CN106442997A - Double-antibody sandwich enzyme-linked immunosorbent assay kit for detection of Streptococcus agalactiae - Google Patents

Double-antibody sandwich enzyme-linked immunosorbent assay kit for detection of Streptococcus agalactiae Download PDF

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CN106442997A
CN106442997A CN201510921656.1A CN201510921656A CN106442997A CN 106442997 A CN106442997 A CN 106442997A CN 201510921656 A CN201510921656 A CN 201510921656A CN 106442997 A CN106442997 A CN 106442997A
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monoclonal antibody
solution
hours
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porous plate
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陈汉忠
梁万文
李旻
王凯
陈明
吴文德
王瑞
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Guangxi University
Guangxi Academy of Fishery Sciences
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Guangxi Academy of Fishery Sciences
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56944—Streptococcus

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Abstract

The invention discloses a double-antibody sandwich enzyme-linked immunosorbent assay (double-antibody sandwich ELISA) kit for detection of Streptococcus agalactiae. The kit is composed of a perforated plate coated with 3A9 monoclonal antibody, a horse radish peroxidase labeled monoclonal antibody 4C12 and a chromogenic substrate 3'.3'.5,5'-tetramethyl benzidine (TMB). The preparation method of the kit includes the steps of: (1) preparing the perforated plate coated with 3A9 monoclonal antibody; (2) preparing the horse radish peroxidase labeled monoclonal antibody 4C12; and (3) preparing the chromogenic substrate 3'.3'.5,5'-tetramethyl benzidine.

Description

The DAS ELISAA test kit of detection streptococcus agalactiae
Technical field
The invention belongs to streptococcicosises immunologic diagnosises, detection technique field, being related to a kind of streptococcus is streptococcus agalactiae DAS ELISAA and its preparation method of test kit.
Technical background
Streptococcus agalactiae（Streptococcus agalactiae）It is a kind of gram positive bacteria with high virulence, energy Infect multiple fresh water and seawater fish, bring hundreds of millions of losses to World Aquaculture every year, in recent years in China south Square tilapia culture zone wreak havoc popular, due to produce on lack effective diagnosis detecting method, cause tilapia streptococcus agalactiae Disease does not obtain effective monitoring and preventing and treating, causes to Rofe fish culture and causes heavy losses, seriously hinders China tilapia The development of aquaculture.At present streptococcus agalactiae disease diagnosis detecting method is mainly had：（1）The form culture identification of routine, physiology The traditional methods such as biochemical identification, these usual degree of accuracy of traditional diagnosis detecting method are relatively low, take time and effort（2）Molecular biology Detection method, mainly has some molecular biology methods such as PCR, LAMP, although these molecular biology method sensitivity are high, But operating technology difficulty is high, basic unit uses certain difficulty.（3）Immunological detection method, immunologic context of detection is a lot, Have inspection antibody, also have inspection antigen, inspection antibody as indirect enzyme-linked immunosorbent assay, it is anti-that this method can only examine circulation Body, but cannot distinguish between Current Infection and previous infection, therefore epidemic disease can not be made with accurate diagnosis.The present invention utilizes us to make The special 3A9 and 4C12 monoclonal antibody of the standby anti-source of fish streptococcus agalactiae with Screening and Identification sets up a kind of new tilapia chain Coccus disease immunologic diagnosises detection method, that is,：The detection method of double antibody sandwich ELISA, the present invention is due to make use of for sieve The special monoclonal antibody of non-fish streptococcus agalactiae, direct detection cause of disease not only can carry out existing disease diagnosis, and due to list Clonal antibody can substantial amounts of be prepared, quality is easily controlled, and therefore can further improve stability, sensitivity and the spy of detection method The opposite sex.
The present invention is clinically to diagnose and detect that Rofe source of fish streptococcus agalactiae disease provides a kind of new method, can be used for The detection of tilapia streptococcus agalactiae, diagnosis and Evaluation of drug treatment etc..
Content of the invention
It is an object of the invention to overcoming the deficiencies in the prior art, provide a kind of the dual anti-of detection streptococcus agalactiae for clinical Body Sandwich ELISA test kit and preparation method thereof, can provide one kind to be used for tilapia agalactia chain for clinical practice The new method of the detection of coccus, diagnosis and Evaluation of drug treatment.
The DAS ELISAA test kit of detection streptococcus agalactiae of the present invention, is coated with The porous plate of 3A9 monoclonal antibody, monoclonal antibody 4C12 of horseradish peroxidase-labeled and chromogenic substrate 3'.3'.5,5'- Tetramethyl benzidine（TMB）.
The present invention is to reach above-mentioned target, have selected patent applicant's preparation, the 3A9 monoclonal antibody of screening as being coated Antibody, described monoclonal antibody by preserving number is:CCTCC NO:The hybridoma of C2014244 produces, and C2014244 hybridizes Oncocyte is saved in Chinese Typical Representative culture and preserves center.
In described test kit, it is coated with the porous plate of 3A9 monoclonal antibody, the monoclonal anti of horseradish peroxidase-labeled Body 4C12 and chromogenic substrate 3'.3'.5,5'- tetramethyl benzidine is each individually deposited respectively.
Further：It is 2.0-200.0 μ g/mL that the every hole of described porous plate is coated 3A9 MAb concentration.
Further：Described test kit also includes chromogenic substrate 3'.3'.5,5'- tetramethyl benzidine.
Further：The consumption that the every hole of described porous plate is coated 3A9 monoclonal antibody is 100 μ L, and concentration is 10.0 μ g/ mL.
