CN103739708A - Infectious haematopoietic necrosis virus resisting monoclonal antibody and application thereof - Google Patents
Infectious haematopoietic necrosis virus resisting monoclonal antibody and application thereof Download PDFInfo
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- CN103739708A CN103739708A CN201310700633.9A CN201310700633A CN103739708A CN 103739708 A CN103739708 A CN 103739708A CN 201310700633 A CN201310700633 A CN 201310700633A CN 103739708 A CN103739708 A CN 103739708A
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Abstract
The invention discloses an infectious haematopoietic necrosis virus resisting monoclonal antibody and application thereof. The infectious haematopoietic necrosis virus resisting monoclonal antibody is generated through the secreting of a mouse hybridoma cell strain with the collection number of CGMCC No. 8556. The infectious haematopoietic necrosis virus resisting monoclonal antibody can be used for detecting an infectious haematopoietic necrosis virus. The invention further discloses an ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting the infectious haematopoietic necrosis virus.
Description
Technical field
The invention belongs to field of immunology, be specifically related to a kind of infectivity resistant hematopoietic necrosis virus's monoclonal antibody, hybridoma cell strain and set up a kind of sandwich ELISA method test kit of the infectious hematopoietic necrosis's of detection poison.
Background technology
Infectious hematopoietic necrosis's poison (Infectious haematopoietic necrosis virus, IHNV) in classification, belong to Rhabdoviridae (Rhabdoviridae), the outer Rhabdovirus of grain (Novirhabdovirus).The cold water fishes such as main infection rainbow trout, rainbow trout (Salmo gairdneri), during acute infection cumulative mortality more than 90-95%, and can vertical transmission to filial generation.Be popular in the earliest North America and Europe some countries, along with the development of aquatic products trade constantly spreads, spread at present the main cold water fish cultivation area to China northeast, Hebei, Shandong, Beijing, Gansu, Qinghai etc., and in partial area outbreak of epidemic, caused larger financial loss.IHN is classified as by International Office of Epizootics (OIE) must notifiable disease, is fish port the 1st class Quarantine Objects, by China, is classified as two class animal epidemics.
This disease there is no methods for the treatment of at present, and the most real effective measure of prevention and control IHN are fries to trans-regional transportation and parent population isolates and carry out the monitoring of continuous effective, finds that virus finds source as early as possible, controls and eliminate cause of disease, controls viral propagation.The diagnostic method of several IHNV that OIE recommends at present comprises cell cultures separation method, immunological method (neutralization test method, indirect fluorescent antibody technique and ELISA method) and molecular biology method (PCR method, PCR probe and sequencing method).Wherein the accuracy of cell cultures isolation technique is higher, but sense cycle is longer, at least needs within 15, just can obtain final result, can not meet the needs of rapid detection; On the basis of molecular biology method based on cell isolation method, for detection of the virus after cell cultures, or the virus that apparent infection fish is carried is done further confirmation; Immunology detection technology based on monoclonal antibody technique, it is the effective way that meets the quick mass detection IHNV of laboratories, have been reported, or the antigen not had and antiserum(antisera), or need comparatively expensive precision instrument, be not suitable for laboratories, could not be applied on a large scale in the monitoring of disease always.Therefore, obtain the monoclonal antibody with stronger specific infectivity resistant hematopoietic necrosis virus, not only there is quick, sensitive feature, and simple to operate, low, simple and efficient to plant and instrument requirement, be applicable to the extensive monitoring of this disease.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of infectivity resistant hematopoietic necrosis virus's monoclonal antibody and application thereof.
For solving the problems of the technologies described above, the present invention adopts following technical proposals:
The present invention has obtained the hybridoma cell strain IHNV-1B10 of energy stably excreting infectivity resistant hematopoietic necrosis virus monoclonal antibody through screening, the Classification And Nomenclature of this hybridoma cell strain is the hybridoma cell strain of infectious hematopoietic necrosis's poison, it 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: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.8556.
