mouse
The mouse of immunity use is SPF level female BALB/c mouse in 5 week age.Every mouse carries out abdominal injection fundamental immunity take the viral suspension of above-mentioned purifying as antigen, and viral suspension mixes with Freund's complete adjuvant 1:1, abdominal injection 0.2ML; Booster immunization after 2 weeks, viral suspension mixes with Freund's incomplete adjuvant 1:1, abdominal injection 0.1ML; Afterwards every 1 week booster immunization 1 time, abdominal injection, viral suspension 0.2ML; After the 4th immunity the 3rd day, de-mouse cervical vertebra to be put to death, the aseptic spleen of getting is for cytogamy.
3. cytogamy
Conventional cell-fusion techniques: get after the spleen of immune mouse and SP2/0 myeloma cell merge, add the thymocyte of mouse and fused cell to cultivate cultivation altogether in system at HAT.
The preparation of the HAT substratum of applying in described technology: by 50 times of concentrated HAT (2ml, GIBCO company) and superfine foetal calf serum (20ml, Hangzhou Sijiqing Biological Engineering Material Co., Ltd.) join in modified form RPMI1640 substratum (80ml, hyclone company) and mix.
4. the screening of hybridoma and clone
Fused cell was cultivated after 10 days, collected cells and supernatant, strangles irido virus carry out indirect ELISA detection as antigen using the glass of gradient centrifugation purification.The positive hybridoma cell strain limiting dilution assay filtering out is cloned.Wherein positive mouse hybridoma cell strain (BIV-3A4) was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC on December 4th, 2013, address is No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute), deposit number is CGMCC No.8555.
5. inducing of ascites
Get female Balb/C mouse in 6～8 week age, the aseptic whiteruss 0.5ml/ of intraperitoneal injection only; After 1 week, intraperitoneal injection positive hybridoma cell; Inoculation hybridoma after 7～10 days, see that mouse web portion obviously expands, tap the abdomen, centrifugal rear collection supernatant ,-80 ℃ frozen for subsequent use.
Embodiment 2: anti-glass is strangled the CHARACTERISTICS IDENTIFICATION of the monoclonal antibody of irido virus
1. the titer of ascites of monoclonal antibody is identified
Method: adopt indirect elisa method to identify the titer of ascites of monoclonal antibody.
Result: monoclonal antibody titer of ascites of the present invention is 1:1 × 10
6, show that hybridoma cell strain has the ability of the high titre antibody of secretion.
2. the hypotype of monoclonal antibody is identified
Method: parting kit (the SBA Clonotyping of the SouthernBiotech of the application U.S.
tMsystem/HRP), according to its specification sheets, the Ig hypotype of monoclonal antibody of the present invention is identified.
Result: the hypotype of monoclonal antibody of the present invention is IgG2b type k chain.
3. the specificity analyses of monoclonal antibody
Method: adopt indirect ELISA method, detect monoclonal antibody and strangle irido virus (BIV), SVCV (spring viremia of carp virus with glass respectively, SVCV), viral hemorrhagic septicemia, VHS virus (Viral haemorrhagic septicaemia virus, and infectious hematopoietic necrosis's poison (Infectious hematopoietic necrosis virus, IHNV) reaction VHSV).Result shows that monoclonal antibody specificity is good, only strangles irido virus with glass and reacts, and does not all react with other virus strain and cell.
The specificity analyses (P/N value) of table 1 monoclonal antibody
| Virus strain | BIV | SVC | VHSV | IHNV |
| P/N value | 16.98 | 1.16 | 1.65 | 086 |
Embodiment 3 detects glass and strangles the composition of the ELISA test kit of irido virus
Detect glass and strangle the consisting of of ELISA test kit of irido virus: the solid phase carrier of monoclonal antibody prepared by coated embodiment 1; Goat-anti glass is strangled the polyclonal antibody of irido virus; The antibody (purchased from sigma company) of the anti-sheep of horseradish peroxidase mark; The substrate reactions liquid of enzyme; Positive control; Negative control; Washings; Reaction terminating liquid.