Further：The monoclonal antibody of described horseradish peroxidase-labeled is 4C12,
The preparation method of described horseradish peroxidase-labeled monoclonal antibody 4C12 is：
1）Prepare mouse ascites monoclonal antibody 4C12 according to a conventional method, purify mouse ascites with caprylic acid one saturated ammonium sulfate method Monoclonal antibody 4C12, with BCA determination of protein concentration test kit detect monoclonal antibody 4C12 concentration as 4.34mg/mL, Monoclonal antibody 4C12 is positioned over and saves backup under -20 DEG C of environment；
2) weigh 5mg horseradish peroxidase to be dissolved in 1ml distilled water；
3) by step 2）The solution obtaining adds the 0.1M NaIO that 0.2ml newly joins4Solution, under ice bath, lucifuge stirs 15 minutes；
4) by step 3）The solution obtaining loads in bag filter, with the sodium-acetate buffer dialysis of 1mM pH4.4, changes liquid within 4 hours Once, period changes liquid 3-5 time；
5) in step 4）Add 20 μ l 0.2M pH9.5 carbonate buffer solutions in the solution obtaining, make the pH of above hydroformylation HRP It is increased to 9.0～9.5, adds 10mg IgG immediately after, ice bath lucifuge stirs 2 hours；
6) by step 5）It is 4mg/ml NaBH that the solution obtaining adds 0.1ml concentration4Liquid, mixes, then places under 4 DEG C of environment 2 hours；
7) by step 6）The solution obtaining loads in bag filter, with 0.15M pH7.4 PBS, changes liquid once within 4 hours, period Change liquid 3-5 time；
8) by step 7）The solution obtaining dropwise is slowly added to equal-volume saturated ammonium sulfate under ice bath stirring, is placed in 4 DEG C of environment Lower 2 hours；
9) by step 8）The solution obtaining is put in centrifuge tube, is centrifuged 30min, supernatant discarded with centrifuge 3000rpm, precipitation is used Semi-saturation ammonium sulfate is washed 1-2 time, and last precipitate is dissolved in the PBS of 0.15M pH7.4；
10) by step 9）The solution that obtains loads in bag filter, with the PBS buffer saline dialysis of 0.15M pH7.4, dispel ammonium from After son (with the detection of Nai Shi reagent), 12000rpm is centrifuged 30 minutes and removes precipitation, and supernatant is enzyme conjugates, i.e. enzyme mark 4C12, stored frozen after enzyme mark 4C12 subpackage, detects with indirect ELISA method, the potency of enzyme mark 4C12 reaches 1：6400, the most suitable dilute Releasing working concentration is 1：1600.
Further：Present invention also offers the DAS ELISAA reagent of detection streptococcus agalactiae Box preparation method, described step is as follows：
A. prepare the porous plate being coated with 3A9 monoclonal antibody
That is made with carbonate buffer solution is coated the diluent that 3A9 monoclonal antibody is diluted to concentration 10.0 μ g/mL by liquid, described It is coated liquid by Na2CO31.59 g、NaHCO32.93 g, plus distilled water is settled to 1000 mL and makes, it is protected under the conditions of 4 DEG C Deposit, then described 3A9 monoclonal antibody diluent is added each in the hole of porous plate, be placed in that to be coated 12-24 under 4 DEG C of environment little When, after the time of being coated expires, the defatted milk powder solution of mass concentration 5% is added each in the hole of porous plate, and sealed at 37 DEG C Close reaction, capping time at least 1-1.5 hour, after capping terminates, wash porous plate with lavation buffer solution, work as porous Unreacted reactant on plate obtains, after being removed, the porous plate being coated with 3A9 monoclonal antibody, and its storage temperature is 4 DEG C；
B. prepare monoclonal antibody 4C12 of horseradish peroxidase (HRP) labelling
According to a conventional method：Prepare mouse ascites monoclonal antibody 4C12 with hybridoma, with caprylic acid one saturated ammonium sulfate method Purification, with BCA determination of protein concentration test kit detect monoclonal antibody 4C12 concentration as 4.34mg/mL, monoclonal antibody Save backup to -20 DEG C；
C. it is equipped with chromogenic substrate 3'.3'.5,5'- tetramethyl benzidine；
D. prepare the DAS ELISAA test kit of detection streptococcus agalactiae,
Described horseradish peroxidase HRP labelling 4C12 step is as follows：
Step 1）Weigh 5mg horseradish peroxidase to be dissolved in 1ml distilled water；
Step 2）In step 1）The 0.1M NaIO that 0.2ml newly joins is added in the solution obtaining4Solution, lucifuge stirring 15 under ice bath Minute；
Step 3）In step 2）The solution obtaining loads in bag filter, is dialysed with the sodium-acetate buffer of 1mM pH4.4,4 hours Change liquid once, period changes liquid 3-5 time；
Step 4）In step 3）Add 20 μ l 0.2M pH9.5 carbonate buffer solutions in the solution obtaining, make above hydroformylation Radix Cochleariae officinalises mistake The pH of oxide enzyme is increased to 9.0～9.5, adds 10mg IgG (slightly to carry through caprylic acid one saturated ammonium sulfate immediately after The 4C12 and 4C12 through Protein G affinitive layer purification), ice bath lucifuge stirs 2 hours；
Step 5）By step 4）The solution obtaining adds the 4mg/ml NaBH that 0.1ml newly joins4Liquid, mixes, then puts 4 DEG C 2 hours；
Step 6）By step 5）The solution obtaining loads in bag filter, with 0.15M pH7.4 PBS, changes liquid once within 4 hours, Period changes liquid 3-5 time；
Step 7）By step 6）The solution obtaining dropwise is slowly added to equal-volume saturated ammonium sulfate under ice bath stirring, puts 4 DEG C 2 Hour；
Step 8）By step 7）The solution obtaining is placed in centrifuge tube, is centrifuged 30min with centrifuge, abandons under the conditions of 3000rpm Remove supernatant, precipitation semi-saturation ammonium sulfate is washed 1-2 time, and last precipitate is dissolved in the PBS of a small amount of 0.15M pH7.4；Step 9） The solution that step 8 is obtained loads in bag filter, with the PBS buffer saline dialysis of 0.15M pH7.4, (uses after removing ammonium ion Nai Shi reagent detects), 12000rpm is centrifuged 30 minutes and removes precipitation, and supernatant is enzyme conjugates, i.e. enzyme mark 4C12, uses When, make 1 with phosphate buffer：1600 times of dilutions.
Further：When described preparation is coated with the porous plate of 3A9 monoclonal antibody, described 3A9 monoclonal antibody dilution The addition of liquid is 100 L/ holes, the addition of the monoclonal antibody 4C12 diluent of described horseradish peroxidase (HRP) labelling Measure as 100 L/ holes, the addition of the defatted milk powder solution of described mass concentration 5% is 100 L/ holes.
Further：Described lavation buffer solution consists of the following composition：NaCl 8.0 g, KH2PO40.2 g, KCl 0.2 G, Na2HPO412H2O 3.58 g, Tween-20 0.5 mL, plus distilled water is settled to 1000 mL and makes lavation buffer solution.
