CN102759619A - Enzyme-linked immunosorbent assay kit of streptococcus suis serotype 2 capsular polysaccharide antibody and application of enzyme-linked immunosorbent assay kit - Google Patents
Enzyme-linked immunosorbent assay kit of streptococcus suis serotype 2 capsular polysaccharide antibody and application of enzyme-linked immunosorbent assay kit Download PDFInfo
- Publication number
- CN102759619A CN102759619A CN2012102452502A CN201210245250A CN102759619A CN 102759619 A CN102759619 A CN 102759619A CN 2012102452502 A CN2012102452502 A CN 2012102452502A CN 201210245250 A CN201210245250 A CN 201210245250A CN 102759619 A CN102759619 A CN 102759619A
- Authority
- CN
- China
- Prior art keywords
- streptococcus suis
- capsular polysaccharide
- enzyme
- solution
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an enzyme-linked immunosorbent assay (ELISA) kit of streptococcus suis serotype 2 capsular polysaccharide antibody and an application of the enzyme-linked immunosorbent assay kit. The enzyme-linked immunosorbent assay kit also comprises an antigen ELISA plate, a diluent A, defatted milk powder, positive control, negative control, a cleaning solution, a goat-anti-pig enzyme labeled secondary antibody, a color development solution A, a color development solution B and a stop solution, wherein the antigen ELISA plate takes the streptococcus suis serotype 2 capsular polysaccharide as the antibody; the positive control is pig positive control serum which is immunized by the streptococcus suis serotype 2; the cleaning solution is phosphate buffer solution (PBS) containing Tween-20; the stop solution is hydrofluoric acid solution; the color development solution A is citrate buffer solution containing hyperol, and the color development solution B is citric acid/sodium citrate buffer solution containing TMB (tetramethylbenzidine) and having pH of 5.0; and the diluent A is PBS containing Tween-20. The enzyme-linked immunosorbent assay kit is applied to detecting the enzyme-linked immunization of the streptococcus suis serotype 2 capsular polysaccharide antibody. The enzyme-linked immunosorbent assay kit has good specificity and high flexibility when being used for the streptococcus suis serotype 2 capsular polysaccharide. The detection time is short, and the result can be obtained within 2 hours generally.
Description
Technical field
The present invention relates to animal epidemic detects; Be specifically related to a kind of enzyme linked immunological kit of streptococcus suis 2-type capsular polysaccharide antibody; The purposes that also relates to a kind of enzyme linked immunological kit of streptococcus suis 2-type capsular polysaccharide antibody simultaneously is applicable to the antibody horizontal of the capsular polysaccharide that detects the streptococcus suis 2-type in the porcine blood serum.
Technical background
Streptococcus suis (streptococcus suis) is a kind of important pathogenic bacteria that can cause people, pig and other zoogenetic infections, morbidity.According to the capsular polysaccharide somatotype, Streptococcus suis has 35 serotypes, i.e. 1/2 type and 1-34 type (research afterwards thinks that 32 and 34 types belong to Streptococcus orisratti).Wherein streptococcus suis 2-type (SS2) is virulence the widest the strongest, the popular serotype.Abroad existing people more than 200 is dead because of the infected pigs streptococcus since nineteen sixty-eight, Holland reported first people infected the meningitis case.In China, should disease since the seventies more than 20 provinces and cities have taken place or are popular in the whole nation, caused economic loss in various degree.Between 6～August in 2005, regional outbreak of epidemic streptococcus suis 2-type such as Ziyang City, Sichuan Province, Neijiang City is sick, and causes 204 people to infect, and 38 people are dead.In other provinces and cities fragmentary generation is arranged also.This shows that this disease extensively exists also seriously popular in China, become a kind of principal disease of current serious harm pig industry development, not only cause the tremendous economic loss, and also caused serious harm for the mankind to pig industry.
In order to effectively prevent and control the streptococcus suis 2-type disease; In the booster immunization prevention; Use diagnosis and detection method special, responsive, quick, that be convenient to operate, strong means and important instrument be provided for effectively prevention and control streptococcus suis 2-type.
The capsular polysaccharide of the streptococcus suis 2-type of application fetches of the present invention and ELISA detection technique make kit have good susceptibility and specificity, greatly the antibody of the streptococcus suis 2-type capsular polysaccharide in the detection porcine blood serum of flux.