Wherein, anti-glass is strangled the monoclonal antibody embedding ELISA batten of irido virus: 100 μ l/ holes after monoclonal antibody proper ratio dilution prepared by embodiment 1,4 ℃ are spent the night.After taking-up, wash plate 3 times with PBST, each 5min, dries.With the bovine serum albumin sealing of 0.01mol/LPBS dilution 3%, every hole 150 μ l, 37 ℃ of sealing 1h.After taking-up, wash plate 3 times with PBST, each 3min, dries, dry rear-80 ℃ of preservations.The preparation process of polyclonal antibody that goat-anti glass is strangled irido virus is as follows: strangle irido virus as antigen with the glass of gradient centrifugation purification, multi-point injection immune goat, be respectively immunity in every 1 week once, immunity 4 times altogether; Get serum, packing ,-80 ℃ of preservations.
The preparation of 10 × PBST: NaCl:80g, Na
2hPO
412H2O:29g, KH
2pO
4: 2g, KCl:2g, tween 20: 5ml, distilled water adds to 1000ml, 4 ℃ of preservations.
The substrate reactions liquid of enzyme: be 10% vitriol (TMB, sigma configure with dimethyl formamide): 150 μ l, H
2o
2: 4 μ l, pH5.0 citric acid-phosphoric acid buffer: 10ml.Matching while using.
Positive control is that glass is strangled irido virus solution, and negative control is fish cell suspension or healthy tissues homogenate.
Washings is: as aforementioned PBST preparation.
Reaction terminating liquid: prepare 2mol/LH with distilled water
2sO
4.
Embodiment 4: detect the double antibodies sandwich ELISA detection method that glass is strangled irido virus
Sandwich ELISA method step:
1. ELISA enzyme plate from the monoclonal antibody of irido virus to coated anti-glass that strangle adds and dilutes by cell culture and virus suspension with 10 multiple proportions with the PBS of 0.01mol/L.The original titre of this virus is 10
6tCID
50mL
-l;
2. anti-glass is strangled to the polyclonal antibody of irido virus, diluted 1000 times, join on enzyme plate;
3. the anti-goat-anti body of horseradish peroxidase mark (is diluted by description of commodity, then joined on enzyme plate;
4. detersive enzyme target;
5. add the substrate reactions liquid of enzyme;
6. termination reaction.
Result is as shown in table 2, and the glass that detects that the sandwich ELISA method of foundation can be special is strangled irido virus, and titre can reach 10
5tCID
50mL
-l.
Table 2 detects glass strangles the detected result (P/N value) of the double antibodies sandwich ELISA method of irido virus
| Virus | P/N value |
| 10 0 | 7.69 |
| 10 -1 | 3.08 |
| 10 -2 | 1.62 |
| 10 -3 | 1.04 |
Embodiment 5: double antibodies sandwich ELISA detection method specificity analyses
Sandwich ELISA method step:
1. ELISA enzyme plate from the monoclonal antibody of irido virus to coated anti-glass that strangle adds titre 10
6tCID
50viral suspension (comprises that glass strangles irido virus (BIV), SVCV (spring viremia of carp virus, SVCV), viral hemorrhagic septicemia, VHS virus (Viral haemorrhagic septicaemia virus, and frog virus 3 type (frogvirus3, FLV3) VHSV);
2. anti-glass is strangled to the polyclonal antibody of irido virus, diluted 1000 times, join on enzyme plate;
3. the anti-goat-anti body of horseradish peroxidase mark is diluted by description of commodity, then join on enzyme plate;
4. detersive enzyme target;
5. add the substrate reactions liquid of enzyme;
6. termination reaction.
Result is as shown in table 3, and the glass that detects that the sandwich ELISA method of foundation can be special is strangled irido virus, and in other experiments, virus strain can not detect.
The specificity analyses (P/N value) of table 3 double antibodies sandwich ELISA detection method
| Virus strain | P/N value |
| BIV | 3.85 |
| FLV3 | 1.05 |
| SVCV | 1.09 |
| VHSV | 1.04 |
Obviously; the above embodiment of the present invention is only for example of the present invention is clearly described; and be not the restriction to embodiments of the present invention; for those of ordinary skill in the field; can also make other changes in different forms on the basis of the above description; here cannot give all embodiments exhaustively, everyly belong to apparent variation or the still row in protection scope of the present invention of variation that technical scheme of the present invention extends out.