Monoclonal antibody 4C12 of described horseradish peroxidase-labeled, preparation method is as follows：According to a conventional method, with hybridization Oncocyte prepares mouse ascites monoclonal antibody 4C12, and this monoclonal antibody by preserving number is：CCTCC NO:C2014248's is miscellaneous Oncocyte is handed over to produce, C2014248 hybridoma is saved in Chinese Typical Representative culture and preserves center.
The culture title of the preservation of described 4C12：A kind of hybridoma of the monoclonal antibody producing anti-tilapia streptococcus agalactiae Cell strain 4C12, depositary institution：China typical culture collection center；Preservation address：Wuhan, China city, Wuhan University；Preservation Date：On January 7th, 2015；Deposit number：CCTCC NO：C2014248.
With the purification of caprylic acid one saturated ammonium sulfate method, monoclonal antibody is detected to obtain with BCA determination of protein concentration test kit The concentration of 4C12 is 4.34mg/mL, and monoclonal antibody saves backup to -20 DEG C.
HRP labelling 4C12 step is as follows：
1. weigh 5mg horseradish peroxidase（HRP）It is dissolved in 1ml distilled water.
2. add the 0.1M NaIO that 0.2ml newly joins in above-mentioned solution4Solution, under ice bath, lucifuge stirs 15 minutes.
3. above-mentioned solution is loaded in bag filter, with the sodium-acetate buffer dialysis of 1mM pH4.4, changes liquid once within 4 hours, Period changes liquid 3-5 time；
4. above-mentioned solution is added 20 μ l 0.2M pH9.5 carbonate buffer solutions, make the pH of above hydroformylation HRP be increased to 9.0～ 9.5, add the 10mg IgG (4C12 that slightly carries through caprylic acid one saturated ammonium sulfate and through Protein G affinity chromatograph immediately after The 4C12 of purification), ice bath lucifuge stirs 2 hours.
5. above-mentioned solution is added the 4mg/ml NaBH that 0.1ml newly joins4Liquid, mixes, then puts 4 DEG C 2 hours.
6. above-mentioned solution is loaded in bag filter, with 0.15M pH7.4 PBS, change liquid once within 4 hours, period changes Liquid 3-5 time..
7. above-mentioned solution is dropwise slowly added to equal-volume saturated ammonium sulfate under ice bath stirring, puts 4 DEG C 2 hours.
8. above-mentioned solution is put in centrifuge tube, be centrifuged 30min, supernatant discarded with centrifuge 3000rpm, precipitation is with half-full Wash 1-2 time with ammonium sulfate, last precipitate is dissolved in the PBS of a small amount of 0.15M pH7.4.
9. above-mentioned solution is loaded in bag filter, dialysed with the PBS buffer saline of 0.15M pH7.4, after removing ammonium ion (with the detection of Nai Shi reagent), 12000rpm is centrifuged 30 minutes and removes precipitation, and supernatant is enzyme conjugates, and that is, enzyme mark 4C12, makes Used time, make 1 with phosphate buffer：1600 times of dilutions.
The preparation of the monoclonal antibody 4C12 related reagent of above-mentioned horseradish peroxidase-labeled：
①0.1M NaIO4：Weigh 241mg sodium metaperiodate to be dissolved in distilled water 10ml.
2. 1mM pH4.4 sodium-acetate buffer:
0.2M NaAc weighs anhydrous sodium acetate 16.4g, plus distilled water 200ml dissolving, constant volume to 1000ml.
0.2M HAc measures 11.55ml glacial acetic acid constant volume to 1000ml.
1mM pH4.4 sodium-acetate buffer：0.2M NaAc 195ml adds 0.2M HAc 305ml and fully mixes.
3. 0.2M pH9.5 carbonate buffer solution:
Na2CO30.32g
NaHCO30.586g
Plus distilled water is to 50ml
Using working solution again with distilled water make 20 times dilution, 0.01M pH9.5 carbonate buffer solution.
4. dilution buffer:0.15M pH7.4 PBS：
The NaH of A .0.2mol/L2PO4：Weigh NaH2PO4·2H2O 31.2g adds distilled water to 1000ml solution；
The Na of B .0.2mol/L2HPO4：Weigh Na2HPO4·12H2O 71.632g adds distilled water to 1000ml solution；
0.15M pH7.4 PBS adds 457.5ml B liquid by the A liquid of 292.5ml and is sufficiently mixed, and adjusts pH to 7.4, and constant volume arrives 1000ml.
⑤NaBH4Solution (4mg/ml):
Facing the used time weighs NaBH44mg is dissolved in 1ml distilled water.
In test kit of the present invention and preparation method, 3A9 monoclonal antibody is essentially protein, can specific recognition agalactia chain Coccus.
The culture of the preservation of described 3A9 is entitled：A kind of hybridization of the monoclonal antibody producing anti-tilapia streptococcus agalactiae Tumor cell strain 3A9, depositary institution：China typical culture collection center；Preservation address：Wuhan, China city, Wuhan University；Preservation Date：On January 7th, 2015；Deposit number：CCTCC NO：C2014244.
The DAS ELISAA test kit of detection streptococcus agalactiae of the present invention, detects that specimen is Infection animal（As tilapia）Serum and brain, liver,spleen,kidney tissue leachate, required instrument be microplate reader, using method is such as Under：
Measuring samples, after 37 DEG C are reacted 1 hour, cleaning mixture are added in being coated with the hole of porous plate of 3A9 monoclonal antibody Washing 3 times removing unreacted reactants, add HRP labelling monoclonal antibody 4C12,37 DEG C effect 1 hour after washing remove not anti- Answer thing, add freshly prepared 3'.3'.5,5'- tetramethyl benzidine develops the color, the color development at room temperature time is to add after 20 minutes to terminate Liquid 2 mol/L H2SO4 50 μ L/ hole terminating reaction, surveys the OD in each hole of described porous plate with microplate reader450Value.
The culture of the preservation of described 3A9 is entitled：A kind of hybridization of the monoclonal antibody producing anti-tilapia streptococcus agalactiae Tumor cell strain 3A9, depositary institution：China typical culture collection center；Preservation address：Wuhan, China city, Wuhan University；Preservation Date：On January 7th, 2015；Deposit number：CCTCC NO：C2014244.