Summary of the invention
The objective of the invention is to be to provide a kind of enzyme linked immunological kit of streptococcus suis 2-type capsular polysaccharide antibody; The advantage of this kit has been to use the capsular polysaccharide of streptococcus suis 2-type as antigen, makes the detection of kit have good sensitivity and specificity.
Another object of the present invention is the application of enzyme linked immunological kit in the enzyme linked immunological that detects streptococcus suis 2-type capsular polysaccharide antibody that has been to provide a kind of streptococcus suis 2-type capsular polysaccharide antibody; With the streptococcus suis 2-type capsular polysaccharide that extracts as antigen coated on polystyrene micropore plate; Adding serum to be checked then hatches; Add goat-anti pig enzyme labelled antibody (available from Southern biotechnology associates (SBA)) again, should occur two kinds of situation during colour developing, if serum to be checked is positive; The goat-anti pig enzyme labelled antibody that adds this moment so will continue with positive serum in antibody response, ELIASA detects numerical value will be high more.If seronegativity to be checked, so goat-anti pig enzyme labelled antibody just not can with streptococcus suis 2-type capsular polysaccharide antigen-reactive on the elisa plate, the numerical value that obtains will be low.This kit specificity is good, susceptibility is high.The present domestic similar business-like product that also do not have.Good prospects for application is arranged.
In order to realize above-mentioned purpose, the present invention adopts following technical measures:
A kind of kit of enzyme linked immunological of streptococcus suis 2-type capsular polysaccharide antibody comprises the antigen ELISA Plate that the streptococcus suis 2-type capsular polysaccharide encapsulates.
Described kit also comprises antigen ELISA Plate, diluent A, skimmed milk power (commercially available Erie milk powder), positive control, negative control, cleansing solution, goat-anti pig ELIAS secondary antibody, colour developing liquid A, colour developing liquid B, stop buffer.
Described antigen ELISA Plate be with the streptococcus suis 2-type capsular polysaccharide as antigen, when encapsulating polystyrene micropore plate, use 0.1mol/L, the sodium bicarbonate solution of pH9.6 is as encapsulating damping fluid.Described Streptococcus suis capsular polysaccharide was from streptococcus suis 2-LT strain (CCTCC NO:M2011282), to extract gained, and described streptococcus suis 2-LT strain has been deposited in Chinese typical culture collection center, CCTCC NO:M2011282 on August 9th, 2011.(number of patent application: 201110234192.9)
The pig positive control serum that described positive control is crossed for the streptococcus suis 2-type bacterial immunity, described negative control was not for infecting and the not immune health pig negative serum of crossing Streptococcus suis.
Described cleansing solution is for containing the PBS damping fluid of 0.05% (volume ratio) Tween-20; Described stop buffer is for containing 0.25% volume ratio hydrofluoric acid solution.
Described colour developing liquid A is the citrate buffer that contains the 50mg/mL carbamide peroxide, and colour developing liquid B liquid is the citric acid/sodium citrate damping fluid that contains 0.2mg/mL TMB pH5.0.
Described diluent A is the PBS damping fluid that contains 0.05% volume ratio Tween-20.
A kind of preparation method of streptococcus suis 2-type capsular polysaccharide, its preparation process are that the lysozyme processing combines with cold phenol extraction.Specifically be divided into for two steps:
One, uses lysozyme facture (Lin Shuqian etc., the preparation of staphylococcus aureus capsular polysaccharide and biological characteristics, the ecology of domestic animals newspaper of improvement; The 27th the 6th phase of volume), uses sterile saline to give a baby a bath on the third day after its birth time the streptococcus suis 2-type bacterium liquid of deactivation, use the 0.01mol/L PBS of original bacteria liquid 1/10 volume that it is resuspended again; The glycocoll that adds final concentration 0.1mol/L; And add final concentration 3mg/L lysozyme (available from Beijing Pu Boxin bio tech ltd), put under 37 ℃ spend the night (8～10 hours), centrifugal 45 minutes of room temperature 14000r/min; Collect supernatant, adding final concentration again and be 25% absolute ethyl alcohol and final concentration is the CaCl of 0.1g/L
2, putting 2～8 ℃ of down effects 2 hours, centrifugal 10 minutes of 12000r/min collects supernatant, adds final concentration then and be 80% absolute ethyl alcohol, puts 2～8 ℃ of effects 12 hours down, centrifugal 10 minutes of 12000r/min, the deposition that obtains is rough capsular polysaccharide.