The infectivity resistant hematopoietic necrosis virus's who is secreted by above-mentioned hybridoma cell strain monoclonal antibody also belongs to protection scope of the present invention.
The present invention also provides a kind of ELISA test kit of the infectious hematopoietic necrosis's of detection poison, and this test kit comprises monoclonal antibody of the present invention.Further, in this test kit, also comprise the solid phase carrier of coated infectivity resistant hematopoietic necrosis virus's polyclonal antibody, the sheep anti-mouse antibody of horseradish peroxidase mark.
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 infectious hematopoietic necrosis's poison solution, and negative control is fish cell suspension or normal Fish Tissue 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 enzyme reaction stop buffer of enzyme.
The present invention also provides a kind of method of the infectious hematopoietic necrosis's of detection poison, and the method comprises the following steps:
(1) by testing sample application of sample in the solid phase carrier of polyclonal antibody that is coated with infectivity resistant hematopoietic necrosis virus;
(2) solid phase carrier monoclonal antibody application of sample claimed in claim 1 being obtained in (1);
(3) solid phase carrier sheep anti-mouse antibody application of sample of horseradish peroxidase mark being obtained in (2);
(4) add the substrate reactions liquid of enzyme;
(5) add enzyme reaction stop buffer;
(6) result is judged.
The present invention also claimed said monoclonal antibody detects the application in infectious hematopoietic necrosis's poison test kit in preparation.And this monoclonal antibody or the application of test kit in detection infectious hematopoietic necrosis poison.
The present invention has the following advantages and effect
1, obtain the downright bad sick monoclonal antibody of infectivity resistant hemocytopoietic organ and secrete this monoclonal antibody hybridoma cell strain.After this hybridoma proliferation, can prepare a large amount of required specific antibodies; After hybridoma injection mouse, the ascites MAb mediated ELISA of generation is tired as 1:2.5 × 10
5; 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 96.2%, and positive rate is 93.4%.
2, in antigen purification mode, it is mainly centrifugal by gradient sucrose that Chinese scholars obtains infectivity resistant hematopoietic necrosis virus antigen, after SDS-PAGE electrophoresis, cut again glue object band, immune mouse after processing, this class methods virus purity is high, but complicated operation, the large and virus stability of medicine ' Bingduxiao ' consumption is difficult to guarantee.The antigen of immune mouse of the present invention is taked the mode of slightly putting forward of differential centrifugation, has protected largely the original conformation of antigen protein, for filtering out monoclonal antibody specific, provides guarantee; On immune programme for children, utilize the principle of immunological tolerance, make immune mouse produce immunological tolerance to cellular constituent in immunizing antigen, thereby improve the purity of virus in immunity, and then strengthen the stimulus intensity of virus antigen to mouse boosting cell, obtained good effect.
3, good, the height of tiring of the specificity of the infectious hematopoietic necrosis of the present invention development poison monoclonal antibody, can 10 infectious hematopoietic necrosis's strains of specific identification, there is good broad spectrum, be applied in infectious hematopoietic necrosis's poison immunological diagnostic reagent, specificity is good, has avoided cross reaction.
The sandwich ELISA method of the detection infectious hematopoietic necrosis poison of 4, setting up, can detect fast and accurately virus in cell suspension and in tissue and whether exist, and monitors in enormous quantities virus disseminating favourable condition is provided for laboratories.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail;
Fig. 1 is that after purifying, recombinant protein Western blotting detects: M: the SDS-PAGE of molecular weight of albumen standard 1, IHNV.
Embodiment
For understanding better the present invention, will further illustrate the solution of the present invention by specific embodiment below, protection scope of the present invention should comprise the full content of claim, but is not limited to this.
The preparation of embodiment 1 infectivity resistant hematopoietic necrosis virus's monoclonal antibody
1. antigen is purified
Antigen used is the strain of IHNV-UK(infectious hematopoietic necrosis poison Britain) by the OIE reference laboratory preservation of Britain Weymouth.Inoculate the EPC(carp epithelioma cell into exponential phase of growth), with M199(containing Earle ' s balanced salt solution, 10% foetal calf serum and 200U/mL penicillin-Streptomycin sulphate, pH7.2-7.6) be placed in 17.5 ℃ of increments.Collect virus, through the centrifugal 30min of 8000r/min, the more centrifugal 2h of 24000r/min.Precipitation is resuspended with 0.5mL0.01M PBS.