The culture title of the preservation of described 4C12：A kind of hybridoma of the monoclonal antibody producing anti-tilapia streptococcus agalactiae Cell strain 4C12, depositary institution：China typical culture collection center；Preservation address：Wuhan, China city, Wuhan University；Preservation Date：On January 7th, 2015；Deposit number：CCTCC NO：C2014248.
The invention has the advantages that：
1. monoclonal antibody 3A9 of the identification streptococcus agalactiae that the present invention uses is us from multiple monoclonal antibodies of preparation Screening and Identification out, therefore makes test kit detection streptococcus agalactiae specific effect of the present invention more preferable.
2. carry out streptococcus agalactiae detection using test kit of the present invention, operational approach is simple, the detection of a sample Only need about 5 hours.Compared with the traditional methods such as traditional form culture identification, Physiology and biochemistry identification, using institute of the present invention State test kit and can carry out early diagnosiss, and there is higher detection sensitivity.
3., compared with conventional indirect ELISA, using test kit of the present invention, Current Infection can be distinguished and previously feel Dye, can be made whether to infect the direct judgement of streptococcus agalactiae, it may also be used for the evaluation of clinical streptococcus agalactiae disease therapeutic effect.
Specific embodiment
Below by embodiment to detection streptococcus agalactiae of the present invention（Streptococcus agalactiae）Double Sandwich ELISA（double antibody sandwich ELISA）Test kit and preparation method and make It is described further with method.
In following embodiments, the material source being related to and preparation method are as follows：
3A9 monoclonal antibody and 4C12 monoclonal antibody use 3A9 hybridoma cell strain respectively（Deposit number：CCTCC NO: C2014244）With 4C12 hybridoma cell strain（Deposit number：CCTCC NO:C2014248）Prepare according to a conventional method;
Horseradish peroxidase（HRP）, chromogenic substrate 3'.3'.5,5'- tetramethyl benzidine, bovine serum albumin are Sigma Products；
IgG is the 4C12 slightly carrying through caprylic acid one saturated ammonium sulfate
Remaining conventional reagent is domestic pure analysis pure level product；
Various test solutions are prepared：
It is coated buffer：Na2CO31.59 g, NaHCO32.93 g, plus distilled water is settled to 1 000 mL, 4 DEG C of guarantors Deposit；
Confining liquid is prepared：Defatted milk powder 5.0 g, plus be coated liquid and be settled to 100 mL.
Dilution buffer is prepared：Weigh KH2PO40.2 g, KCl 0.2 g, Na2HPO4·12H2O 3.58 g, NaCl 8.0 g, plus tri-distilled water is settled to 1000 mL, latter 4 DEG C of sterilizing saves backup.
Lavation buffer solution is prepared：NaCl 8.0 g, KH2PO40.2 g, KCl 0.2 g, Na2HPO4·12H2O 3.58 G, Tween-20 0.5 mL, plus distilled water is settled to 1000 mL.
Substrate buffer solution is prepared：Na2HPO4·12H2O 3.68 g, citric acid 0.93 g, plus tri-distilled water is settled to 100 mL.
3'.3'.5,5'- tetramethyl benzidine（TMB）Mother liquor：Weigh TMB（Tetramethyl benzidine）200mg, adds 100 mL anhydrous alcohol solutions.
Nitrite ion is prepared：Take TMB mother solution 500 μ L, substrate buffer solution 10mL, 0.75% H2O2 32 μ L mixes.
Terminate liquid is prepared：Measure the dense H2SO4 of 2M 22.2 mL, distilled water 177.8 mL mixes（Operation is careful）.
Porous plate is the enzyme mark detection porous plate in commercially available 96 holes.
Embodiment 1
In the present embodiment, detection streptococcus agalactiae DAS ELISAA examination is prepared using following processing step Agent box.
（1）Preparation is coated with the porous plate of 3A9 monoclonal antibody
It is coated the diluent that 3A9 monoclonal antibody is diluted to concentration 10.0 μ g/mL by liquid with carbonate buffer solution, then by institute State each in the hole that 3A9 monoclonal antibody diluent adds porous plate, be placed in 4 DEG C and be coated at least 12 hours, after the time of being coated expires, The defatted milk powder solution of mass concentration 5% is added each in the hole of porous plate, and carry out capping at 37 DEG C, during capping Between at least more than 1 hour, after capping terminates, wash porous plate with lavation buffer solution, when the unreacted reactant quilt on porous plate The porous plate being coated with 3A9 monoclonal antibody is obtained, its storage temperature is 4 DEG C after removal.
（2）Prepare monoclonal antibody 4C12 of horseradish peroxidase (HRP) labelling
According to a conventional method：Prepare mouse ascites monoclonal antibody 4C12 with hybridoma, with caprylic acid one saturated ammonium sulfate method Purification, with BCA determination of protein concentration test kit detect monoclonal antibody 4C12 concentration as 4.34mg/mL, monoclonal antibody Save backup to -20 DEG C.
HRP labelling 4C12 step is as follows：
1. weigh 5mg horseradish peroxidase to be dissolved in 1ml distilled water.
2. add the 0.1M NaIO that 0.2ml newly joins in upper liquid4Solution, under ice bath, lucifuge stirs 15 minutes.
3. above-mentioned solution is loaded in bag filter, with the sodium-acetate buffer dialysis of 1mM pH4.4, changes liquid once within 4 hours, Period changes liquid 3 times.
4. plus 20 μ l 0.2M pH9.5 carbonate buffer solutions, the pH of above hydroformylation HRP is made to be increased to 9.0～9.5, then Add the 10mg IgG (4C12 that slightly carries through caprylic acid one saturated ammonium sulfate and through Protein G affinitive layer purification immediately 4C12), ice bath lucifuge stirs 2 hours.
Plus the 4mg/ml NaBH that newly joins of 0.1ml 5.4Liquid, mixes, then puts 4 DEG C 2 hours.
6. above-mentioned liquid is loaded in bag filter, with 0.15M pH7.4 PBS, change liquid once within 4 hours, period changes liquid 3 Secondary.
7. dropwise it is slowly added to equal-volume saturated ammonium sulfate under ice bath stirring, put 4 DEG C 2 hours.
8. 3000rpm centrifugation 30min, supernatant discarded.Precipitation semi-saturation ammonium sulfate is washed 1-2 time, and last precipitate is dissolved in In the PBS of a small amount of 0.15M pH7.4.