Two, with cold phenol facture (Lin Shuqian etc., the preparation of staphylococcus aureus capsular polysaccharide and biological characteristics, ecology of domestic animals newspaper, the 27th the 6th phase of volume), rough polysaccharide is carried out purifying.The rough polysaccharide that obtains is resuspended with 10ml saturated acetic acid sodium, the phenol of adding precooling, jolting is 30 minutes fast, and 8 ℃ of 4000r/min are centrifugal; Collect supernatant, triplicate, again with gained the supernatant bag filter of packing into dialysed 48 hours with the NaCl solution of 1mol/L; Collect dialyzed solution, add final concentration then and be 55% absolute ethyl alcohol, put 2～8 ℃ of effects 24 hours down; Centrifugal 10 minutes of 12000r/min gets the dry back of deposition with the 10mlPBS dissolving, is refining capsular polysaccharide.
The application of a kind of kit of enzyme linked immunological of streptococcus suis 2-type capsular polysaccharide antibody in the enzyme linked immunological that detects streptococcus suis 2-type capsular polysaccharide antibody the steps include:
1) skimmed milk power is dissolved in the diluent A, the final concentration that makes its skimmed milk power is 5%, as the dilution of serum to be checked and control serum.
2) get antigen coated microplate (being coated with streptococcus suis 2-type capsular polysaccharide detection of antigens plate); Earlier wash plate 2 times, will dilute serum to be checked, negative control sera and positive control serum well again and respectively get 100 μ l and join in the antigen coated plate hole with the good cleansing solution of dilution.Serum to be checked is established 1 hole, and negative control and positive control are respectively established 2 holes.Shake gently and spare sample (not overflowing) in the hole, put 37 ℃ of following incubations 30 minutes.
3) discard solution in the hole, every hole adds the good cleansing solution 200 μ l of dilution, leave standstill 3 minutes after, discard cleansing solution, and do at the thieving paper arsis, repeat to wash plate 5 times, do at the thieving paper arsis for the last time.
4) every hole adds goat-anti pig ELIAS secondary antibody 100 μ l, puts 37 ℃ of following incubations (same step 3) of method of washing plate after 30 minutes 5 times.
5) every hole adds colour developing liquid A, colour developing liquid B each (about 50 μ L), mixing, room temperature (18～25 ℃, below identical) lucifuge colour developing 10 minutes.Every hole adds one of stop buffer (about 50 μ L), measures every hole OD630nm value of reading with ELIASA in 10 minutes.
Kit criterion of the present invention is: on ELIASA, survey each hole OD630nm value, the condition that test is set up is; Positive control hole OD630nm value all should >=0.8, and＜2.0; Negative control hole OD630nm value all should＜0.3.If sample OD630nm value >=0.35 is judged to the positive; If sample OD630nm value＜0.35 then is judged to feminine gender.
The present invention has the following advantages and effect:
1. the present domestic similar business-like product that also do not have
2. owing to the use of streptococcus suis 2-type capsular polysaccharide, specificity is good, and is highly sensitive.
3. simple to operate, detection time is short, can go out the result in general 2 hours;
Description of drawings
Fig. 1 is a kind of typical curve synoptic diagram of drawing during with phenol sulfuric acid process mensuration capsular polysaccharide.
Horizontal ordinate is sugared concentration among the figure, and ordinate is the OD value.
Fig. 2 carries out the quality testing synoptic diagram for a kind of use spectrophotometer to capsular polysaccharide.
Horizontal ordinate is a wavelength among the figure, and ordinate is a light absorption value.
Embodiment
In order to make the present invention be more prone to understand, will further set forth embodiments of the invention below.In conjunction with implementing the present invention is done further description and demonstration.But present embodiment is not a limitation of the present invention.
Embodiment 1: the preparation of antigen
The extraction of 1 antigen
1.1 microbe growth
Adopt the TSA solid medium to go down to posterity and increase bacterium; The 3rd generation bacterial classification optimize in the fluid nutrient medium with the TSB that certain inoculum concentration is inoculated in optimization, cultivated 12～14 hours down for 37 ℃, standard bacterium opacity tube stops cultivating when surveying bacteria concentration to 1.5 * 109/ml; The formaldehyde of adding 5/1000ths stirs deactivation.
