CN107389921B - A kind of preparation method and application of rabbit-anti grouper serum immune globulin HRP labelled antibody - Google Patents
A kind of preparation method and application of rabbit-anti grouper serum immune globulin HRP labelled antibody Download PDFInfo
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Abstract
The invention discloses a kind of preparation method and applications of rabbit-anti grouper serum immune globulin HRP labelled antibody, belong to antibody production techniques field.The enzyme antibody conjugate that antibody of the present invention is made of HRP and rabbit-anti grouper serum immune globulin antibody, can specifically bind, and the substrate of HRP can be made to develop the color with grouper serum immune globulin.HRP labelled antibody prepared by the present invention is with high purity, enzymatic activity is high, conjugate mark rate is suitable for, and the dilution for carrying out ELISA test is big, and high sensitivity；It can be used for detecting viral disease, parasitic diseases and the bacteriosis of grouper.
Description
Technical field
The invention belongs to antibody production techniques fields, and in particular to a kind of rabbit-anti grouper serum immune globulin HRP mark
Remember the preparation method and application of antibody.
Background technique
Grouper is the high-quality fish of top grade in current marine fish.With China's Artificial Rearing of Epinephelus coioides
Mature and Epinephelus coioides seed industrialization large-scale production, becomes important marine fish culture kind.But it is sick in recent years
Evil takes place frequently, and causes huge economic loss to the aquaculture industry of grouper.
Virus, helminth and bacterium infection are very harmful to prelarva and juvenile fish, cause to marine fish culture industry huge
Loss.
Currently, generalling use PCR method detection lithosporic fish virus, helminth and bacterium infection, PCR method is by detecting fish body
The expression of viral gene in brain and eyes, Lai Jianding disease infect；However, parent population is as nursery in the nursery of grouper
Main body, it is impossible to by parent population kill detected again by PCR method；Therefore, a kind of basis for guaranteeing fish body normal vitality is needed
On, the method that detects disease.
Summary of the invention
In order to solve the problems in the existing technology, the technical scheme is that
A kind of rabbit-anti grouper serum immune globulin HRP labelled antibody, it is by HRP and rabbit-anti grouper sero-immunity
The enzyme antibody conjugate of globulin antibody composition, can specifically bind, and can make HRP's with grouper serum immune globulin
Substrate colour developing.
On the basis of above scheme, the grouper is Epinephelus coioides.
On the basis of above scheme, the enzyme antibody of the HRP and rabbit-anti grouper serum immune globulin antibody composition
Conjugate is that the antibody of the fish immunoglobulin of purifying is added after aoxidizing HRP with sodium periodate to be coupled, and has been coupled
It is restored at rear addition protective agent and with sodium borohydride.
On the basis of above scheme, the enzyme antibody of the HRP and rabbit-anti grouper serum immune globulin antibody composition
The preparation method of conjugate, steps are as follows:
A. 6mg HRP is taken to be dissolved in 1mL distilled water, 0.05M sodium periodate solution 1mL, after mixing, 4 DEG C of refrigerator standings are kept away
Light 25min；
B. 0.2mol/L glycol water 0.8mL is added after taking out, after being placed at room temperature for 25min, rabbit containing 15mg is added
The aqueous solution 5mL of anti-grouper serum immune globulin antibody, dialysed overnight after mixing；
C. it is sucked out and adds 7mg/mL NaBH44 DEG C of refrigerator 2h are set in solution 0.5mL, mixing；
D. plus in equal volume saturated ammonium sulfate solution, after 4 DEG C of standing 1h, 3 000r/min low-temperature centrifugation 30min abandon supernatant；
E. it by after the precipitating PBS dissolution of 0.02mol/L pH7.4, is fitted into bag filter, is put into 0.02mol/L pH7.4
Phosphate buffer solution (PBS) in, 4 DEG C dialysis；
F. after dialysing, it is centrifuged 30min in 10000r/min, supernatant is collected up to the enzyme conjugates of antibody, equivalent is added
Glycerol mixes, and packing is stored in -20 DEG C.
