CN101846685A - Enzyme-linked immunoassay kit for chlamys ferreri blood cells and preparation method thereof - Google Patents
Enzyme-linked immunoassay kit for chlamys ferreri blood cells and preparation method thereof Download PDFInfo
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- CN101846685A CN101846685A CN201010191097A CN201010191097A CN101846685A CN 101846685 A CN101846685 A CN 101846685A CN 201010191097 A CN201010191097 A CN 201010191097A CN 201010191097 A CN201010191097 A CN 201010191097A CN 101846685 A CN101846685 A CN 101846685A
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Abstract
The invention discloses an enzyme-linked immunoassay kit for chlamys ferreri blood cells and a preparation method thereof. The kit comprises an enzyme labeled plate, confining solution, washing solution, enzyme labeled antibody, pNPP substrate developing solution, phosphate buffer solution, blood cell anticoagulant, chlamys ferreri blood cell monoclonal antibody and a blood cell standard sample. The preparation method for the kit comprises the following steps of: preparing the chlamys ferreri blood cell standard sample, preparing the blood cell monoclonal antibody, optimizing an optimal using proportion of the antigen and the antibody, and establishing a detection result assessment criteria. Detection results can assess the possibility that the chlamys ferreri to be detected is subjected to pathogenic infection or environmental stress and the like by judging the established assessment criteria so as to provide basis for monitoring the health condition of the chlamys ferreri.
Description
Technical field
The present invention relates to enzyme linked immunological (ELISA) detection kit of a kind of using monoclonal antibody detection by quantitative Chlamys farreri (Chlamys farreri) haemocyte and preparation method thereof, belong to shellfish molecular immunology technical field.
Background technology
Shellfish lacks immunoglobulin (Ig) and specific immunity, and its immune defense process is to finish by the humoral factor in haemocyte and the hemolymph is collaborative.Haemocyte by self-dissolving, assemble, engulf, mode such as packing, exocytosis, oxidation kill and wound, reach identification, wrap up and remove the purpose of external foreign matter; Haemocyte can also be nursed one's health auxiliary humoral factor immunity by discharging materials such as bacteriolysin, agglutinin, opsonin, and it is resisted in the process that external environment stimulates and the exotic disease pathogenic microorganism is attacked shellfish and plays key effect.
Chlamys farreri is the important economic species of the coastal shellfish culture of northern China.Disease takes place frequently in recent years, becomes one of factor of restriction Chlamys farreri aquaculture sustainable development gradually.Present Chlamys farreri breeding way such as adopt that marine raft formula is raised in cages, but bigger to the generation of disease, prevention and popular control difficulty in wide waters more, thereby the monitoring of shellfish health status seemed particularly important.Have and discover that the variation of envirment factor and the invasion and attack of cause of disease can cause the marked change of scallop haemocyte quantity.This seminar discovers that scallop is after hunger, high temperature or cause of disease stimulate, and its haemocyte quantity will be starkly lower than the normal scallop that does not stimulate; In addition, Ma Hongming etc. [hi-tech communication, 2006:16 (7), 745-751] also report scallop behind salinity bust or bacterial infection, and its haemocyte quantity can die-off; Some foreign scholars also find in other shellfish researchs: shellfish is through Cu
2+, Pb
2+, do to reveal, after anoxics etc. stimulated, haemocyte quantity can be starkly lower than a certain normal value, some in addition dead.Therefore, the change of haemocyte quantity can be used as the detection index that an assessment scallop is subjected to pathogen infection or environment-stress etc.
The method that detects the haemocyte variation of quantity at present mainly contains blood counting chamber method and flow cytometer method.Blood counting chamber method error is big, subjectivity strong, be not suitable for extensive sample detection; The flow cytometer method costs an arm and a leg, and consumptive material expense height needs special technician's operation and not strong to the specific aim of sample, easily thinks the impurity fragment by mistake haemocyte.Monoclonal antibody (hereinafter to be referred as: monoclonal antibody) have characteristics such as high specificity, sensitivity height and often be applied in the quantitative examination of the location of immunocyte and cause of disease.