1.2 the extraction purifying of capsular polysaccharide
1.2.1 the extraction of capsular polysaccharide
Use the lysozyme facture of improvement, use sterile saline to give a baby a bath on the third day after its birth time the streptococcus suis 2-type bacterium of deactivation, use the 0.01mol/LPBS of original bacteria liquid 1/10 volume that it is resuspended again; The glycocoll that adds final concentration 0.1mol/L, and add final concentration 3mg/L lysozyme, put under 37 ℃ spend the night (8～10 hours); Centrifugal 45 minutes of room temperature 14000r/min collects supernatant, and adding final concentration again and be 25% absolute ethyl alcohol and final concentration is the CaCl2 of 0.1g/L; Put 2～8 ℃ and act on 2 hours down, centrifugal 10 minutes of 12000r/min collects supernatant; Add final concentration then and be 80% absolute ethyl alcohol; Put 2～8 ℃ and act on 12 hours down, centrifugal 10 minutes of 12000r/min, the deposition that obtains is rough capsular polysaccharide.
1.2.2 the purifying of capsular polysaccharide
With cold phenol facture, rough polysaccharide is carried out purifying.The rough polysaccharide that obtains is resuspended with 10ml saturated acetic acid sodium, the phenol of adding precooling, jolting is 30 minutes fast, and 8 ℃ of 4000r/min are centrifugal; Collect supernatant, triplicate, again with gained the supernatant bag filter of packing into dialysed 48 hours with the NaCl solution of 1mol/L; Collect dialyzed solution, add final concentration then and be 55% absolute ethyl alcohol, put 2～8 ℃ of effects 24 hours down; Centrifugal 10 minutes of 12000r/min gets the dry back of deposition with the 10mlPBS dissolving, is refining capsular polysaccharide.
2. the mensuration of antigen
2.1 adopt the phenolsulfuric acid method to survey capsular polysaccharide concentration, precision takes by weighing 105 ℃ of glucose 1g that are dried to constant weight, adds water-solublely to separate and be settled in the 100ml volumetric flask, promptly obtains the glucose stock solution, accurate absorption stock solution 0; 2,4,6,8,10; 12,14,16ml puts respectively in the 100ml volumetric flask, adds water to scale, shakes up; Be the glucose application liquid, draw above-mentioned application liquid 1ml respectively in test tube, add 8% phenol solution 1ml, add concentrated sulphuric acid 5ml behind the mixing rapidly; Shake up, 40 ℃～50 ℃ water-bath 30min are that control tube is measured at the 490nm place with No. 1 pipe, draw out the standard regression equation.
The capsular polysaccharide antigen of purifying carries out full wavelength scanner to extracting also 2.2 utilize spectrophotometer, to measure the content and the purity of polysaccharide in the antigen.
3. result
3.1 with Excel2003 statistical software analyzing and processing, get regression equation, and require coefficient R 2 >=0.985 according to the result.Y=0.4163x+0.0024 (R2=0.9987) (see figure 1).
3.2 capsular polysaccharide antigenic quality testing result
See shown in Figure 2; 260 do not have the absorption peak of protein and nucleic acid with the 280nm place in the collection of illustrative plates; 200nm left and right sides absorption value very high (100 times of capsular polysaccharide diluted samples); This is the characteristic absorption crest of polysaccharide, proves that heteroproteins in the CPS antigen that obtains and residual nucleic acid content are lower, and the capsular polysaccharide antigen purity is higher.
Embodiment 2: the preparation of antigen coated microplate
It is 1.25 μ g/ml that capsular polysaccharide antigen is used the carbonate buffer solution dilution of pH9.6, is added in the polystyrene micropore plate by 100 μ L/ holes, puts and encapsulates spend the night (14～18 hours) under 2～8 ℃; Take out next day, discards the antigen coated liquid in the hole, and every hole adds the sealing of 120 μ l confining liquids (seeing note 1); Put 37 ℃ of down effects 1 hour, discard in the hole behind the confining liquid, every again hole adds 37 ℃ of effects of 120 μ l protective agents (seeing note 1) 1 hour; Discard in the hole after the protective agent, clap and do, put 37 ℃ of dryings again 2 hours; Seal film with tinfoil paper antigen coated microplate is sealed, put 2～8 ℃ and preserve down.
