CN1779462A - Hyperimmune serum against Vibrio parahaemolyticus, its preparation and inspecotion for astacin pathogenic bacterium - Google Patents
Hyperimmune serum against Vibrio parahaemolyticus, its preparation and inspecotion for astacin pathogenic bacterium Download PDFInfo
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- CN1779462A CN1779462A CN 200410091369 CN200410091369A CN1779462A CN 1779462 A CN1779462 A CN 1779462A CN 200410091369 CN200410091369 CN 200410091369 CN 200410091369 A CN200410091369 A CN 200410091369A CN 1779462 A CN1779462 A CN 1779462A
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- vibrio parahaemolytious
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Abstract
A method for preparing high immune serum resisting parahemolytic vibrio uses germ suspension of parahemolytic vibrio to carry out immune on male rabbit and a method of utilizing prepared high immune serum to detect prawn corpus rubrum disease-producing germ includes enveloping standard antigen; blocking enzyme labeled board; making competitive reaction of antiserum on antigen to be tested; making enzyme labeled antibody reaction, color display reaction and ending reaction; and determining absorbance value.
Description
Technical field
The present invention relates to a kind of method that detects prawn kwashiorkor pathogen, specifically, relate to the preparation method of the hyper-immune serum of a kind of anti-vibrio parahaemolytious, and use prepared hyper-immune serum to detect the method for vibrio parahaemolytious in the prawn body with enzyme-linked immunosorbent assay (competitive ELISA).
Background technology
The caused kwashiorkor of vibrio parahaemolytious (V.parahaemolyticus) all has generation from the shrimp seedling to one-tenth each stage of shrimp in prawn, the course of disease is long, the cumulative mortality height.Because this a kind of obvious performance after being ill takes place prawn is " corpus hemorrhagicum ", do not having under the detection method condition of science, culturing the dealer often waits this disease and has disease that " corpus hemorrhagicum " show equally and obscure mutually and make false judgment with " taura syndrome ", and then owing to the measure of adopting is not suited the medicine to the illness, affect treatment opportunity adversely, caused more serious loss.Prawn is taking place all can to show the corpus hemorrhagicum phenomenon in appearance under the situations such as bacteroidal vibriosis, viral " taura syndrome " and envirment factor sudden change.Therefore, setting up cause of disease detection method fast and accurately, accurately judge and differentiate the prawn Different Kinds of Pathogens, accomplish to suit the remedy to the case, is one of effective measures of control disease of prawn.
Up to now, the detection technique of prawn kwashiorkor cause of disease vibrio parahaemolytious mainly is divided into three aspects: the one, and utilization bacteriology is conventional to be separated and authentication method.By purifying cultivation repeatedly, get bacterium colony and carry out the Physiology and biochemistry evaluation, perhaps carry out 16S rRNA gene order homology analysis, the result is searched comparison respectively in bacteriology handbook and gene database judge, this method required time is long, and the cost height does not possess the actual value of applying.The 2nd, the PCR detection technique is set the Auele Specific Primer of this bacterium, in suitable reaction system, increase, and then analysing amplified result.Sensitive high, special characteristics such as strong that this method has, but expense is high and personnel's experimental skill had relatively high expectations etc. limited the application in practice of this method.The 3rd, have the immunology detection technology that high degree of specificity is set up according to antigen-antibody reaction.The fluorescence antibody detection technique is wherein arranged, and is label with the fluorescein, combines as standard reagent with known antibodies, is used to detect unknown antigen, can present the specific antigen-antibody compound of fluorescence under fluorescent microscope and have the position.This technology has high specificity, highly sensitive characteristics, but the fluorescent microscope price is higher at present, and the grass-roots unit that is difficult in actual production promotes.Be exactly enzyme-linked immunosorbent assay in addition, this technology with antigen or antibodies, forms the enzyme labeling thing with the enzyme thing that serves as a mark, and enzyme runs into corresponding substrate and forms coloured product, thus the existence of reflection antigen or antibody.This technology high specificity, highly sensitive, required instrument and equipment is simple, the test sample cost is low, be suitable for applying in vast production grass-roots unit, use this method, for the early diagnosis of prawn kwashiorkor cause of disease, effectively control disease takes place, and aspects such as reduction and the infection of removing prawn vibrio parahaemolytious have the actual application value more outstanding than other method.