Claims (10)
1. anti-glass is strangled a monoclonal antibody for irido virus, is to be CGMCC No.8555 by deposit number hybridoma cell strain secretion produces.
2. secretion produces anti-glass and strangles the hybridoma cell strain of the monoclonal antibody of irido virus, and its deposit number is CGMCC No.8555.
3. monoclonal antibody claimed in claim 1 is strangled the application in irido virus at detection glass.
4. monoclonal antibody claimed in claim 1 or hybridoma cell strain claimed in claim 2 detect glass in preparation and strangle the application in irido virus test kit.
5. detect glass and strangle an ELISA test kit for irido virus, it is characterized in that, described test kit comprises the solid phase carrier of coated monoclonal antibody claimed in claim 1.
6. ELISA test kit according to claim 5, is characterized in that, described test kit also comprises that goat-anti glass strangles the polyclonal antibody of irido virus, and the antibody of the anti-sheep of enzyme mark.
7. ELISA test kit according to claim 6, is characterized in that, described enzyme is horseradish peroxidase.
8. according to the ELISA test kit described in claim 5 or 6, it is characterized in that, described test kit also comprises negative control and positive control.
9. ELISA test kit according to claim 8, is characterized in that, described test kit also comprises substrate reactions liquid, washings and reaction terminating liquid.
10. detect glass and strangle a method for irido virus, it is characterized in that, the method comprises the following steps:
(1) by testing sample application of sample in the solid phase carrier that is coated with monoclonal antibody claimed in claim 1;
(2) the polyclonal antibody application of sample of goat-anti glass being strangled to irido virus is in the solid phase carrier of step (1) acquisition;
(3) solid phase carrier antibody application of sample of the anti-sheep of enzyme mark being obtained in step (2);
(4) detect enzyme labelling thing, whether determine existence that glass in testing sample strangles irido virus or the amount existing, thereby determine that existence that glass strangles irido virus whether.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104774262A (en) * | 2015-04-21 | 2015-07-15 | 中国检验检疫科学研究院 | Anti-andrias davidianus ranavirus monoclonal antibody, and preparation method and application thereof |
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| JPH06153979A (en) * | 1992-11-16 | 1994-06-03 | Maruha Corp | Monoclonal antibody against fish iridovirus, hybridoma for producing the antibody and production method |
| CN101735309A (en) * | 2008-11-17 | 2010-06-16 | 黄金城 | Iridovirus antigen active polypeptide and application thereof |
| CN103288954A (en) * | 2013-06-04 | 2013-09-11 | 大连海洋大学 | Monoclonal antibody with rock bream iridovirus ORF049L recombinant proteins and preparation method thereof |
-
2014
- 2014-04-25 CN CN201410169309.3A patent/CN103910795B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH06153979A (en) * | 1992-11-16 | 1994-06-03 | Maruha Corp | Monoclonal antibody against fish iridovirus, hybridoma for producing the antibody and production method |
| CN101735309A (en) * | 2008-11-17 | 2010-06-16 | 黄金城 | Iridovirus antigen active polypeptide and application thereof |
| CN103288954A (en) * | 2013-06-04 | 2013-09-11 | 大连海洋大学 | Monoclonal antibody with rock bream iridovirus ORF049L recombinant proteins and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| WHITTINGTON R.J.等: "Detection of antibodies against iridoviruses in the serum of the amphibian Bufo marinus", 《JOURNAL OF VIROLOGICAL METHODS》 * |
| 朱春华等: "中华鳖虹彩病毒单克隆抗体的制备及其抗原表位的初步分析", 《水产学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104774262A (en) * | 2015-04-21 | 2015-07-15 | 中国检验检疫科学研究院 | Anti-andrias davidianus ranavirus monoclonal antibody, and preparation method and application thereof |
| CN104774262B (en) * | 2015-04-21 | 2017-10-20 | 中国检验检疫科学研究院 | Anti- giant salamander irido virus monoclonal antibody and its preparation and application |
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