9. above-mentioned solution is loaded in bag filter, dialysed with the PBS buffer saline of 0.15M pH7.4, after removing ammonium ion (with the detection of Nai Shi reagent), 12000rpm is centrifuged 30 minutes and removes precipitation, and supernatant is enzyme conjugates, and that is, enzyme mark 4C12, makes Used time, use dilution buffer（Ibid）Make 1：1600 times of dilutions.
（3）Prepare 3'.3'.5,5'- tetramethyl benzidine substrates nitrite ion 20000 μ L with substrate buffer solution.
Described chromogenic substrate 3'.3'.5,5'- tetramethyl benzidine is commercial goods, can directly buy from market.
In said method, preparation be coated with 3A9 monoclonal antibody porous plate when, described 3A9 monoclonal antibody diluent plus Entering amount is every hole 100 μ L, and the addition of the monoclonal antibody 4C12 diluent of described horseradish peroxidase (HRP) labelling is 100 L/ holes, the addition of the defatted milk powder solution of described mass concentration 5% is 100 L/ holes.
In test kit of the present invention and preparation method, 3A9 monoclonal antibody is essentially protein, and energy specific recognition is no Streptococcus lactis (Lister) Lohnis 1909.554..
The DAS ELISAA test kit of detection streptococcus agalactiae of the present invention, detects that specimen is Infection animal（As tilapia）Serum and brain, liver,spleen,kidney tissue leachate, required instrument be microplate reader, using method is such as Under：
Measuring samples, after 37 DEG C are reacted 1 hour, cleaning mixture are added in being coated with the hole of porous plate of 3A9 monoclonal antibody Washing 3 times removing unreacted reactants, add HRP labelling monoclonal antibody 4C12,37 DEG C effect 1 hour after washing remove not anti- Answer thing, add freshly prepared 3'.3'.5,5'- tetramethyl benzidine develops the color, the color development at room temperature time is to add after 20 minutes to terminate Liquid 2 mol/L H2SO4 50 μ L/ hole terminating reaction, surveys the OD in each hole of described porous plate with microplate reader450Value.
With detection streptococcus agalactiae DAS ELISAA test kit manufactured in the present embodiment to agalactia Streptococcus and negative serum are detected, operation is as follows：
By streptococcus agalactiae and negative serum dilution buffer（Ibid）It is diluted to the extension rate in table 1；Buffered with carbonic acid Liquid（Ibid）3A9 monoclonal antibody is diluted to the diluent of concentration 10.0 μ g/mL；By enzyme mark 4C12 dilution buffer（With On）Make 1：1600 times of dilutions；Blank is dilution buffer（Ibid）.
It is separately added into the agalactia hammer of different extension rates in each row hole of the porous plate being coated with 3A9 monoclonal antibody Bacterium, negative serum and comparison dilution salt buffer（PBS）.Addition is every hole 100 μ L.After 37 DEG C of reactions 1 hour, use Cleaning mixture washs 3 removing unreacted reactants, adds monoclonal antibody 4C12 of HRP labelling, and 37 DEG C of effects were washed and removed after 1 hour Remove unreacted reactant, add freshly prepared 3'.3'.5,5'- tetramethyl benzidine develops the color, the color development at room temperature time is to add after 20 minutes Enter terminate liquid 2 mol/L H2SO4 50 μ L/ hole terminating reaction, surveys the OD in each hole of described porous plate with microplate reader450Value.Detection knot Fruit is shown in Table 1：
Table 1 testing result
The reduction of streptococcus agalactiae concentration, OD from the testing result of table 1 can be seen that with detection specimen450Value also drops therewith Low, the OD of blank450Value is then far below the OD of detection streptococcus agalactiae450Value.
Embodiment 2
Different from embodiment 1 it is to use negative and positive marginal value（OD450Meansigma methodss）Have or not agalactia hammer as judging to detect in sample The standard of bacterium.
In the present embodiment, detection streptococcus agalactiae DASELISA immunoadsorption is prepared using following processing step and surveys Determine test kit.
（1）Preparation is coated with the porous plate of 3A9 monoclonal antibody
It is coated liquid with carbonate buffer solution（Ibid）3A9 monoclonal antibody is diluted to the diluent of concentration 10.0 μ g/mL, so Afterwards described 3A9 monoclonal antibody diluent is added each in the hole of porous plate, be placed in 4 DEG C and be coated at least 12 hours, be coated the time After at the expiration, the defatted milk powder solution of mass concentration 5% is added each in the hole of porous plate, and carry out capping at 37 DEG C, closing In at least more than 1 hour response time, after capping terminates, use lavation buffer solution（Ibid）Washing porous plate, when on porous plate Unreacted reactant be removed after obtain and be coated with the porous plate of 3A9 monoclonal antibody, its storage temperature is 4 DEG C（Typically 4 DEG C Refrigerator store is standby）.
（2）Prepare monoclonal antibody 4C12 of horseradish peroxidase (HRP) labelling
According to a conventional method：Prepare mouse ascites monoclonal antibody 4C12 with hybridoma, with caprylic acid one saturated ammonium sulfate method Purification, with BCA determination of protein concentration test kit detect monoclonal antibody 4C12 concentration as 4.34mg/mL, monoclonal antibody Save backup to -20 DEG C.
HRP labelling 4C12 step is as follows：
1. weigh 5mgHRP to be dissolved in 1ml distilled water.
2. add the 0.1M NaIO that 0.2ml newly joins in upper liquid4Solution, under ice bath, lucifuge stirs 15 minutes.
3. above-mentioned solution is loaded in bag filter, with the sodium-acetate buffer dialysis of 1mM pH4.4, changes liquid once within 4 hours, Period changes liquid 5 times.
4. plus 20 μ l 0.2M pH9.5 carbonate buffer solutions, the pH of above hydroformylation HRP is made to be increased to 9.0～9.5, then Add the 10mg IgG (4C12 that slightly carries through caprylic acid one saturated ammonium sulfate and through Protein G affinitive layer purification immediately 4C12), ice bath lucifuge stirs 2 hours.
Plus the 4mg/ml NaBH that newly joins of 0.1ml 5.4Liquid, mixes, then puts 4 DEG C 2 hours.
6. above-mentioned liquid is loaded in bag filter, with 0.15M pH7.4 PBS, change liquid once within 4 hours, period changes liquid 5 Secondary.