On the basis of above scheme, the preparation method of the rabbit-anti grouper serum immune globulin antibody, step is such as
Under:
1) grouper serum immune globulin purifies
A. it kills fish and takes blood, standing takes serum, ammonium sulfate is added into serum, makes its final concentration of 10%, 10000r/
Min is centrifuged 5min, abandons precipitating；
B. ammonium sulfate is added into the solution after above-mentioned centrifugation, makes its final concentration of 30%, 10000r/min centrifugation
5min abandons supernatant；
C. above-mentioned precipitating is dissolved with PBS buffer solution, Bradford method measures protein concentration；
2) preparation and purification of grouper immunoglobulin rabbit antibody
A. adjuvant system is added in the fish immunoglobulin of 2mg purifying to mix, for rabbit to be immunized, between being immunized twice
Every 2 weeks；After the 4th is immune, ear vein takes blood, with ELISA qualitative determination, measures potency and reaches 40000 or more, kill rabbit and take entirely
Blood；
B. the rabbit blood after will be immune takes serum after standing, and serum is centrifuged 30min in 20000g, takes supernatant that proteinA is added
Affine absorption is carried out in filler, and twice with PBS buffer solution rinsing, the glycine elution for being later 2.5 with pH uses 1.5M immediately
PH is the pH to 7.4 in 8.0 Tris buffer with eluent；Finally at 4 DEG C with PBS buffer solution dialyse in and after elution
Liquid, the solution after dialysis are the antibody-solutions of fish serum immunoglobulin.
A kind of application of rabbit-anti grouper serum immune globulin HRP labelled antibody, for detecting the viral of grouper
Disease, parasitic diseases and bacteriosis.
On the basis of above scheme, the viral disease is nervous necrosis virus infection；The parasitic diseases
It is infected for stimulation cryptonucleus insect；The bacteriosis is vibrio parahaemolytious infection.
A kind of kit detecting grouper nervous necrosis virus includes above-mentioned rabbit-anti grouper serum immune globulin
HRP labelled antibody.
On the basis of above scheme, the kit of the detection grouper nervous necrosis virus, further includes antigen；It is described
Antigen is the pathogenic microorganism albumen for causing grouper viral disease, parasitic diseases and bacteriosis.
On the basis of above scheme, detection method is as follows:
A. it after antigen being diluted to 5 μ g/mL with PBS buffer solution, is added in elisa plate, 37 DEG C are incubated for 2h or 4 DEG C of coating
Overnight；Tap water board-washing is three times；
B. the antigenic solution in elisa plate is outwelled, confining liquid closing, 37 DEG C of incubation 1h in wet box are added；
C. it outwells confining liquid and is added fish serum to be measured, 37 DEG C of incubation 1h in wet box, tap water board-washing 6 times；
D. 1: 2000 diluted rabbit-anti grouper serum immune globulin HRP labelled antibody, 37 DEG C of incubations in wet box are added
1h；
E. it outwells rabbit-anti grouper serum immune globulin HRP labelled antibody solution, after tap water board-washing 6 times, is added aobvious
The colour developing of color liquid, room temperature, which is protected from light, stands 15min；
F. the H of 2M is added2SO4Color development stopping reads the OD value under 492nm wavelength in microplate reader.
The Epinephelus coioides (Epinepheluscoioides) are Epinephelus, derive from the small hill path aquatic science and technology in Xiamen
Co., Ltd's aquatic farm.
Beneficial effects of the present invention:
HRP labelled antibody prepared by the present invention is with high purity, enzymatic activity is high, conjugate mark rate is suitable for, and carries out ELISA test
Dilution it is big, and high sensitivity.
Labelled antibody prepared by the present invention can be used for the monitoring and detection of many diseases of ablen, and advantage mainly has 1.
Have the advantages that monoclonal antibody does not have, can detecte the infection of a variety of cause of diseases of grouper, such as parasitic infection, bacterium infection
And the infection of virosis, and monoclonal antibody can only be detected for a kind of cause of disease；2. can not only infection to cause of disease into
Row qualitative (whether infecting), but also can substantially determine the degree of pathogen infection, as it detects potency after infecting 6 days and 15 days
Difference, potency is higher, illustrates that pathogen infection is more serious.3. standard curve can be done for the infection of certain cause of disease, then detect,
The occurrence and development of related disease can be monitored in real time.