Summary of the invention
At the problems referred to above, an object of the present invention is to provide enzyme linked immunological (ELISA) detection kit that a kind of high specificity, degree of accuracy height, the anti-chlamys ferreri blood cells monoclonal antibody of application highly sensitive, good reproducibility detect the haemocyte number change, in the hope of assessing the possibility that cultured scallop is subjected to pathogen infection or environment-stress etc., for monitoring scallop health status provides foundation.
Another object of the present invention provides the preparation method of above-mentioned chlamys ferreri blood cells ELISA detection kit.
ELISA detection kit of the present invention comprises: ELISA Plate, confining liquid, cleansing solution, enzymic-labelled antibody, pNPP substrate colour developing liquid, phosphate buffer (PBS), the blood cell anticoagulation agent is characterized in that described kit also comprises anti-chlamys ferreri blood cells monoclonal antibody, haemocyte standard model.
Wherein said anti-chlamys ferreri blood cells monoclonal antibody is the culture supernatant by the monoclonal hybridoma 1F7 that collects anti-chlamys ferreri blood cells, cross Protein G-agarose affinity chromatography post, concentrate, dialysis, purifying make, and it is characterized in that described monoclonal antibody can combine with Chlamys farreri all kinds haemocyte (transparent haemocyte, granular hemocyte) specificity;
Described haemocyte standard model is by collecting the haemocyte of healthy scallop, makes behind resuspended, ultrasonic disruption;
Described confining liquid is for containing the PBS of 3.0% (w/v) bovine serum albumin(BSA);
Described cleansing solution is for containing the PBS of 0.1% (v/v) Tween-20;
Described enzymic-labelled antibody is the goat anti-mouse igg antibody of alkaline phosphatase (AP) mark;
Described blood cell anticoagulation agent is the PBS that contains 0.02M EDTA.
The preparation method of ELISA detection kit of the present invention comprises the following steps:
1. preparation haemocyte standard model: get healthy Chlamys farreri, quantitatively extract hemolymph, centrifugal after, gained haemocyte precipitation is resuspended with the blood cell anticoagulation agent of equivalent, through ultrasonic disruption, as the haemocyte standard model of this kit;
2. the monoclonal antibody for preparing anti-chlamys ferreri blood cells: with the haemocyte standard model is the antigen immune mouse, and Fusion of Cells through the indirect immunofluorescence screening, is cloned, and obtains the monoclonal hybridoma of anti-chlamys ferreri blood cells, and its culture supernatant promptly contains monoclonal antibody; Choose 1 strain Immunofluorescence Reactions result and be strong positive, and the monoclonal antibody that can combine with Chlamys farreri all kinds haemocyte specificity is as the chlamys ferreri blood cells monoclonal antibody of this kit;
3. determine the best usage ratio of antigen and antibody: above-mentioned haemocyte standard model and haemocyte monoclonal antibody are carried out gradient dilution with phosphate buffer respectively, obtain the suitable reaction ratio of antigen-antibody and then the best usage ratio of definite antigen and antibody by ELISA chessboard titrimetry;
4. set up the testing result evaluation criteria: respectively by the stimulation of high temperature and cause of disease, the ELISA method of using the haemocyte monoclonal antibody detects the number change of haemocyte under each incentive condition; According to the statistical analysis method, formulate scallop health status and the critical range that is subjected to environment-stress with the result of normal temperature and high temperature stimulation test group; Result with contrast and pathogen infection experimental group formulates the critical range that scallop is subjected to environment-stress and pathogen infection; Between the two, be the scope that is subjected to environment-stress; Thereby set up evaluation criteria.