Embodiment 3: the preparation of control serum
1. the preparation of Streptococcus suis positive control serum
Select healthy piglet in 4 ages in week for use.With being concentrated into 3,000,000,000/ml after the deactivation of streptococcus suis 2-type (LT-1 strain) bacterium liquid, add for the first time after the emulsification of equivalent complete Freund's adjuvant as immunogene; For the second time with add for the third time after the emulsification of equivalent incomplete Freund's adjuvant as immunogene; The 4th immunity do not add adjuvant, directly with the inactivated bacterial liquid that concentrates as immunogene, musculi colli injection three times, 2ml for the first time, 4ml for the second time, 6ml for the third time, the 4th intravenous injection 3ml, each immunity is 2 weeks at interval.The 4th immunity blood sampling in back 10 days detects antibody titer with agar gel diffusion test, reaches more than the 1:16, gathers blood from arteria carotis; Separation of serum, adding 0.04% merthiolate is anticorrosion in serum, 0.22 μ m membrane filtration degerming; Aseptic subpackaged, every pipe 1ml puts-70 ℃ and preserves down.
2. the preparation of Streptococcus suis negative control sera
Select healthy piglet in 4 ages in week for use, detect all negative through streptococcus suis 2-type agar gel diffusion test and streptococcus suis 2-type PCR test.Arteria carotis is gathered blood, separation of serum, and it is anticorrosion in serum, to add 0.04% (volume ratio) merthiolate, 0.22 μ m membrane filtration degerming.Aseptic subpackaged, every pipe 1ml puts-70 ℃ and preserves down.
Embodiment 4: the kit assembling:
A kind of kit of enzyme linked immunological of streptococcus suis 2-type capsular polysaccharide antibody comprises streptococcus suis 2-type capsular polysaccharide antigen coated microplate.
Described kit also comprises ELISA Plate, diluent A, skimmed milk power, positive control, negative control, cleansing solution, goat-anti pig ELIAS secondary antibody, colour developing liquid A, colour developing liquid B, stop buffer.
Described antigen coated microplate be with the streptococcus suis 2-type capsular polysaccharide as antigen, when encapsulating polystyrene micropore plate, use 0.1mol/L, the sodium bicarbonate solution of pH9.6 is as encapsulating damping fluid.Described Streptococcus suis capsular polysaccharide was from streptococcus suis 2-LT strain (CCTCC NO:M2011282), to extract gained, and described streptococcus suis 2-LT strain has been deposited in Chinese typical culture collection center, CCTCC NO:M2011282 on August 9th, 2011.
The pig positive control serum that described positive control is crossed for the streptococcus suis 2-type bacterial immunity, described negative control was not for infecting the health pig negative serum of Streptococcus suis.
Described cleansing solution is the PBS damping fluid that contains 0.05% volume ratio Tween-20; Described stop buffer is for containing 0.25% volume ratio hydrofluoric acid solution.
Described colour developing liquid A is the citrate buffer that contains the 50mg/mL carbamide peroxide, and colour developing liquid B liquid is the citric acid/sodium citrate damping fluid that contains 0.2mg/mL TMB pH5.0.
Described diluent A is the PBS damping fluid that contains 0.05% volume ratio Tween-20.
The assembling of kit contains:
(1) 2 of antigen coated microplates (96 holes/piece)
(2) negative control sera 1 pipe (1ml/ pipe)
(3) positive control serum 1 pipe (1ml/ pipe)
(4) 1 bottle of goat-anti pig ELIAS secondary antibody (20ml/ bottle)
(5) skimmed milk power 1 pipe (2.5g/ pipe)
(6) 1 bottle of diluent A (50ml/ bottle)
(7) 1 bottle of substrate solution A (10ml/ bottle)
(8) 1 bottle of substrate solution B (10ml/ bottle)
(9) 1 bottle of stop buffer (10ml/ bottle)
(10) 20 times of 1 bottle of concentrated cleaning solutions (30ml/ bottle)
(11) serum dilution plate is 2
(12) instructions is 1 part
The preparation of the positive, negative control:
Streptococcus suis positive serum adding 0.04% (volume ratio) Sodium Mercurothiolate that screening is obtained is anticorrosion, 0.22 μ m membrane filtration degerming.Aseptic subpackaged, every pipe 1ml is as positive control; With the pig standard female serum that screening obtains, adding 0.04% (volume ratio) Sodium Mercurothiolate is anticorrosion, 0.22 μ m membrane filtration degerming.Aseptic subpackaged, every pipe 1ml is as negative control.