Detailed description of the invention
The standard curve of Fig. 1 nervous necrosis virus infection；
The standard curve that Fig. 2 stimulates cryptonucleus insect to infect；
The standard curve of Fig. 3 vibrio parahaemolytious infection.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment 1
1. the preparation of rabbit-anti grouper serum immune globulin antibody
1) grouper serum immune globulin purifies
A. it kills fish and takes blood, standing takes serum, ammonium sulfate is added into serum, makes its final concentration of 10%, 10000r/
Min is centrifuged 5min, abandons precipitating；
B. ammonium sulfate is added into the solution after above-mentioned centrifugation, makes its final concentration of 30%, 10000r/min centrifugation
5min abandons supernatant；
C. above-mentioned precipitating is dissolved with PBS buffer solution, Bradford method measures protein concentration；
2) preparation and purification of grouper immunoglobulin rabbit antibody
A. adjuvant system is added in the fish immunoglobulin of 2mg purifying to mix, for rabbit to be immunized, between being immunized twice
Every 2 weeks；After the 4th is immune, ear vein takes blood, with ELISA qualitative determination, measures potency and reaches 40000 or more, kill rabbit and take entirely
Blood；
B. the rabbit blood after will be immune takes serum after standing, and serum is centrifuged 30min in 20000g, takes supernatant that proteinA is added
Affine absorption is carried out in filler, and twice with PBS buffer solution rinsing, the glycine elution for being later 2.5 with pH uses 1.5M immediately
PH is the pH to 7.4 in 8.0 Tris buffer with eluent；Finally at 4 DEG C with PBS buffer solution dialyse in and after elution
Liquid, the solution after dialysis are the antibody-solutions of fish serum immunoglobulin.
The Epinephelus coioides (Epinepheluscoioides) are Epinephelus, derive from the small hill path aquatic science and technology in Xiamen
Co., Ltd's aquatic farm.
2. the HRP of rabbit-anti grouper serum immune globulin antibody is marked
A. 6mg HRP is taken to be dissolved in 1mL distilled water, 0.05M sodium periodate solution 1mL, after mixing, 4 DEG C of refrigerator standings are kept away
Light 25min；
B. 0.2mol/L glycol water 0.8mL is added after taking out, after being placed at room temperature for 25min, rabbit containing 15mg is added
The aqueous solution 5mL of anti-grouper serum immune globulin antibody, dialysed overnight after mixing；
C. it is sucked out and adds 7mg/mL NaBH44 DEG C of refrigerator 2h are set in solution 0.5mL, mixing；
D. plus in equal volume saturated ammonium sulfate solution, after 4 DEG C of standing 1h, 3 000r/min low-temperature centrifugation 30min abandon supernatant；
E. it by after the precipitating PBS dissolution of 0.02mol/L pH7.4, is fitted into bag filter, is put into 0.02mol/L pH7.4
Phosphate buffer solution (PBS) in, 4 DEG C dialysis,
F. after dialysing, it is centrifuged 30min in 10 000r/min, supernatant is collected up to the enzyme conjugates of antibody, equivalent is added
Glycerol mixes, and packing is stored in -20 DEG C.
The Quality Identification of 3.HRP label rabbit-anti grouper serum immune globulin antibody
Using microplate reader, HRP label rabbit-anti grouper serum immune globulin antibody is detected at 280nm and 403nm
OD value；It is as follows to calculate enzyme content, IgG content, mole ratio, conjugate mark rate calculation formula:
Enzyme content (mg/mL)=OD403nm×0.4mg/mL
IgG amount (mg/mL)=(OD280nm-OD403nm×0.3)×0.62mg/L
Gram molecule ratio=(enzyme content/IgG amount) × 4
Conjugate mark rate=(OD403nm/OD280Nm) × 100%
Using the enzymatic activity of o-dianisidine method measurement marker, Salmonella method measures optimal use concentration.
When the mark rate of conjugate is between 30-60%, mole ratio in 1.0-2.0, enzymatic activity > 1000U/mg,
It is complied with standard when thinner ratio > 1: 1000 when ELISA.
OD is measured using microplate reader280And OD403Respectively 2.01 and 1.12；It is calculated according to above-mentioned formula:
Enzyme content is 0.448mg/L, IgG content 1.038mg/L, mole ratio 1.726, conjugate mark rate are
55.72%；Thinner ratio when enzymatic activity > 1000U/mg, ELISA is 1: 2000.
Embodiment 2
A method of detection grouper nervous necrosis virus
A kind of kit detecting grouper nervous necrosis virus includes above-mentioned rabbit-anti grouper serum immune globulin
HRP labelled antibody；It further include antigen, the antigen is grouper nervous necrosis virus Coat albumen.