7. dropwise it is slowly added to equal-volume saturated ammonium sulfate under ice bath stirring, put 4 DEG C 2 hours.
8. 3000rpm centrifugation 30min, supernatant discarded.Precipitation semi-saturation ammonium sulfate is washed 2 times, and last precipitate is dissolved in few In the PBS of amount 0.15M pH7.4.
9. above-mentioned solution is loaded in bag filter, dialysed with the PBS buffer saline of 0.15M pH7.4, after removing ammonium ion (with the detection of Nai Shi reagent), 12000rpm is centrifuged 30 minutes and removes precipitation, and supernatant is enzyme conjugates, and that is, enzyme mark 4C12, makes Used time, use dilution buffer（Ibid）Make 1：1600 times of dilutions.
（3）Prepare 3'.3'.5,5'- tetramethyl benzidine substrates nitrite ion 20000 μ L with substrate buffer solution.
Described chromogenic substrate 3'.3'.5,5'- tetramethyl benzidine is commercial goods, can directly buy from market.
In said method, preparation be coated with 3A9 monoclonal antibody porous plate when, described 3A9 monoclonal antibody diluent plus Entering amount is every hole 100 μ L, and the addition of the monoclonal antibody 4C12 diluent of described horseradish peroxidase (HRP) labelling is 100 L/ holes, the addition of the defatted milk powder solution of described mass concentration 5% is 100 L/ holes.
In test kit of the present invention and preparation method, 3A9 monoclonal antibody is essentially protein, and energy specific recognition is no Streptococcus lactis (Lister) Lohnis 1909.554..
The DAS ELISAA test kit of detection streptococcus agalactiae of the present invention, detects that specimen is Infection animal（As tilapia）Serum and brain, liver,spleen,kidney tissue leachate, required instrument be microplate reader, using method is such as Under：
Measuring samples, after 37 DEG C are reacted 1 hour, cleaning mixture are added in being coated with the hole of porous plate of 3A9 monoclonal antibody Washing 3 times removing unreacted reactants, add HRP labelling monoclonal antibody 4C12,37 DEG C effect 1 hour after washing remove not anti- Answer thing, add freshly prepared 3'.3'.5,5'- tetramethyl benzidine develops the color, the color development at room temperature time is to add after 20 minutes to terminate Liquid 2 mol/L H2SO4 50 μ L/ hole terminating reaction, surveys the OD in each hole of described porous plate with microplate reader450Value.
With detection streptococcus agalactiae DAS ELISAA test kit manufactured in the present embodiment to agalactia Streptococcus and negative serum are detected, operation is as follows：
By streptococcus agalactiae and negative serum dilution buffer（Ibid）It is diluted to the extension rate in table 1；Buffered with carbonic acid Liquid（Ibid）3A9 monoclonal antibody is diluted to the diluent of concentration 10.0 μ g/mL；By enzyme mark 4C12 dilution buffer（With On）Make 1：1600 times of dilutions；Blank is dilution buffer（Ibid）.
It is separately added into the agalactia hammer of different extension rates in each row hole of the porous plate being coated with 3A9 monoclonal antibody Bacterium, negative serum and comparison dilution salt buffer（PBS）.Addition is every hole 100 μ L.After 37 DEG C of reactions 1 hour, use Cleaning mixture washs 3 removing unreacted reactants, adds monoclonal antibody 4C12 of HRP labelling, and 37 DEG C of effects were washed and removed after 1 hour Remove unreacted reactant, add freshly prepared 3'.3'.5,5'- tetramethyl benzidine develops the color, the color development at room temperature time is to add after 20 minutes Enter terminate liquid 2 mol/L H2SO4 50 μ L/ hole terminating reaction, surveys the OD in each hole of described porous plate with microplate reader450Value.
According to the detection streptococcus agalactiae DAS ELISAA test kit detection behaviour described in embodiment 2 Make method, be separately added into in each row hole of the porous plate being coated with 3A9 monoclonal antibody negative serum, positive control serum and Blank（PBS, ibid）Detection sample, detects 10 parts of negative serum samples, and positive control serum and blank, often Part repeats 3 holes, the results are shown in Table 2, calculates its OD450Meansigma methodss and standard variance be respectively：Meansigma methodss（X）For 0.1484, Standard deviation（S）For 0.0201, the numerical value of blank is 0.088 ± 0.001, is calculated according to formula X+3S：Positive and cloudy Property the marginal value that judges of result as 0.21, that is, in the case that yin and yang attribute comparison is set up, OD450 just can sentence more than 0.21 It is set to streptococcus agalactiae positive, otherwise, be judged to feminine gender.
The measurement result of table 2 negative sample
Table 5-5 Results of Detection the negative sera
Sample number 1 2 3 4 5 6 7 8 9 10
OD value 0.131 0.125 0.123 0.156 0.159 0.164 0.146 0.151 0.14 0.189
Judge streptococcus agalactiae negative and positive marginal value in detection sample（OD450Meansigma methodss）Determination.According to the inspection described in embodiment 1 Survey streptococcus agalactiae DAS ELISAA test kit detection operational approach, to being coated with 3A9 monoclonal anti It is separately added into negative serum, positive control serum and blank in each row hole of the porous plate of body（PBS, ibid）Detection sample Product, detect 10 parts of negative serum samples, and positive control serum and blank, every part of repetition 3 holes, the results are shown in Table 2, calculate Its OD450Meansigma methodss and standard variance.Yin and yang attribute marginal value=negative sample OD450Meansigma methodss+3 × standard variance.It is computed the moon Property serum OD450Meansigma methodss（X）=0.08427, standard variance（S）=0.015252.So, yin and yang attribute marginal value=0.08427+3 × 0.015252=0.130 is therefore set to 0.130 fasciola gigantica antigen negative and positive marginal value in judgement detection sample.With described During detection Fasciola gigantica DAS ELISAA test kit detection sample, situation about setting up in negative and positive comparison Under, OD450More than 0.130 it is determined that being fasciola gigantica antigen positive, otherwise, it is judged to feminine gender.