Advantage of the present invention be with haemocyte as antigen coated in ELISA Plate, using the haemocyte monoclonal antibody intuitively expresses the haemocyte content of sample to be checked by the method for high efficiency enzymic-labelled antibody colour developing, high specificity, the degree of accuracy height, highly sensitive, the parallel detection, repeatability and the good stability that can be used for small amount of sample, a plurality of samples.Testing result can be assessed the possibility that scallop is subjected to pathogen infection or environment-stress etc., plays the effect that gives warning in advance, and makes things convenient for the raiser to take prevention and control measure early.
Description of drawings
Fig. 1 is the result that chlamys ferreri blood cells suspension of the present invention and monoclonal antibody 1F7 reaction indirect immunofluorescence detect.
The ELISA testing result of chlamys ferreri blood cells sample when Fig. 2 is 15 ℃ and 25 ℃ for water temperature of the present invention.
Fig. 3 is 25 ℃ for water temperature of the present invention, chlamys ferreri blood cells sample ELISA testing result when being subjected to pathogen infection.
Fig. 4 is the ELISA testing result of the chlamys ferreri blood cells sample of Qingdao in 2009 of the present invention 3～Dec area breed.
Shown in Figure 1: A is the result of fluoroscopic examination under the dark field; B is the result that the original position differential interference detects under the bright-field; Wherein G is a granular hemocyte; H is transparent haemocyte.
Embodiment
The invention will be further described below in conjunction with accompanying drawing and by specific embodiment.
Embodiment 1: the preparation of chlamys ferreri blood cells ELISA detection kit
1. prepare the haemocyte standard model
Get 30～40 vigor health, normal (the suitable living environment: 15 ℃ of water temperatures of physique, salinity 31ppt, the bait abundance, no pathogenic microorganism etc.) Chlamys farreri, extracting hemolymph 50ml from closed shell flesh, is 1: 1 blood cell anticoagulation agent (the PBS:0.02M EDTA that contains 0.02M EDTA, 0.14M NaCl with precooling by volume, 3mM KCl, 8mM Na
2HPO
4, 1.5mM KH
2PO
4, pH 7.4) and mixing, mixed liquor is through 3000r/min, 4 ℃ of centrifugal 10min, gained haemocyte precipitation, resuspended with the 50ml anti-coagulants, again through ultrasonic disruption, be the haemocyte standard model, packing ,-80 ℃ are frozen.
2. the monoclonal antibody for preparing anti-chlamys ferreri blood cells
(1) animal immune: get 100 μ l haemocyte standard models as antigen and isopyknic Freund's complete adjuvant mixing, fully after the emulsification, from the abdominal cavity multi-point injection emulsion of BALB/c mouse totally 100 μ l; First immunisation is carried out booster immunization (adjuvant is used incomplete Freund instead, the same first immunisation in immunizing dose and position) after two weeks; After one week, carry out secondary booster immunization (the haemocyte standard model 100 μ l of no adjuvant, tail vein injection); After one week, the immunity of increasing (immune substance, dosage and position are with the secondary booster immunization); After three days, merge;
(2) screening of Fusion of Cells and haemocyte positive hybridoma: with the splenocyte of immune mouse and myeloma cell [Zhu Liping according to a conventional method, old serum etc. immunology is used test method [M] always. Beijing: People's Medical Officer Press, 2000,23-74] carry out Fusion of Cells, cultivation, and indirect immunofluorescence (to be antigen) screening without fixing scallop blood cell suspension, obtain the positive hybridoma cell of anti-scallop haemocyte; With limiting dilution assay positive hybridoma cell is cloned again, obtain the positive monoclonal hybridoma of anti-scallop haemocyte.Collect the culture supernatant (promptly containing monoclonal antibody) of positive monoclonal hybridoma, further filter out the monoclonal antibody 1F7 of 1 strain immunofluorescence result for strong positive by indirect immunofluorescence, it can combine (Fig. 1) with transparent haemocyte of Chlamys farreri and the equal specificity of granular hemocyte.Monoclonal antibody 1F7 is after SABC detects, and the result shows itself and other histocyte no cross reaction of Chlamys farreri;
(3) collection of haemocyte monoclonal antibody and purifying: the culture supernatant 250ml that collects hybridoma 1F7, cross the affinity column (HiTrap Protein G Sepharose Column) of AmershamPhamacia Biotech company, concentrate, dialysis, freeze-drying, PBS is resuspended, packing ,-80 ℃ frozen.