The preparation of standard curve:
It selects size for 100 tail of the Epinephelus coioides young of 3-4cm, is randomly divided into two groups, every group 50, test group is injected
5×106The 30 μ L of nervous necrosis virus of copies/ μ L, control group inject the physiological saline of equivalent sterilizing.Respectively at the of test
3,6,9,12 and 15 days, every group of potency for taking 6 fish blood drawings to detect nervous necrosis virus antibody in serum at random.The nerve is bad
Dead virus is RGNNV serotype.
Detection method is as follows:
A. it after antigen being diluted to 5 μ g/mL with PBS buffer solution, is added in elisa plate, 37 DEG C are incubated for 2h or 4 DEG C of coating
Overnight；Tap water board-washing is three times；
B. the antigenic solution in elisa plate is outwelled, confining liquid closing, 37 DEG C of incubation 1h in wet box are added；
C. it outwells confining liquid and is added fish serum to be measured, 37 DEG C of incubation 1h in wet box, tap water board-washing 6 times；
D. 1: 2000 diluted rabbit-anti grouper serum immune globulin HRP labelled antibody, 37 DEG C of incubations in wet box are added
1h；
E. it outwells rabbit-anti grouper serum immune globulin HRP labelled antibody solution, after tap water board-washing 6 times, is added aobvious
The colour developing of color liquid, room temperature, which is protected from light, stands 15min；
F. the H of 2M is added2SO4Color development stopping reads the OD value under 492nm wavelength in microplate reader.
With the logarithm (log of nervous necrosis virus antibody titer in serum2) it is ordinate, the time is abscissa, is made
Standard curve is as shown in Figure 1.
As seen from Figure 1, over time, nervous necrosis virus antibody content gradually rises in Epinephelus coioides serum
Height, in a linear relationship, linear formula y=0.676x+8.104, R2=0.9998 degree of fitting is preferable, can be used as standard curve use
In quantitative analysis, degree of viral infectivity is determined.
Embodiment 3
A method of detection grouper stimulates cryptocaryoniosis.
A kind of kit of detection grouper stimulation cryptocaryoniosis, includes above-mentioned rabbit-anti grouper serum immune globulin
HRP labelled antibody；It further include antigen, the antigen is cryptocaryon irritans larva polypide total protein.
The extraction of cryptocaryon irritans larva polypide total protein
It is extracted according to holoprotein extracts kit (being purchased from Beijing Suo Laibao Science and Technology Ltd., article No. BC3640) specification
Cryptocaryon irritans larva polypide total protein.
The preparation of standard curve:
It selects size for 120 tail of the Epinephelus coioides young of 4-6cm, is randomly divided into two groups, every group of 60 tails, every 20 tail is put into
In cylinder equipped with 40L aeration seawater；Test group impregnates 3 × 103The cryptocaryon irritans larva of a/tail fish, control group do not do any
Processing, other conditions are consistent.Respectively at the 3rd, 6,9,12 and 15 day of test, every group was taken at random in 6 fish blood drawing detection serum
Stimulate cryptonucleus insect polypide total protein antibody titer.
Detection method is the same as embodiment 2.
With the logarithm (log of serum moderate stimulation cryptonucleus insect polypide total protein antibody titer2) it is ordinate, the time is horizontal seat
Mark, it is as shown in Figure 2 to make standard curve.
As seen from Figure 2, over time, Epinephelus coioides serum moderate stimulation cryptonucleus insect polypide total protein antibody content
It gradually rises, in a linear relationship, linear formula y=0.8267x+5.214, R2=0.9991 degree of fitting is preferable, can be used as mark
Directrix curve is used for quantitative analysis, determines the gradient of infection of stimulation cryptonucleus insect.
Embodiment 4
A method of detection grouper vibrio parahaemolytious infection.
A kind of kit of detection grouper vibrio parahaemolytious infection, includes above-mentioned rabbit-anti grouper serum immune globulin
HRP labelled antibody；It further include antigen, the antigen is vibrio parahaemolytious bacterial protein.
The extraction of vibrio parahaemolytious bacterial protein
It is extracted according to holoprotein extracts kit (being purchased from Beijing Suo Laibao Science and Technology Ltd., article No. BC3640) specification
Vibrio parahaemolytious bacterial protein.