Embodiment 5: the application of kit
The application of a kind of kit of enzyme linked immunological of capsular polysaccharide antibody in the enzyme linked immunological that detects streptococcus suis 2-type capsular polysaccharide antibody the steps include:
1) skimmed milk power is dissolved in the diluent A, the final concentration that makes its skimmed milk power is 5%, as the dilution of serum to be checked and control serum.
2) get antigen coated microplate (being coated with streptococcus suis 2-type capsular polysaccharide detection of antigens plate); Earlier wash plate 2 times, will dilute serum to be checked, negative control sera and positive control serum well again and respectively get 100 μ l and join in the antigen coated plate hole with the good cleansing solution of dilution.Serum to be checked is established 1 hole, and negative control and positive control are respectively established 2 holes.Shake gently and spare sample (not overflowing) in the hole, put 37 ℃ of following incubations 30 minutes.
3) discard solution in the hole, every hole adds the good cleansing solution 200 μ l of dilution, leave standstill 3 minutes after, discard cleansing solution, and do at the thieving paper arsis, repeat to wash plate 5 times, do at the thieving paper arsis for the last time.
4) every hole adds goat-anti pig ELIAS secondary antibody 100 μ l, puts 37 ℃ of following incubations (same step 3) of method of washing plate after 30 minutes 5 times.
5) every hole adds colour developing liquid A, colour developing liquid B each (about 50 μ L), mixing, room temperature (18～25 ℃, below identical) lucifuge colour developing 10 minutes.Every hole adds one of stop buffer (about 50 μ L), measures every hole OD630nm value of reading with ELIASA in 10 minutes.
The situation of institute's result of use experiment is following in the application:
The test of 1 specificity:
China's pig main diseases viral disease (PRV, PPV, HCV, PRRSV, FMDV) positive serum is detected testing result all negative (table 1) with streptococcus suis 2-type capsular polysaccharide antibody assay kit.
The sero-fast testing result of table 1 China pig main diseases viral disease
How high antibody assay kit detects testing result all negative (table 2) with the streptococcus suis 2-type pod membrane with China's pig The main pathogenic fungi positive serum (APP, HPS, the sick positive serum of C group streptococcus).
The sero-fast testing result of table 2 China pig The main pathogenic fungi
2, the detection of susceptibility:
With detecting with streptococcus suis 2-type capsular polysaccharide antibody assay kit behind 10 parts of different Streptococcus suis positive serum 1:5, the continuous doubling dilution of 1:10～1:1280, can find out that from table 3 kit susceptibility is good.
The positive serum testing result of the different extension rates of table 3
Claims (3)
1. the enzyme linked immunological kit of a streptococcus suis 2-type capsular polysaccharide antibody; Comprise the antigen ELISA Plate that the streptococcus suis 2-type capsular polysaccharide encapsulates, it is characterized in that: described kit also comprises antigen ELISA Plate, diluent A, skimmed milk power, positive control, negative control, cleansing solution, goat-anti pig ELIAS secondary antibody, colour developing liquid A, colour developing liquid B, stop buffer;
Described antigen ELISA Plate be with the streptococcus suis 2-type capsular polysaccharide as antigen, when encapsulating polystyrene micropore plate, use 0.1mol/L, the sodium bicarbonate solution of pH9.6 is as encapsulating damping fluid;
The pig positive control serum that described positive control is crossed for the streptococcus suis 2-type bacterial immunity;
Described negative control was not for infecting and the not immune health pig negative serum of crossing Streptococcus suis;
Described cleansing solution is the PBS damping fluid that contains 0.05% volume ratio Tween-20;
Described stop buffer is for containing 0.25% volume ratio hydrofluoric acid solution;
Described colour developing liquid A is the citrate buffer that contains the 50mg/mL carbamide peroxide, and colour developing liquid B liquid is the citric acid/sodium citrate damping fluid that contains 0.2mg/mL TMB pH5.0;
Described diluent A is the PBS damping fluid that contains 0.05% volume ratio Tween-20.