Summary of the invention
The object of the present invention is to provide a kind of enzyme-linked immunosorbent assay that detects prawn kwashiorkor pathogen vibrio parahaemolytious.
In order to finish above-mentioned purpose, the present invention at first provides the preparation method of the hyper-immune serum of a kind of anti-vibrio parahaemolytious, and this method comprises: with concentration is 1 * 10
7～1 * 10
9The vibrio parahaemolytious bacteria suspension immunity body weight of cfu/mL is 1.5～2.5 kilograms a male rabbit, per injection 1～3mL bacteria suspension, during first immunisation, in bacteria suspension, add isopyknic Freund's complete adjuvant, adopt the injection system of sole 4～6, first immunisation is carried out 3～5 times immunity once more after 15～20 days, immunity is spaced apart 10～15 days, each immunity in the immunity all adds isopyknic incomplete Freund in bacteria suspension once more, adopt the subcutaneous injection system in back at 4～6, last immunity blood sampling after 7～10 days, separation of serum obtains the hyper-immune serum of anti-vibrio parahaemolytious.
In the preparation process of above-mentioned anti-vibrio parahaemolytious hyper-immune serum, preferably using vibrio parahaemolytious concentration is 1 * 10
8The bacteria suspension of cfu/mL, first immunisation carry out 5 times immunity once more after 20 days, each immunity is spaced apart 15 days in the immunity once more.
The present invention also provides the hyper-immune serum of the anti-vibrio parahaemolytious that obtains according to above-mentioned preparation method.
The hyper-immune serum of the anti-vibrio parahaemolytious of prepared according to the methods of the invention can be used to detect the vibrio parahaemolytious in the prawn body.
The competitive ELISA detection method that the present invention sets up at first is to carry out on the basis of the anti-vibrio parahaemolytious hyper-immune serum of preparation.The competitive ELISA detection method of vibrio parahaemolytious comprises in the prawn body provided by the present invention:
(1) dilutes antigen with coating buffer, coated elisa plate, oven dry;
(2) wash plate with cleansing solution;
(3) with the confining liquid sealing, wash plate;
(4) hyper-immune serum of the anti-vibrio parahaemolytious that makes according to above-mentioned preparation method that adds determined antigen and times dilution in 1: 6000～1: 11000 is in ELISA Plate, and plate is washed in competitive reaction;
(5) add ELIAS secondary antibody, wash plate;
(6) add colour developing liquid, the lucifuge colour developing;
(7) use the stop buffer color development stopping;
(8) measure OD with microplate reader
492Value.
In above-mentioned detection method, be 10 preferably with the coating buffer compound concentration
6The vibrio parahaemolytious bacteria suspension of cfu/mL, coated elisa plate.
In above-mentioned detection method, preferably 1: 10000 times of dilution of the hyper-immune serum of employed anti-vibrio parahaemolytious, serum and determined antigen mixed in equal amounts be in 37 ℃ of competitive reactions 1 hour.
In above-mentioned detection method, employed reagent is respectively:
The carbonate buffer solution of coating buffer: pH9～10;
Confining liquid: the phosphate buffer that contains pH7～8 of 0.5% skimmed milk;
Cleansing solution: the phosphate buffer that contains pH7～8 of 0.05%Tween-20;
Colour developing liquid: Na
2HPO
4The mixed liquor of solution, citric acid solution and o-phenylenediamine faces the time spent and adds H
2O
2
Stop buffer: H
2SO
4
In above-mentioned detection method, employed positive control is the vibrio parahaemolytious of pure culture, and employed negative control is a sterile saline, is conversed the vibrio parahaemolytious content of testing sample by the typical curve that obtains according to positive control.
The competitive ELISA detection method of vibrio parahaemolytious can be used for the vibrio parahaemolytious in the mass detection prawn body in the prawn body of the present invention.
Product advantage applies of the present invention exists:
Hyper-immune serum high specificity, the reaction that the detects vibrio parahaemolytious height of tiring; Detection time is short, can detect the result in general 10 hours; Easy to operation, can be standardized as product; Detect with low costly, be suitable for promoting; Can carry out mass detection, be used for epidemiology survey.