Table 2 negative serum, positive control serum OD450 value measurement result450
Blood serum sample Meansigma methodss Blood serum sample Meansigma methodss
1 0.085±0.004 6 0.092±0.016
2 0.092±0.004 7 0.110±0.001
3 0.085±0.011 8 0.049±0.004
4 0.074±0.001 9 0.084±0.002
5 0.085±0.006 10 0.086±0.001
Blank Blank 0.049±0.004 Positive control Positive 1.501±0.089
Embodiment 3
It is to use negative and positive marginal value from embodiment 1 and the different of embodiment 2（OD450Meansigma methodss）To judge to streptococcus agalactiae Specific detection effect.
According to the detection streptococcus agalactiae DAS ELISAA test kit detection side described in embodiment 1 Method, detection streptococcus agalactiae, Vibrio anguillarum（Vibrio anguillarum）, Aeromonas hydrophila（A.hydrophila）, fluorescence Pseudomonass（P.Fluorescens）, Cavia porcelluss Zymomonas mobiliss（Aeromonas caviae）, Pseudomonas aeruginosa （P.Aeruginosa）And Pseudomonas stutzeri（Pseudomonas stutzer）And use SP2/0 cell（Myeloma cell）Make Negative control, carries out specificity verification mensure, the results are shown in Table 3.
Table 3 testing result
T
Bacterial concentration（CFU/ml） 1.5×108
Streptococcus agalactiae 1.021±0.072
Vibrio anguillarum 0.088±0.007
Aeromonas hydrophila 0.130±0.003
Pseudomonas fluorescens 0.120±0.053
Aeromonas caviae 0.132±0.013
Pseudomonas aeruginosa 0.123±0.009
Pseudomonas stutzeri 0.115±0.017
Negative control 0.121±0.043
From the results shown in Table 3, the OD of streptococcus agalactiae450It is worth for 1.021 ± 0.072, higher than negative and positive marginal value（Negative and positive Marginal value is 0.21）, judge for positive, and Vibrio anguillarum, Aeromonas hydrophila, pseudomonas fluorescens, Cavia porcelluss Zymomonas mobiliss, Aerugo be false The OD that Zymomonas mobiliss, Pseudomonas stutzeri detect450Value is all far below negative and positive marginal value it was demonstrated that with described detection agalactia hammer Bacterium DAS ELISAA test kit detection Vibrio anguillarum, Aeromonas hydrophila, pseudomonas fluorescens, Cavia porcelluss are single When born of the same parents bacterium, Pseudomonas aeruginosa, Pseudomonas stutzeri, there is not cross reaction with these antigens, described detection agalactia is described Streptococcus DAS ELISAA test kit has good detection specificity.
Embodiment 4
Different from embodiment 1-3 it is to use negative and positive marginal value（OD450Meansigma methodss）To judge to have in detected difference clinical sample No streptococcus agalactiae.
Using the method for streptococcus agalactiae recurrent infection, with tilapia artificial challenge's streptococcus agalactiae, infective dose is 1.5 ×105CFU（The bacterial community sum containing in every milliliter of sample）/ tail, infection 12 hours after collection infection tilapia liver, Spleen, according to the detection streptococcus agalactiae DAS ELISAA kit test method described in embodiment 1 The tissue leachate sample of 30 parts of liver spleens of detection, the results are shown in Table 4.
Knowable to table 4 testing result, according to the critical OD of negative and positive of experiment determination450Value（See embodiment 2）For 0.21, test Negative and positive comparison and blank set up, in the tissue leachate sample of the 30 parts of liver spleens being detected, can again liver or（With） Spleen detects streptococcus agalactiae, and detection data the results are shown in Table 4.
Table 4 testing result
Fish is numbered Hepatic tissue leachate OD450 value Spleen organizes leachate OD450 value
1 0.859 0.122
2 0.226 0.115
3 1.117 0.168
4 0.363 0.148
5 0.398 0.128
6 0.155 0.643
7 0.152 0.359
8 0.143 0.652
9 0.262 0.281
10 0.129 0.336
11 0.429 0.194
12 0.092 0.192
13 0.118 0.270
14 0.215 0.189
15 0.117 0.244
16 0.094 0.219
17 0.309 0.267
18 0.882 0.151
19 0.079 0.412
20 0.066 0.250
21 0.080 0.318
22 0.113 1.154
23 0.084 0.406
24 0.068 0.210
25 0.846 0.404
26 0.210 0.580
27 0.135 0.253
28 0.103 0.570
29 0.186 0.377
30 0.155 0.643
Negative 0.105 0.145
Negative 0.155 0.130
The present invention is not limited to above-described specific embodiment, the foregoing is only present pre-ferred embodiments, and Not in order to limit the present invention, all any modification, equivalent and improvement made within present invention spirit and principle etc., all should It is included within the scope of the present invention.

Claims (7)

1. a kind of detection streptococcus agalactiae DAS ELISAA test kit it is characterised in that：Described examination Agent box is by being coated with the porous plate of 3A9 monoclonal antibody, monoclonal antibody 4C12 of horseradish peroxidase-labeled.
2. the DAS ELISAA test kit of detection streptococcus agalactiae according to claim 1, its It is characterised by：It is 2.0-200.0 μ g/mL that the every hole of described porous plate is coated 3A9 MAb concentration.
3. the DAS ELISAA test kit of detection streptococcus agalactiae according to claim 1, its It is characterised by：Described test kit also includes chromogenic substrate 3'.3'.5,5'- tetramethyl benzidine.
4. the DAS ELISAA test kit of detection streptococcus agalactiae according to claim 2, its It is characterised by：The consumption that the every hole of described porous plate is coated 3A9 monoclonal antibody is 100 μ L, and concentration is 10.0 μ g/mL.