2. the enzyme linked immunological kit of a kind of streptococcus suis 2-type capsular polysaccharide antibody according to claim 1 is characterized in that: the preparation method of described streptococcus suis 2-type capsular polysaccharide the steps include:
A, use the lysozyme facture of improvement, use sterile saline to give a baby a bath on the third day after its birth time the streptococcus suis 2-type bacterium of deactivation, use the 0.01mol/L PBS of original bacteria liquid 1/10 volume that it is resuspended again; The glycocoll that adds final concentration 0.1mol/L; And add final concentration 3mg/L lysozyme, put and spent the night under 37 ℃ 8～10 hours, centrifugal 45 minutes of room temperature 14000r/min; Collect supernatant, adding final concentration again and be 25% absolute ethyl alcohol and final concentration is the CaCl of 0.1g/L
2, putting 2～8 ℃ of down effects 2 hours, centrifugal 10 minutes of 12000r/min collects supernatant, adds final concentration then and be 80% absolute ethyl alcohol, puts 2～8 ℃ of effects 12 hours down, centrifugal 10 minutes of 12000r/min, the deposition that obtains is rough capsular polysaccharide;
B, with cold phenol facture, rough polysaccharide is carried out purifying, the rough polysaccharide that obtains is resuspended with 10ml saturated acetic acid sodium; The phenol that adds precooling, jolting 30 minutes, 8 ℃ of 4000r/min are centrifugal; Collect supernatant, triplicate, again with gained the supernatant bag filter of packing into dialysed 48 hours with the NaCl solution of 1mol/L; Collect dialyzed solution, add final concentration then and be 55% absolute ethyl alcohol, put 2～8 ℃ of effects 24 hours down; Centrifugal 10 minutes of 12000r/min gets the dry back of deposition with the 10mlPBS dissolving, is capsular polysaccharide.
3. the application of the kit of the enzyme linked immunological of the described a kind of streptococcus suis 2-type capsular polysaccharide antibody of claim 1 in the enzyme linked immunological that detects streptococcus suis 2-type capsular polysaccharide antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012102452502A CN102759619A (en) | 2012-07-16 | 2012-07-16 | Enzyme-linked immunosorbent assay kit of streptococcus suis serotype 2 capsular polysaccharide antibody and application of enzyme-linked immunosorbent assay kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012102452502A CN102759619A (en) | 2012-07-16 | 2012-07-16 | Enzyme-linked immunosorbent assay kit of streptococcus suis serotype 2 capsular polysaccharide antibody and application of enzyme-linked immunosorbent assay kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102759619A true CN102759619A (en) | 2012-10-31 |
Family
ID=47054137
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012102452502A Pending CN102759619A (en) | 2012-07-16 | 2012-07-16 | Enzyme-linked immunosorbent assay kit of streptococcus suis serotype 2 capsular polysaccharide antibody and application of enzyme-linked immunosorbent assay kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102759619A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106093439A (en) * | 2016-06-01 | 2016-11-09 | 中国农业科学院兰州兽医研究所 | A kind of Streptococcus suis indirect hemagglutination detection kit and application thereof |
| CN114755406A (en) * | 2022-03-28 | 2022-07-15 | 南京农业大学 | Streptococcus suis 9 type ELISA antibody detection method and kit thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH11171899A (en) * | 1997-12-04 | 1999-06-29 | Chisso Corp | Monoclonal antibody resistant to erysipelothrix rhusiopathiae and its utilization |
| CN1709505A (en) * | 2005-07-13 | 2005-12-21 | 北京绿竹生物制药有限公司 | Polyvalent bacteria capsule polysaccharide-protein conjugate combined vaccine |
| CN101724085A (en) * | 2008-10-22 | 2010-06-09 | 上海生物制品研究所 | Method for purifying capsular polysaccharide |
| CN102353783A (en) * | 2011-06-29 | 2012-02-15 | 武汉科前动物生物制品有限责任公司 | Kit for detecting pig pseudorabies virus antibodies and block enzyme-linked immuno sorbent assay (ELISA) method |
-
2012
- 2012-07-16 CN CN2012102452502A patent/CN102759619A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH11171899A (en) * | 1997-12-04 | 1999-06-29 | Chisso Corp | Monoclonal antibody resistant to erysipelothrix rhusiopathiae and its utilization |