Description of drawings
Fig. 1 is sample OD
492The typical curve of value is an ordinate with the absorbance, is horizontal ordinate with the logarithm value of standard specimen concentration.
Embodiment
The competitive ELISA detection method of vibrio parahaemolytious in the prawn body that the present invention sets up at first is to carry out on the basis of the hyper-immune serum for preparing anti-vibrio parahaemolytious.The competitive ELISA detection method of vibrio parahaemolytious comprises in the prawn body that the present invention preferably adopts:
(1) be 10 with the coating buffer compound concentration
6The vibrio parahaemolytious bacteria suspension of cfu/mL, coated elisa plate, 100 μ L/ holes, 60 ℃ of oven dry;
(2) wash plate three times with cleansing solution, each concussion washing 1 minute;
(3) with confining liquid capping plate, 200 μ L/ holes, 37 ℃ were sealed 1 hour, and washed plate;
(4) get the musculature of prawn with sterile manner, adding equal-volume physiological saline grinds, add 50 μ L lapping liquids to wrapping by good every hole, add 50 μ L in addition and be 1: 10000 times hyper-immune serum with the anti-vibrio parahaemolytious of the inventive method preparation with confining liquid dilution, set positive control and negative control simultaneously, plate is washed in 37 ℃ of competitive reactions 1 hour;
(5) add ELIAS secondary antibody, 100 μ L/ holes, 37 ℃ of senses are done 1 hour, wash plate;
(6) add colour developing liquid, 100 μ L/ holes, 37 ℃ of lucifuges developed the color 10 minutes;
(7) with the stop buffer color development stopping in 50 μ L/ holes;
(8) measure OD with microplate reader
492Value is used positive control OD
492Value drawing standard curve; Extension rate and the OD that surveys per sample
492Be worth, converse the vibrio parahaemolytious content of testing sample by typical curve.
Employed reagent preferably is respectively in the competitive ELISA detection method of vibrio parahaemolytious in prawn body of the present invention:
The carbonate buffer solution of coating buffer: pH9.6,0.05M;
Confining liquid: contain the pH7.4 of 0.5% skimmed milk, the phosphate buffer of 0.01M;
Cleansing solution: contain the pH7.4 of 0.05%Tween-20, the phosphate buffer of 0.01M;
Colour developing liquid: with the Na of 10.3mL, 0.2M
2HPO
4The 0.1M citric acid solution of solution and 9.7mL mixes, and adds the o-phenylenediamine of 10mg, faces 30% the H that the time spent adds 15 μ L
2O
2
The H of stop buffer: 2M
2SO
4
In the competitive ELISA detection method of vibrio parahaemolytious, employed positive control is the vibrio parahaemolytious through the nutrient agar pure culture in above-mentioned prawn body, and negative control is a sterile saline.
As the vibrio parahaemolytious of the pure culture of positive control, concentration is set at respectively: 10
1Cfu/mL, 10
2Cfu/mL, 10
3Cfu/mL, 10
4Cfu/mL, 10
5Cfu/mL, 10
6Cfu/mL is according to positive control OD
492Value is an ordinate with the absorbance, is horizontal ordinate with the logarithm value of standard specimen concentration, does typical curve, referring to Fig. 1.
Mode by the following examples specify the anti-vibrio parahaemolytious of the present invention hyper-immune serum the preparation method and use the hyper-immune serum competitive ELISA of prepared anti-vibrio parahaemolytious to detect the method for vibrio parahaemolytious in the prawn body.
Preparation embodiment (preparation of the hyper-immune serum of anti-vibrio parahaemolytious)
With concentration is 1 * 10
8The vibrio parahaemolytious bacteria suspension immunity body weight of cfu/mL is the male rabbit about 2 kilograms, per injection 2mL bacteria suspension.During first immunisation, bacteria suspension adds isopyknic Freund's complete adjuvant, adopts the mode of 4 injections of sole, and first immunisation is carried out 5 times immunity once more after 20 days, immunity is spaced apart 15 days, in Mian Yi each immunity, bacteria suspension all adds isopyknic incomplete Freund once more, adopts the mode immunity of subcutaneous 6 injections in back, blood sampling after 7 days of last immunity, separation of serum obtains the hyper-immune serum of anti-vibrio parahaemolytious, and is standby.