5. the DAS ELISAA test kit of detection streptococcus agalactiae according to claim 1, its It is characterised by：The step of the preparation method of described horseradish peroxidase-labeled monoclonal antibody 4C12 is,
1）Prepare mouse ascites monoclonal antibody 4C12 according to a conventional method, purify mouse ascites with caprylic acid one saturated ammonium sulfate method Monoclonal antibody 4C12, with BCA determination of protein concentration test kit detect monoclonal antibody 4C12 concentration as 4.34mg/mL, Monoclonal antibody 4C12 is positioned over and saves backup under -20 DEG C of environment；
2) weigh 5mg horseradish peroxidase to be dissolved in 1ml distilled water；
3) by step 2）The solution obtaining adds the 0.1M NaIO that 0.2ml newly joins4Solution, under ice bath, lucifuge stirs 15 minutes；
4) by step 3）The solution obtaining loads in bag filter, with the sodium-acetate buffer dialysis of 1mM pH4.4, changes liquid within 4 hours Once, period changes liquid 3-5 time；
5) in step 4）Add 20 μ l 0.2M pH9.5 carbonate buffer solutions in the solution obtaining, make the pH of above hydroformylation HRP It is increased to 9.0～9.5, adds 10mg IgG immediately after, ice bath lucifuge stirs 2 hours；
6) by step 5）It is 4mg/ml NaBH that the solution obtaining adds 0.1ml concentration4Liquid, mixes, then places 2 under 4 DEG C of environment Hour；
7) by step 6）The solution obtaining loads in bag filter, with 0.15M pH7.4 PBS, changes liquid once within 4 hours, period Change liquid 3-5 time；
8) by step 7）The solution obtaining dropwise is slowly added to equal-volume saturated ammonium sulfate under ice bath stirring, is placed in 4 DEG C of environment Lower 2 hours；
9) by step 8）The solution obtaining is put in centrifuge tube, is centrifuged 30min, supernatant discarded with centrifuge 3000rpm, precipitation is used Semi-saturation ammonium sulfate is washed 1-2 time, and last precipitate is dissolved in the PBS of 0.15M pH7.4；
10) by step 9）The solution that obtains loads in bag filter, with the PBS buffer saline dialysis of 0.15M pH7.4, dispel ammonium from After son, 12000rpm is centrifuged 30 minutes and removes precipitation, and supernatant is enzyme conjugates, i.e. enzyme mark 4C12, after enzyme mark 4C12 subpackage Stored frozen.
6. a kind of preparation method of the DAS ELISAA test kit of detection streptococcus agalactiae, its feature exists In its preparation process is as follows：
（1）Preparation is coated with the porous plate of 3A9 monoclonal antibody
That is made with carbonate buffer solution is coated the diluent that 3A9 monoclonal antibody is diluted to concentration 10.0 μ g/mL by liquid, described It is coated liquid by Na2CO31.59 g、NaHCO32.93 g, plus distilled water is settled to 1000 mL and makes, it is protected under the conditions of 4 DEG C Deposit, then described 3A9 monoclonal antibody diluent is added each in the hole of porous plate, be placed in and be coated 12 hours -24 under 4 DEG C of environment Hour, after the time of being coated expires, the defatted milk powder solution of mass concentration 5% is added each in the hole of porous plate, and carry out at 37 DEG C Capping, at least 1 hour -1.5 hours capping time, after capping terminates, washs porous plate with lavation buffer solution, Obtain the porous plate being coated with 3A9 monoclonal antibody after the unreacted reactant on porous plate is removed, its storage temperature is 4 ℃；
（2）Monoclonal antibody 4C12 of preparation horseradish peroxidase-labeled
According to a conventional method：Prepare mouse ascites monoclonal antibody 4C12 with hybridoma, with caprylic acid one saturated ammonium sulfate method Purification, with BCA determination of protein concentration test kit detect monoclonal antibody 4C12 concentration as 4.34mg/mL, monoclonal antibody Save backup to -20 DEG C；
（3）It is equipped with chromogenic substrate 3'.3'.5,5'- tetramethyl benzidine according to a conventional method；
（4）It is equipped with lavation buffer solution, lavation buffer solution consists of the following composition：NaCl 8.0 g, KH2PO40.2 g, KCl 0.2 g, Na2HPO4·12H2O 3.58 g, Tween-20 0.5 mL, plus distilled water is settled to 1000 mL to make washing slow Rush liquid；
（5）According to a conventional method lavation buffer solution is dispensed into the vial that 20ML sterilized, the list of horseradish peroxidase-labeled Clonal antibody 4C12 and chromogenic substrate are dispensed in epoxy resin small test tube pipe respectively, are then coated with 3A9 monoclonal with preparation The porous plate of antibody is combined into test kit together；
Described horseradish peroxidase-labeled 4C12 step is as follows：
Step 1）Weigh 5mg horseradish peroxidase to be dissolved in 1ml distilled water；
Step 2）Add the 0.1M NaIO4 solution that 0.2ml newly joins in upper liquid, under ice bath, lucifuge stirs 15 minutes；
Step 3）Above-mentioned solution is loaded in bag filter, with the sodium-acetate buffer dialysis of 1mM pH4.4, changes liquid once within 4 hours, Period changes liquid 3-5 time；
Step 4）Plus 20 μ l 0.2M pH9.5 carbonate buffer solutions, so that the pH value of above hydroformylation horseradish peroxidase is increased to 9.0～9.5, add 10mg IgG immediately after, ice bath lucifuge stirs 2 hours；
Step 5）Plus the 4mg/ml NaBH that 0.1ml newly joins4Liquid, mixes, then puts 4 DEG C 2 hours；
Step 6）Above-mentioned liquid is loaded in bag filter, with 0.15M pH7.4 PBS, changes liquid once within 4 hours, period changes liquid 3-5 time；
Step 7）Dropwise it is slowly added to equal-volume saturated ammonium sulfate under ice bath stirring, be placed in 4 DEG C of environment lower 2 hours；
Step 8）By step 7）The solution obtaining is placed in 4 DEG C of environment solution of lower 2 hours and is placed in the interior centrifuge of centrifuge tube and exists It is centrifuged 30min, supernatant discarded, precipitation semi-saturation ammonium sulfate is washed 1-2 time, and precipitate is dissolved in 0.15M under the conditions of 3000rpm In the PBS of pH7.4；
Step 9）By step 8）The solution obtaining loads in bag filter, with the PBS buffer saline dialysis of 0.15M pH7.4, removes After ammonium ion, 12000rpm is centrifuged 30 minutes and removes precipitation, and supernatant is enzyme conjugates, i.e. enzyme mark 4C12, during use, uses phosphorus Phthalate buffer makees 1：1600 times of dilutions.
7. the DAS ELISAA test kit of a kind of detection streptococcus agalactiae according to claim 6 Preparation method it is characterised in that：When described preparation is coated with the porous plate of 3A9 monoclonal antibody, described 3A9 monoclonal antibody The addition of diluent is 100 L/ holes, the addition of the monoclonal antibody 4C12 diluent of described horseradish peroxidase-labeled For 100 L/ holes, the addition of the defatted milk powder solution of described mass concentration 5% is 100 L/ holes.
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Application publication date: 20170222