3. determine the best usage ratio of antigen and antibody
The haemocyte standard model presses 2 with PBS
0, 2
-1, 2
-2, 2
-3, 2
-4, 2
-5, 2
-6With 2
-7The gradient dilution bag by in the ELISA Plate hole, every hole 100 μ l, sample pipetting volume 6 holes of each gradient, 4 ℃ are spent the night; Add confining liquid (with 3.0% bovine serum albumin solution of PBS preparation) sealing 1h; Haemocyte monoclonal antibody (one is anti-) is pressed 2 with PBS
0, 2
-1, 2
-2, 2
-3, 2
-4With 2
-5Gradient dilution, join in the corresponding antigen hole by the chessboard titrimetry, every hole 100 μ l are hatched 1.5h for 37 ℃; Add enzymic-labelled antibody (AP two is anti-) 100 μ l again, hatch 1h for 37 ℃; Add pNPP substrate colour developing liquid 100 μ l at last, hatch 30min for 37 ℃, with microplate reader in 405nm place reading.The result shows (table 1): haemocyte standard model concentration and haemocyte monoclonal antibody concentration combination are 2
02
0, 2
02
-1, 2
-12
0, 2
-12
-2, 2
-22
0, 2
-22
-1The time OD
405nmValue generally is higher than other various combinations, is the suitable reaction ratio of antigen-antibody.Consider problems such as actual amount, saving cost, select 2
-12
-2(being 2 times of antigen diluents, 4 times of monoclonal antibody dilutions) is as the best usage ratio of kit antigen-antibody.
Table 1: the OD of each antigen, the combination of antibody gradient
405nmValue, runic is OD
405nmValue 〉=0.5600.
4. set up the testing result evaluation criteria
(1) design of high temperature and cause of disease stimulation test: get 100 scallops under the condition of water temperature 15 ℃ (normal temperature) and 25 ℃ (high temperature) (50 every group) respectively, cultured 7 days; Get 100 scallops and inject scallop acute viral necrobiotic virus (AVNV) (AVNV) crude extract respectively, every 100 μ l, other gets the sterile saline of 100 scallops injection 2% and organizes in contrast, in water temperature is respectively to culture 15 days under 25 ℃ the condition.Observe the health status of each experimental group scallop every day, 5～6 scallops of every group of sampling quantitatively extract hemolymph, centrifugal after, gained haemocyte precipitation is resuspended with the equivalent anti-coagulants, through ultrasonic disruption, packing ,-80 ℃ frozen, stand-by;
(2) ELISA detects: respectively will through temperature and cause of disease stimulate each day sample and the haemocyte standard model with the 2-1 dilution as antigen, bag is by in the ELISA Plate hole, every hole 100 μ l, 4 ℃ are spent the night; Add confining liquid and hatch 1h in 37 ℃; Add the haemocyte monoclonal antibody of 2-2 dilution, every hole 100 μ l are hatched 1.5h for 37 ℃; Add enzymic-labelled antibody (AP two is anti-), every hole 100 μ l are hatched 1h for 37 ℃; Add pNPP substrate colour developing liquid at last, every hole 100 μ l are hatched 30min for 37 ℃, in 405nm place reading.Treat the OD of haemocyte standard model
405nmValue reaches at 0.6 o'clock, can stop reading, reads the OD that respectively organizes sample
405nmValue;
(3) ELISA result: water temperature is the OD in 15 ℃ the experimental group 7 days
405nmValue is positioned at 0.591～0.625, does not have significant difference between each value; Water temperature is the OD in 25 ℃ the experimental group 7 days
405nmValue is positioned at 0.489～0.586, and each value is compared with the minimum (0.591) of 15 ℃ of groups, and except that the 7th day the no significant difference of value (0.586), each value of the 1st～6 day all significantly is lower than 0.591 (Fig. 2).Under 25 ℃ of water temperature conditions, the OD in the control group (injecting normal saline) 15 days