Application Example (competitive ELISA of vibrio parahaemolytious detects in the prawn body)
Get 5 tail prawns (sample 1～5), aseptic musculature of getting 1 gram prawn adds 1mL physiological saline and grinds in each sample, and is stand-by.Is 10 with coating buffer (carbonate buffer solution of pH9.6,0.05M) with the dilution of standard vibrio parahaemolytious bacteria suspension
6Cfu/mL, coated elisa plate, 100 μ L/ holes, 60 ℃ of oven dry; Wash plate three times with the phosphate buffer of the pH7.4 that contains 0.05%Tween-20,0.01M, 300 μ L/ holes, each concussion washing 1 minute dries plate; With the phosphate buffer capping plate of the pH7.4 that contains 0.5% skimmed milk, 0.01M, 200 μ L/ holes, 37 ℃ were sealed 1 hour, and washed plate three times; Aseptic musculature of getting prawn, adding equal-volume physiological saline grinds, add 50 μ L lapping liquids to wrapping by good every hole, add 50 μ L in addition and be the hyper-immune serum of anti-vibrio parahaemolytious of 1: 10000 times above-mentioned preparation embodiment preparation with the phosphate buffer dilution of the pH7.4, the 0.01M that contain 0.5% skimmed milk, established standards positive contrast of vibrio parahaemolytious bacteria suspension simultaneously and the negative contrast of physiological saline, 37 ℃ of competitive reactions 1 hour are washed plate three times; Add ELIAS secondary antibody, 100 μ L/ holes, 37 ℃ of senses are done 1 hour, wash plate three times; Add o-phenylenediamine-hydrogen peroxide colour developing liquid, 100 μ L/ holes, 37 ℃ of lucifuges developed the color 10 minutes; 2M H with 50 μ L/ holes
2SO
4The stop buffer color development stopping is measured OD with microplate reader
492Value, extension rate, and OD per sample
492Value (seeing Table 1) is conversed the vibrio parahaemolytious content of testing sample by typical curve.
Table 1 sample OD
492 Value
| Sample | Sample |
| 1 | | | | | |
| OD 492Value | 0.197 | 0.240 | 0.882 | 1.043 | 1.247 |
492nm absorbance per sample can be read the concentration of respective sample from curve, and calculating separately according to extension rate again, concentration is respectively:
Sample 1: have 6.32 * 10 in every gram musculature
5Individual vibrio parahaemolytious;
Sample 2: have 3.99 * 10 in every gram musculature
5Individual vibrio parahaemolytious;
Sample 3: have 2 * 10 in every gram musculature
2Individual vibrio parahaemolytious;
Sample 4: 16 vibrio parahaemolytious are arranged in every gram musculature;
Sample 5: every gram musculature is interior less than 10 vibrio parahaemolytious.
Whole testing process was finished in 10 hours.
Claims (8)
1. the preparation method of the hyper-immune serum of an anti-vibrio parahaemolytious, this method comprises:
With concentration is 1 * 10
7～1 * 10
9The vibrio parahaemolytious bacteria suspension immunity body weight of cfu/mL is 1.5～2.5 kilograms a male rabbit, per injection 1～3mL bacteria suspension, during first immunisation, in bacteria suspension, add isopyknic Freund's complete adjuvant, adopt the injection system of sole 4～6, first immunisation is carried out 3～5 times immunity once more after 15～20 days, immunity is spaced apart 10～15 days, each immunity in the immunity all adds isopyknic incomplete Freund in bacteria suspension once more, adopt the subcutaneous injection system in back at 4～6, last immunity blood sampling after 7～10 days, separation of serum obtains the hyper-immune serum of anti-vibrio parahaemolytious.
2. the preparation method of the hyper-immune serum of anti-vibrio parahaemolytious according to claim 1, wherein to use vibrio parahaemolytious concentration be 1 * 10 to immunizing antigen
8The bacteria suspension of cfu/mL, first immunisation carry out 5 times immunity once more after 20 days, each immunity is spaced apart 15 days in the immunity once more.
3. the hyper-immune serum of the anti-vibrio parahaemolytious that preparation method according to claim 1 and 2 obtains.