| CN1709505A (en) * | 2005-07-13 | 2005-12-21 | 北京绿竹生物制药有限公司 | Polyvalent bacteria capsule polysaccharide-protein conjugate combined vaccine |
| CN101724085A (en) * | 2008-10-22 | 2010-06-09 | 上海生物制品研究所 | Method for purifying capsular polysaccharide |
| CN102353783A (en) * | 2011-06-29 | 2012-02-15 | 武汉科前动物生物制品有限责任公司 | Kit for detecting pig pseudorabies virus antibodies and block enzyme-linked immuno sorbent assay (ELISA) method |
Non-Patent Citations (1)
| Title |
|---|
| 杜文金等: "II型猪链球菌S2株在兔中免疫原性的研究", 《中国预防兽医学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106093439A (en) * | 2016-06-01 | 2016-11-09 | 中国农业科学院兰州兽医研究所 | A kind of Streptococcus suis indirect hemagglutination detection kit and application thereof |
| CN106093439B (en) * | 2016-06-01 | 2018-05-15 | 中国农业科学院兰州兽医研究所 | A kind of Streptococcus suis indirect hemagglutination detection kit and its application |
| CN114755406A (en) * | 2022-03-28 | 2022-07-15 | 南京农业大学 | Streptococcus suis 9 type ELISA antibody detection method and kit thereof |
| CN114755406B (en) * | 2022-03-28 | 2023-10-24 | 南京农业大学 | Streptococcus suis type9 ELISA antibody detection method and kit thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104407137B (en) | A kind of CSFV velogen strain and low virulent strain differentiate Test paper | |
| Howard et al. | An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to bovine viral diarrhoea virus (BVDV) in cattle sera | |
| CN103048459B (en) | Immune detection reagent for detecting respiratory syncytial virus | |
| CN103278635B (en) | Animal brucellosis competitive ELISA (enzyme linked immunosorbent assay) antibody detection kit | |
| CN105296441B (en) | A kind of bovine viral diarrhea virus strain and its application | |
| CN110642945B (en) | General type foot-and-mouth disease virus structural protein antibody and blocking ELISA detection kit thereof | |
| CN102305859B (en) | Activated toxic antibody kit for detecting porcine reproductive and respiratory syndrome virus | |
| CN103308684A (en) | ELISA (enzyme-linked immuno sorbent assay) detection kit of porcine pseudorabies virus IgM antibody | |
| CN103308688A (en) | ELISA (Enzyme-Linked Immunosorbent Assay) detection kit for porcine circovirus type 2 IgM antibody and preparation method of ELISA detection kit | |
| CN101672849A (en) | Deer epidemic hemorrhage competitive enzyme-linked immunosorbent assay kit and preparation method and use thereof | |
| CN104569428B (en) | A kind of enterovirns type 71 killed vaccine antigen enzyme-linked immunologic detecting kit | |
| CN106771182A (en) | Brucella abortus IgM subclass antibodies indirect ELISA testing kits | |
| CN101348777B (en) | Influenza A virus ELISA nucleoprotein capture antigen diagnose reagent kit and special monoclonal antibody therefor | |
| CN106596958B (en) | Brucellosis CF-ELISA antibody assay kits | |
| CN103698514B (en) | Detect the ELISA kit of pig Lawsonia intracellularis antibody | |
| CN101936997A (en) | Human anti-rabies virus IgG antibody ELISA test kit | |
| CN105348386B (en) | The monoclonal antibody cocktail and detection kit of the O-shaped virus of resistant to foot and mouth disease | |
| CN102759619A (en) | Enzyme-linked immunosorbent assay kit of streptococcus suis serotype 2 capsular polysaccharide antibody and application of enzyme-linked immunosorbent assay kit | |
| CN102286093A (en) | Anaplasma phagocytophilum antiagen and kit containing same | |
| CN102721812B (en) | Indirect ELISA (enzyme-linked immuno-sorbent assay) kit for detecting nephropathogenic avian infectious bronchitis virus and antibody thereof | |
| CN102809653A (en) | Preparation and application of ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting novel bunyavirus antigen | |
| CN103293311A (en) | Indirectly competitive ELISA (Enzyme Linked Immunosorbent Assay) immune kit for detecting porcine reproductive and respiratory syndrome virus | |
| CN103063838A (en) | Primer and kit for identifying foot-and-mouth disease and immunity of entry animal | |
| CN105838682A (en) | Specific monoclonal antibody cell strain for bovine viral diarrhea virus and applications of specific monoclonal antibody cell strain | |
| Sugiyama et al. | An immune adherence assay for discrimination between etiologic agents of hemorrhagic fever with renal syndrome |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20121031 |