405nmValue is positioned at 0.486～0.621; Pathogen infection group scallop in the 2nd～9 day is sharply dead, and final survival rate is 44%; OD in 15 days
405nmValue is positioned at 0.261～0.571, and wherein each value (0.261～0.446) of the 3rd～9 day is low especially, and the minimum (0.486) that significantly is lower than control group (Fig. 3);
(4) analyzing and testing result sets up evaluation criteria: in conjunction with the result of Fig. 2, choose maximal value (0.549) in the 1st～6 day each value of 25 ℃ of experimental group, be the critical range of environment-stress and health status between 0.549 and 0.591; In conjunction with the result of Fig. 3, in the 3rd～9 day each value of pathogen infection group, choose maximal value (0.446), be the critical range of environment-stress and pathogen infection between 0.446 and 0.486.Through the significance of difference and statistical result analysis, the environment-stress of this kit and the critical range of health status are 0.57 ± 0.025, and scallop is a health status when being higher than this scope; The critical range of environment-stress and pathogen infection is 0.47 ± 0.019, and scallop is the pathogen infection state when being lower than this scope.Scallop is the environment-stress state during scope between the two.
Embodiment 2: the concrete using method of chlamys ferreri blood cells ELISA detection kit
1. sample pre-treatments to be checked: quantitatively extracting hemolymph with sterilizing syringe from closed shell flesh, is 1: 1 mixing with the agent of precooling blood cell anticoagulation immediately by volume.Mixed liquor is in 3000r/min, 4 ℃ of centrifugal 10min, and gained haemocyte precipitation is used with the anti-coagulants of hemolymph equivalent resuspended, behind ultrasonic disruption, is sample to be checked;
2. envelope antigen: with above-mentioned sample to be checked and haemocyte standard model with PBS with 2
-1Dilution is added in the ELISA Plate hole, every hole 100 μ l, and 4 ℃ of bags are spent the night;
3. sealing: discard liquid in the hole, add 200 μ l cleansing solutions (PBST) in every hole, jiggle 5min, outwell cleansing solution, 3 times repeatedly.After washing, in every hole, add 200 μ l confining liquids, hatch 1h for 37 ℃;
4. anti-hatching: discard liquid in the hole, add 200 μ l cleansing solutions in every hole, 5min/ time, wash 3 times.After washing, add 100 μ l in every hole and use PBS with 2
-2The haemocyte monoclonal antibody of dilution is hatched 1.5h for 37 ℃;
5. two anti-hatching: discard liquid in the hole, add 200 μ l cleansing solutions in every hole, 5min/ time, wash 3 times.After washing, add 100 μ l enzymic-labelled antibodies in every hole, hatch 1h for 37 ℃;
6. colour developing reading: discard liquid in the hole, add 200 μ l cleansing solutions in every hole, 5min/ time, wash 3 times.After washing, add 100 μ l pNPP substrates colour developing liquid in every hole, in 37 ℃ hatch 30min after with microplate reader reading under the 405nm wavelength.Treat the OD of haemocyte standard model
405nmValue reaches at 0.6 o'clock, can stop reading, reads the OD of sample to be checked
405nmValue;
7. the judgement of testing result: press the appended evaluation criteria of kit, as the OD of sample to be checked
405nmValue is lower than at 0.47 ± 0.019 o'clock and is judged to be the pathogen infection state that is subjected to; Be judged to be the environment-stress state that is subjected in the time of between being positioned at 0.47 ± 0.019 and 0.57 ± 0.025; Be higher than at 0.57 ± 0.025 o'clock and be judged to be health status.