The preparation of standard curve:
It selects size for 100 tail of the Epinephelus coioides young of 4-6cm, is randomly divided into two groups, every group 50, test group is injected
108Vibrio parahaemolytious (being purchased from Chinese industrial Microbiological Culture Collection administrative center number CICC 23924) 50 μ L of cfu, control
The physiological saline of group injection equivalent sterilizing.Respectively at the 3rd, 6,9,12 and 15 day of test, every group took 6 fish blood drawing detections at random
Vibrio parahaemolytious bacterial protein antibody titer in serum.
Detection method is the same as embodiment 2.
With the logarithm (log of vibrio parahaemolytious bacterial protein antibody titer in serum2) it is ordinate, the time is horizontal seat
Mark, it is as shown in Figure 3 to make standard curve.
As seen from Figure 3, over time, vibrio parahaemolytious bacterial protein antibody content in Epinephelus coioides serum
It gradually rises, in a linear relationship, linear formula y=0.8613x+6.628, R2=0.9989 degree of fitting is preferable, can be used as mark
Directrix curve is used for quantitative analysis, determines the gradient of infection of vibrio parahaemolytious.
The above described is only a preferred embodiment of the present invention, being not that the invention has other forms of limitations, appoint
What those skilled in the art changed or be modified as possibly also with the technology contents of the disclosure above equivalent variations etc.
Imitate embodiment.But without departing from the technical solutions of the present invention, according to the technical essence of the invention to above embodiments institute
Any simple modification, equivalent variations and the remodeling made, still fall within the protection scope of technical solution of the present invention.
Claims (7)
1. a kind of rabbit-anti grouper serum immune globulin HRP labelled antibody, it is characterised in that: it is by HRP and rabbit-anti lithosporic
The enzyme antibody conjugate of fish serum immune globulin antibody composition, can specifically bind with grouper serum immune globulin,
And the substrate of HRP can be made to develop the color；
The preparation method of the rabbit-anti grouper serum immune globulin antibody, steps are as follows:
1) grouper serum immune globulin purifies
A. kill fish and take blood, standing takes serum, and ammonium sulfate is added into serum, make its final concentration of 10%, 10000 r/min
It is centrifuged 5min, abandons precipitating；
B. ammonium sulfate is added into the solution after above-mentioned centrifugation, makes its final concentration of 30%, 10000 r/min centrifugation 5min,
Abandon supernatant；
C. the resulting precipitating of step b is dissolved with PBS buffer solution, Bradford method measures protein concentration；
2) preparation and purification of grouper immunoglobulin rabbit antibody
A. adjuvant system is added in the grouper serum immune globulin of 2mg purifying to mix, it is every that it is immunized twice for rabbit to be immunized
Between be spaced 2 weeks；After the 4th is immune, ear vein takes blood, with ELISA qualitative determination, measures potency and reaches 40000 or more, kill rabbit and take
Whole blood；
B. the rabbit blood after will be immune takes serum after standing, and serum is centrifuged 30min in 20000g, takes supernatant that proteinA is added and fills out
Affine absorption is carried out in material, and twice with PBS buffer solution rinsing, the glycine elution for being later 2.5 with pH uses 1.5M immediately
PH is the pH to 7.4 in 8.0 Tris buffer with eluent；Finally at 4 DEG C with PBS buffer solution dialyse in and after elution
Liquid, the solution after dialysis are the antibody-solutions of rabbit-anti grouper serum immune globulin.
2. rabbit-anti grouper serum immune globulin HRP labelled antibody according to claim 1, it is characterised in that: the stone
Spot fish is Epinephelus coioides.
3. the antibody of rabbit-anti grouper serum immune globulin HRP label according to claim 1 or claim 2, it is characterised in that: institute
The enzyme antibody conjugate for stating HRP and rabbit-anti grouper serum immune globulin antibody composition is to carry out oxygen to HRP with sodium periodate
After change, the rabbit-anti grouper serum immune globulin antibody that purifying is added is coupled, and protective agent is added after the completion of coupling and is used in combination
Sodium borohydride is restored.