2. animal immune
Immunity is SPF level female BALB/c mouse in 5 week age with mouse.Every mouse carries out abdominal injection fundamental immunity using the virus after purifying as antigen, and virus is mixed with Freund's complete adjuvant 1:1, abdominal injection; Booster immunization after 2 weeks, virus is mixed with Freund's incomplete adjuvant 1:1, abdominal injection; Afterwards every 1 week booster immunization 1 time, abdominal injection, virus injection 4mg/ is only; 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 and cloning
Spleen and the SP2/0 cell of getting immune mouse merge, as antigen, carry out indirect ELISA detection with infectious hematopoietic necrosis's poison of gradient centrifugation purification, cloning is carried out with limiting dilution assay in the positive hole filtering out, until obtain the hybridoma cell strain of the anti-hematopoietic necrosis virus's of energy stably excreting infectivity monoclonal antibody, Hybridoma Cell Culture supernatant antibody titer is 1:160, called after infectious hematopoietic necrosis poison hybridoma cell strain 1B10, this 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, BeiJing, China city institute), deposit number is CGMCC No.8556.
4. the preparation of monoclonal antibody
Pre-treatment female BALB/c mouse in normal 5 week age, the aseptic paraffin oil 0.5mL/ of abdominal injection only.7d pneumoretroperitoneum is inoculated the hybridoma 1 × 10 of exponential phase of growth
6～2 × 10
6/ only, the healthy state of close observation mouse and ascites sign, after about 7d～12d, put to death mouse, extracting ascites, and low-speed centrifugal is collected supernatant, and packing postposition-80 ℃ save backup.
Embodiment 2: the Biological characteristics of infectivity resistant hematopoietic necrosis virus monoclonal antibody
1. the mensuration of tiring
Method: use the concentration coated elisa plate of infectious hematopoietic necrosis's poison 15 μ g/mL of purifying, adopt indirect elisa method to identify the titer of ascites of monoclonal antibody.
Result: monoclonal antibody titer of ascites of the present invention is 1:2.5 × 10
5, 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 IgG1 type k chain.
3. the specificity analyses of monoclonal antibody
With purifying infectious hematopoietic necrosis poison and concentrated EPC cell pyrolysis liquid, carry out immunoblot experiment, through SDS-PAGE and Western blot, analyze, monoclonal antibody prepared by the present invention for antigenic determinant be positioned on the protein band of Mt42000, this protein band has confirmed it is the nucleoprotein (see figure 1) of infectious hematopoietic necrosis's poison.
4. the cross reaction of monoclonal antibody
Method: adopt indirect ELISA method, detect monoclonal antibody respectively with infectious hematopoietic necrosis's poison (IHNV), SVCV (spring viremia of carp virus, SVCV), viral hemorrhagic septicemia, VHS virus (Viral haemorrhagic septicaemia virus, VHSV) and 8 kinds of fish cells and normal fish tissue have no cross reaction.
Result: monoclonal antibody specificity is good, only reacts with infectious hematopoietic necrosis's poison, does not all react with other virus strain and cell.
The two sandwich ELISA detection kit of embodiment 3 infectious hematopoietic necrosis's poison
1, the composition of the two sandwich ELISA detection kit of infectious hematopoietic necrosis's poison
Consisting of of infectious hematopoietic necrosis's two sandwich ELISA detection kit of poison: the solid phase carrier that 1) has wrapped infectivity resistant hematopoietic necrosis virus polyclonal antibody; 2) monoclonal antibody that prepared by embodiment 1; 3) sheep anti-mouse antibody of horseradish peroxidase mark (purchased from sigma company); 4) the substrate reactions liquid of enzyme; 5) positive control and negative control; 6) washings; 7) enzyme reaction stop buffer.