1. sample collecting and processing: choose Qingdao of Shandong province sand mouth area, each sampling back was got 20 scallops again at random and is handled sample by the method for sample pre-treatments to be checked among the embodiment 2 from the scallop of being gathered to 100 of every middle of a month random acquisition scallops between Dec in 2009 3;
2. sample detection: by the concrete using method of this kit that is provided among the embodiment 2 the haemocyte sample of gathering in per month is carried out ELISA and detect;
3. interpretation of result (Fig. 4): the OD that detected to regional its haemocyte of the Chlamys farreri ELISA of sand mouth breed in Dec in 2009 3
405nmValue is respectively 0.623,0.648, and 0.598,0.685,0.634,0.402,0.385,0.476,0.487 and 0.542.Wherein each value in 3～July all is higher than 0.57 ± 0.025 (critical range of environment-stress and health status), and this, water temperature was suitable in period, the bait abundance, and scallop is grown quick, and sexual gland is full, and especially June, scallop is in health status; 8, each value in September all is lower than 0.47 ± 0.019 (critical range of environment-stress and pathogen infection), this scallop end breeding period in period, and physique is comparatively weak, and water temperature raises in addition, and cause of disease propagation is accelerated, and very easily is in the state that is subjected to pathogen infection; Each value in 10～Dec all is positioned between 0.47 ± 0.019 and 0.57 ± 0.025, and this, scallop was in a convalescence behind the pathogen infection in period, and the shortage day by day of bait food is in the environment-stress state substantially in addition.The present invention is by the detection to the chlamys ferreri blood cells seasonal variety, and the scallop situation of its testing result and actual production is identical substantially, is applicable to the assessment of scallop health status in the production practices.
Claims (10)
1. the enzyme-linked immunologic detecting kit of a chlamys ferreri blood cells, comprise ELISA Plate, confining liquid, cleansing solution, enzymic-labelled antibody, pNPP substrate colour developing liquid, phosphate buffer, the blood cell anticoagulation agent is characterized in that described kit also comprises anti-chlamys ferreri blood cells monoclonal antibody, haemocyte standard model.
2. chlamys ferreri blood cells enzyme-linked immunologic detecting kit according to claim 1, it is characterized in that: described anti-chlamys ferreri blood cells monoclonal antibody is the culture supernatant by the monoclonal hybridoma 1F7 that collects anti-chlamys ferreri blood cells, cross Protein G-agarose affinity chromatography post, concentrate, dialysis, purifying make.
3. chlamys ferreri blood cells enzyme-linked immunologic detecting kit according to claim 1, it is characterized in that: described chlamys ferreri blood cells standard model is by extracting the hemolymph of healthy Chlamys farreri, the centrifugal haemocyte that obtains, resuspended, ultrasonic disruption makes.
4. chlamys ferreri blood cells enzyme-linked immunologic detecting kit according to claim 1 is characterized in that: described confining liquid is for containing the phosphate buffer of 3.0% (w/v) bovine serum albumin(BSA).
5. chlamys ferreri blood cells enzyme-linked immunologic detecting kit according to claim 1 is characterized in that: described cleansing solution is for containing the phosphate buffer of 0.1% (v/v) Tween-20.
6. chlamys ferreri blood cells enzyme-linked immunologic detecting kit according to claim 1 is characterized in that: described enzymic-labelled antibody is the goat anti-mouse igg antibody of alkali phosphatase enzyme mark.
7. chlamys ferreri blood cells enzyme-linked immunologic detecting kit according to claim 1 is characterized in that: described blood cell anticoagulation agent is the phosphate buffer that contains 0.02M EDTA.