4. the detection method of prawn kwashiorkor pathogen vibrio parahaemolytious, this method comprises:
(1) dilutes antigen with coating buffer, coated elisa plate, oven dry;
(2) wash plate with cleansing solution;
(3) with the confining liquid sealing, wash plate;
(4) hyper-immune serum of the described anti-vibrio parahaemolytious of claim 3 of adding determined antigen and times dilution in 1: 6000～1: 11000 is in ELISA Plate, and plate is washed in competitive reaction;
(5) add ELIAS secondary antibody, wash plate;
(6) add colour developing liquid, the lucifuge colour developing;
(7) use the stop buffer color development stopping;
(8) measure OD with microplate reader
492Value.
5. the detection method of prawn kwashiorkor pathogen vibrio parahaemolytious according to claim 4 is 10 with the coating buffer compound concentration wherein
6The vibrio parahaemolytious bacteria suspension of cfu/mL, coated elisa plate.
6. the detection method of prawn kwashiorkor pathogen vibrio parahaemolytious according to claim 4, wherein the hyper-immune serum of employed anti-vibrio parahaemolytious is 1: 10000 times of dilution, serum and determined antigen mixed in equal amounts were in 37 ℃ of competitive reactions 1 hour.
7. the detection method of prawn kwashiorkor pathogen vibrio parahaemolytious according to claim 4, wherein employed reagent is respectively:
The carbonate buffer solution of coating buffer: pH9～10;
Confining liquid: the phosphate buffer that contains pH7～8 of 0.5% skimmed milk;
Cleansing solution: the phosphate buffer that contains pH7～8 of 0.05%Tween-20;
Colour developing liquid: Na
2HPO
4The mixed liquor of solution, citric acid solution and o-phenylenediamine faces the time spent and adds H
2O
2
Stop buffer: H
2SO
4
8. the detection method of prawn kwashiorkor pathogen vibrio parahaemolytious according to claim 4, wherein employed positive control is the vibrio parahaemolytious of pure culture, employed negative control is a sterile saline, is conversed the vibrio parahaemolytious content of testing sample by the typical curve that obtains according to positive control.
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|---|---|---|---|
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|---|---|---|---|
| CN 200410091369 CN1779462A (en) | 2004-11-24 | 2004-11-24 | Hyperimmune serum against Vibrio parahaemolyticus, its preparation and inspecotion for astacin pathogenic bacterium |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102836428A (en) * | 2012-09-12 | 2012-12-26 | 淮海工学院 | Inactivated vaccine preparation method for egg yolk antibody for resisting litopenaeus vannamei red body disease |
| CN107807235A (en) * | 2017-11-01 | 2018-03-16 | 郑州欧柯奇仪器制造有限公司 | Clenbuterol ELISA kit, its preparation method and its application |
| CN108003240A (en) * | 2017-12-04 | 2018-05-08 | 西安德轩驰生物科技有限公司 | A kind of multi-joint antiidiotype Yolk antibody vaccine of mariculture fish and preparation method thereof |
| CN110129237A (en) * | 2019-05-30 | 2019-08-16 | 浙江大学 | A kind of novel K serotype vibrio parahaemolytious and its application |
-
2004
- 2004-11-24 CN CN 200410091369 patent/CN1779462A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102836428A (en) * | 2012-09-12 | 2012-12-26 | 淮海工学院 | Inactivated vaccine preparation method for egg yolk antibody for resisting litopenaeus vannamei red body disease |
| CN102836428B (en) * | 2012-09-12 | 2014-01-08 | 淮海工学院 | Inactivated vaccine preparation method for egg yolk antibody for resisting litopenaeus vannamei red body disease |
| CN107807235A (en) * | 2017-11-01 | 2018-03-16 | 郑州欧柯奇仪器制造有限公司 | Clenbuterol ELISA kit, its preparation method and its application |
| CN108003240A (en) * | 2017-12-04 | 2018-05-08 | 西安德轩驰生物科技有限公司 | A kind of multi-joint antiidiotype Yolk antibody vaccine of mariculture fish and preparation method thereof |
| CN108003240B (en) * | 2017-12-04 | 2020-06-05 | 西安德轩驰生物科技有限公司 | Multi-connected anti-idiotype egg yolk antibody vaccine for marine cultured fish and preparation method thereof |
| CN110129237A (en) * | 2019-05-30 | 2019-08-16 | 浙江大学 | A kind of novel K serotype vibrio parahaemolytious and its application |
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