1) wrapped the preparation of the solid phase carrier of infectivity resistant hematopoietic necrosis virus polyclonal antibody: take infectious hematopoietic necrosis's poison (ATCC VR-714) of gradient centrifugation purification as antigen, immune goat.Prepare the first EPC cell debris of serum and adsorb, then purify by saturated ammonium sulphate method method.The sheep polyclonal antibody of purifying is diluted to protein content 2.5 μ g/ml with coated damping fluid, coated 96 hole ELISA enzyme plates (U.S. Corning company), 100 μ l/ holes, 4 ℃ are spent the night.After taking-up, with washings, wash 3 times, each 5min, dries, dry rear with vacuum bag-80 ℃ preservation.
2) the substrate reactions liquid of enzyme: 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.
3) positive control and negative control
Positive control: infectious hematopoietic necrosis's poison solution, negative control is fish cell suspension or healthy tissues homogenate.
4) washings: NaCl80g, Na2HPO412H
2o29g, KH
2pO
42g, KCl2g, tween 20 5ml, distilled water 1L, 4 ℃ of preservations.Distilled water 1:10 dilution, matching while using.
5) enzyme reaction stop buffer: H
2sO
4(96%) 22.2mL, distilled water 177.8mL, room temperature is placed.
2, the using method of the two sandwich ELISA detection kit of infectious hematopoietic necrosis's poison
1) to the ELISA enzyme plate of the IgG that is coated with goat-anti IHNV, add sample, positive control and negative control, 37 ℃ of effect 1h.
2) washings is washed plate 3 times, each 3min.
3) by second antibody, the infectivity resistant hematopoietic necrosis virus's that prepared by embodiment 1 monoclonal antibody, dilutes 1600 times, joins on enzyme plate 37 ℃ of effect 1h.
4) washings is washed plate 3 times, each 3min.
5) sheep anti-mouse antibody of horseradish peroxidase mark is diluted by description of commodity, then join on enzyme plate, 37 ℃ of effect 1.5h.
6) washings is washed plate 3 times, each 3min.
7) add the substrate reactions liquid of enzyme, 100 μ L/ holes, lucifuge.
6) add stop buffer termination reaction, 150 μ L/ holes.
7) read 450nm absorbance value.
8) result is judged
The application of the two sandwich ELISA detection kit of embodiment 4 infectious hematopoietic necrosis's poison
Specific assay
In order to verify the specificity of the two sandwich ELISA test kits of infectious hematopoietic necrosis's poison of the present invention, use the test kit of embodiment 3 to detect 10 strain infectious hematopoietic necrosis poison (cell culture fluid), establish SVCV (SVCV), viral hemorrhagic septicemia, VHS virus (VHSV) is done cross reaction checking simultaneously.
Table 1 test kit specific assay
| IHNV2012074 | P/N | 5.53 | IHNV2012191 | P/N | 5.03 |
| ? | TCID 50 | 10 -5.2 | ? | TCID 50 | 10 -5.2 |
| IHNV2012075 | P/N | 5.97 | IHNV2012196 | P/N | 6.28 |
| ? | TCID 50 | 10 -4.5 | ? | TCID 50 | 10 -5.5 |
| IHNV2012096 | P/N | 3.34 | IHNV2013066 | P/N | 6.62 |
| ? | TCID 50 | 10 -4.8 | ? | TCID 50 | 10 -4.8 |
| IHNV2012169 | P/N | 6.83 | IHNV2013071 | P/N | 8.1 |
| ? | TCID 50 | 10 -4.8 | ? | TCID 50 | 10 -4.5 |
| IHNV2012171 | P/N | 4.93 | INHV-UK | P/N | 4.52 |
| ? | TCID 50 | 10 -4.8 | ? | TCID 50 | 10 -4.5 |
| VHSV | P/N | 0.92 | SVCV | P/N | 0.95 |
| ? | TCID 50 | 10 -5.5 | ? | TCID 50 | 10 -5.5 |
Result shows, this test kit can specificly detect infectious hematopoietic necrosis's poison (P/N value is between 3.34 to 8.1) of 10 strain different titers, with VHSV, the SVCV of Rhabdovirus, cross reaction does not occur, and has shown good specificity.