8. the preparation method of the described chlamys ferreri blood cells enzyme-linked immunologic detecting kit of claim 1, it is characterized in that: described method may further comprise the steps: preparation chlamys ferreri blood cells standard model; With the haemocyte standard model is the antigen immune mouse, preparation haemocyte monoclonal antibody; Choose 1 strain Immunofluorescence Reactions result and be strong positive, and the monoclonal antibody that can combine with all kinds haemocyte specificity is as the anti-chlamys ferreri blood cells monoclonal antibody of the described kit of claim 1; Optimize the best usage ratio of antigen and antibody; Set up the evaluation criteria of testing result.
9. the preparation method of chlamys ferreri blood cells enzyme-linked immunologic detecting kit according to claim 8, it is characterized in that: described 1 strain Immunofluorescence Reactions result is strong positive, and the monoclonal antibody that can combine with all kinds haemocyte specificity is monoclonal antibody 1F7.
10. the preparation method of chlamys ferreri blood cells enzyme-linked immunologic detecting kit according to claim 8, it is characterized in that: described testing result evaluation criteria is the stimulation by high temperature and cause of disease, gather the haemocyte sample under each incentive condition, enzyme linked immunosorbent assay detects the number change of its haemocyte, the gained result obtains to be subjected to the critical range of pathogen infection and environment-stress to determine to form by the statistical method analysis.
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| CN102269764A (en) * | 2011-05-12 | 2011-12-07 | 中国海洋大学 | Enzyme-linked immunodetection kit of scallop granular cells and application thereof |
| CN102321578A (en) * | 2011-07-13 | 2012-01-18 | 中国水产科学研究院东海水产研究所 | Purification method of marine bivalve shellfish blood cell |
| CN102844331A (en) * | 2010-06-03 | 2012-12-26 | 中国海洋大学 | Monoclonal antibody capable of resisting chlamys farrei particle blood corpuscle and preparation method thereof |
| CN104117227A (en) * | 2013-04-28 | 2014-10-29 | 北京华安麦科生物技术有限公司 | Deoxynivalenol immune affinity column and preparation method and use thereof |
| CN104198715A (en) * | 2014-09-12 | 2014-12-10 | 青岛农业大学 | Enzyme-linked immunoassay kit for portunustrituberculatus blood cell and preparing method thereof |
| CN107817259A (en) * | 2017-10-30 | 2018-03-20 | 中国水产科学研究院黄海水产研究所 | A kind of method of seawater blood clam section shellfish blood cell collection and Ultrastructural observation |
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| CN102844331A (en) * | 2010-06-03 | 2012-12-26 | 中国海洋大学 | Monoclonal antibody capable of resisting chlamys farrei particle blood corpuscle and preparation method thereof |
| CN102269764A (en) * | 2011-05-12 | 2011-12-07 | 中国海洋大学 | Enzyme-linked immunodetection kit of scallop granular cells and application thereof |
| CN102321578A (en) * | 2011-07-13 | 2012-01-18 | 中国水产科学研究院东海水产研究所 | Purification method of marine bivalve shellfish blood cell |
| CN102321578B (en) * | 2011-07-13 | 2013-04-17 | 中国水产科学研究院东海水产研究所 | Purification method of marine bivalve shellfish blood cell |
| CN104117227A (en) * | 2013-04-28 | 2014-10-29 | 北京华安麦科生物技术有限公司 | Deoxynivalenol immune affinity column and preparation method and use thereof |
| CN104117227B (en) * | 2013-04-28 | 2016-08-03 | 北京华安麦科生物技术有限公司 | A kind of deoxynivalenol immune affinity column and its production and use |
| CN104198715A (en) * | 2014-09-12 | 2014-12-10 | 青岛农业大学 | Enzyme-linked immunoassay kit for portunustrituberculatus blood cell and preparing method thereof |
| CN107817259A (en) * | 2017-10-30 | 2018-03-20 | 中国水产科学研究院黄海水产研究所 | A kind of method of seawater blood clam section shellfish blood cell collection and Ultrastructural observation |
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Application publication date: 20100929 |