4. the antibody of rabbit-anti grouper serum immune globulin HRP label according to claim 3, it is characterised in that: described
The preparation method of the enzyme antibody conjugate of HRP and rabbit-anti grouper serum immune globulin antibody composition is as follows:
A. it takes 6mg HRP to be dissolved in 1mL distilled water, 1 mL of 0.05M sodium periodate solution is added, after mixing, 4 DEG C of refrigerator standings are kept away
Light 25min；
B. 0.2 mol/L glycol water 0.8mL is added after taking out, after being placed at room temperature for 25min, the stone of rabbit-anti containing 15mg is added
The aqueous solution 5mL of spot fish serum immune globulin antibody, dialysed overnight after mixing；
C. 7mg/mL NaBH is added into the solution after dialysis44 DEG C of refrigerator 2h are set in solution 0.5mL, mixing；
D. after taking out plus isometric saturated ammonium sulfate solution, after 4 DEG C of standing 1h, 3 000 r/min low-temperature centrifugation 30min, in abandoning
Clearly；
E. it by after the precipitating PBS dissolution of 0.02 mol/L pH7.4, is fitted into bag filter, is put into 0.02 mol/L pH7.4's
In PBS, 4 DEG C of dialysis；
F. after dialysing, it is centrifuged 30min in 10000r/min, supernatant is collected up to the enzyme conjugates of antibody, equivalent glycerol is added,
It mixes, packing is stored in -20 DEG C.
5. the kit of a kind of detection grouper viral disease, parasitic diseases and bacteriosis, it is characterised in that: packet
Containing rabbit-anti grouper serum immune globulin HRP labelled antibody described in claim 1.
6. the kit of grouper viral disease, parasitic diseases and bacteriosis is detected according to claim 5,
It is characterized by also including antigens；The antigen is to cause grouper viral disease, parasitic diseases and bacteriosis
Pathogenic microorganism albumen.
7. according to the detection grouper viral disease of claim 5 or 6, the reagent of parasitic diseases and bacteriosis
Box, it is characterised in that: detection method is as follows:
A. it after antigen being diluted to 5 μ g/mL with PBS buffer solution, is added in elisa plate, 37 DEG C are incubated for 2h or 4 DEG C of coating overnight；
Tap water board-washing is three times；
B. the antigenic solution in elisa plate is outwelled, confining liquid closing, 37 DEG C of incubation 1h in wet box are added；
C. it outwells confining liquid and is added fish serum to be measured, 37 DEG C of incubation 1h in wet box, tap water board-washing 6 times；
D. 1: 2000 diluted rabbit-anti grouper serum immune globulin HRP labelled antibody, 37 DEG C of incubation 1h in wet box are added；
E. it outwells rabbit-anti grouper serum immune globulin HRP labelled antibody solution, after tap water board-washing 6 times, developing solution is added
Colour developing, room temperature, which is protected from light, stands 15min；
F. the H of 2M is added2SO4Color development stopping reads the OD value under 492nm wavelength in microplate reader.
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| CN113376381A (en) * | 2021-06-11 | 2021-09-10 | 中国水产科学研究院黄海水产研究所 | Breeding method of high-antibody-level parent strain for nervous necrosis of grouper |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104198741A (en) * | 2014-07-23 | 2014-12-10 | 福建省淡水水产研究所 | Tilapia streptococcus agalactiae IgM antibody capture ELISA detection kit |
| CN104497139A (en) * | 2014-12-10 | 2015-04-08 | 敖云霞 | Method for purifying egg yolk immunoglobulin IgY |
| CN104597247A (en) * | 2014-12-09 | 2015-05-06 | 中国海洋大学 | Danio reeio vitellogenin sandwich ELISA kit, detection method and application thereof |
-
2017
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104198741A (en) * | 2014-07-23 | 2014-12-10 | 福建省淡水水产研究所 | Tilapia streptococcus agalactiae IgM antibody capture ELISA detection kit |
| CN104597247A (en) * | 2014-12-09 | 2015-05-06 | 中国海洋大学 | Danio reeio vitellogenin sandwich ELISA kit, detection method and application thereof |
| CN104497139A (en) * | 2014-12-10 | 2015-04-08 | 敖云霞 | Method for purifying egg yolk immunoglobulin IgY |
Non-Patent Citations (2)
| Title |
|---|
| 美洲黑石斑鱼血清 IgM 纯化及其兔抗血清部分特性;郑磊 等;《上海海洋大学学报》;20110731;第20卷(第4期);494-498 * |
| 辣根过氧化物酶(HRP)3种方法标记兔抗牛IgG效果的比较;高琳 等;《中国兽医杂志》;19991231;第25卷(第11期);23-24 * |
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