2. with the coincidence rate of isolation of virus test kit more of the present invention
In order to verify the coincidence rate compared with the gold method that the two sandwich ELISA diagnostic kits of infectious hematopoietic necrosis of the present invention poison recommend with OIE, simultaneously with test kit of the present invention and viral separation method to detecting with a collection of 14 samples.
Detected result shows, in 14 parts of tissue samples, viral separation method detects 1 part of positive, and all the other 13 parts are negative sample; Test kit of the present invention adopts fish setup action to detect sample, detects 3 parts positive (1 positive detecting containing viral separation method); Test kit of the present invention adopts cell culture fluid as detecting sample, detect 1 part positive, in full accord with viral separation method detected result.
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 (9)
1. infectivity resistant hematopoietic necrosis virus's monoclonal antibody is by deposit number, to be the hybridoma cell strain secretion generation of CGMCC No.8556.
2. secretion produces the hybridoma cell strain of infectivity resistant hematopoietic necrosis virus's monoclonal antibody, and its deposit number is CGMCC No.8556.
3. an ELISA test kit that detects infectious hematopoietic necrosis's poison, is characterized in that, described test kit comprises monoclonal antibody claimed in claim 1.
4. test kit according to claim 3, is characterized in that, described test kit also comprises the solid phase carrier of coated infectivity resistant hematopoietic necrosis virus's polyclonal antibody, the sheep anti-mouse antibody of horseradish peroxidase mark.
5. test kit according to claim 4, is characterized in that, described test kit also comprises positive control and negative control.
6. test kit according to claim 5, is characterized in that, described positive control is infectious hematopoietic necrosis's poison solution, and negative control is fish cell suspension or normal Fish Tissue homogenate.
7. a method that detects infectious hematopoietic necrosis's poison, is characterized in that, comprises the following steps:
(1) by testing sample application of sample in the solid phase carrier of polyclonal antibody that is coated with infectivity resistant hematopoietic necrosis virus;
(2) solid phase carrier monoclonal antibody application of sample claimed in claim 1 being obtained in (1);
(3) solid phase carrier sheep anti-mouse antibody application of sample of horseradish peroxidase mark being obtained in (2);
(4) add the substrate reactions liquid of enzyme;
(5) add enzyme reaction stop buffer;
(6) result is judged.
8. monoclonal antibody claimed in claim 1 detects the application in infectious hematopoietic necrosis's poison test kit in preparation.
9. monoclonal antibody claimed in claim 1 is in the application detecting in infectious hematopoietic necrosis's poison.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106749647A (en) * | 2016-02-18 | 2017-05-31 | 北京市农林科学院 | Salmon trout IHNV monoclonal antibodies and detection kit |
| CN108409857A (en) * | 2018-01-24 | 2018-08-17 | 四川农业大学 | The preparation and its application of rainbow trout infectivity resistant Hematopoietic Necrosis's disease Yolk antibody |
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| JPH0315396A (en) * | 1989-03-07 | 1991-01-23 | Sapporo Breweries Ltd | Monoclonal antibody against pathogenic virus of fishes and its production |
| CN102702350A (en) * | 2012-05-17 | 2012-10-03 | 中国检验检疫科学研究院 | Monoclonal antibody of virus-resistance viral hemorrhagic septicemia virus G proteins and application of monoclonal antibody |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106749647A (en) * | 2016-02-18 | 2017-05-31 | 北京市农林科学院 | Salmon trout IHNV monoclonal antibodies and detection kit |
| CN108409857A (en) * | 2018-01-24 | 2018-08-17 | 四川农业大学 | The preparation and its application of rainbow trout infectivity resistant Hematopoietic Necrosis's disease Yolk antibody |
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