CN101571539A - Elisa kit for detecting cephalo-type medicine and application thereof - Google Patents
Elisa kit for detecting cephalo-type medicine and application thereof Download PDFInfo
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Abstract
The invention provides an elisa kit for detecting cephalo-type medicine, containing: an ELIAS plate peridium of which is provided with peridium source, an enzyme label, cephalo-type medicine specific antibody working solution (contained when the peridium on the ELIAS plate is antigen and the enzyme label is enzyme label anti antibody or when the peridium on the ELIAS plate is anti antibody and the enzyme label is enzyme label antigen), ceftiofur standard solution, substrate developer, stop solution, concentrated cleaning solution and concentrated complex solution. The invention also discloses a method applying the elisa kit for detecting cephalo-type medicine, including: firstly sample preprocessing is carried out, then the elisa kit is used for detection, and finally the detection result is analyzed. The elisa kit provided by the invention can be applied to detecting residual quantity of cephalo-type medicine in samples such as animal tissue (chicken, pork, fish and shrimp) and milk, etc, the operation is easy, the cost is low, the sensitivity is high, and the elisa kit is applied to on-site supervision and numerous sample screening.
Description
Technical field
The present invention relates to the enzyme linked immunosorbent detection technology, be specifically related to a kind of enzyme linked immunological kit that is used to detect cephalosporins medicine, it is particularly suitable for the residual detection of cephalosporins medicine in chicken, pork, fish, shrimp tissue and the milk sample.
Technical background
Cephalosporins medicine is a kind of microbiotic of wide spectrum, has good antibacterial activity and pharmacokinetics characteristics, and is effective to the bacterium of many Gram-positives and Gram-negative and product beta-lactamase.Cephalosporins medicine is to adopt the mode killing bacteria that destroys bacteria cell wall.Because the check and analysis method sample pre-treatment of using at present and measurement operation is loaded down with trivial details and expense is higher makes to apply to be restricted.
The conventional sense method of cephalosporins medicine residual quantity mainly contains high performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry method (LC-MS/MS), paper chromatography etc., because complicated instrument and equipment and loaded down with trivial details process and to reviewer's high professional qualification requirement are not suitable for the examination of on-site supervision and great amount of samples.
Summary of the invention
The objective of the invention is to provides a kind of enzyme linked immunological kit that cephalosporins medicine detects that is used at above-mentioned deficiency, and it is simple to operate, is fit to the screening of on-the-spot batch samples.
Kit of the present invention, it contains:
(1) is coated with the ELISA Plate (coating antigen is antigen, antibody or antiantibody) of coating antigen;
(2) enzyme labeling thing (being enzyme labeling haptens, enzymic-labelled antibody or enzyme labeling antiantibody);
(3) cephalosporins medicine specific antibody working fluid (when envelope antigen on the ELISA Plate and enzyme labeling thing are to contain when bag is the enzyme labeling haptens by antiantibody and enzyme labeling thing on enzyme labeling antiantibody or the ELISA Plate);
(4) Ceftiofur standard solution;
(5) substrate colour developing liquid;
(6) stop buffer;
(7) concentrated cleaning solution;
(8) concentrate redissolution liquid.
Cephalosporins medicine of the present invention comprises that Ceftiofur, ceftriaxone, CTX etc. have the medicine of common molecular skeleton pharmacologically active.
The enzyme linked immunological kit of detection cephalosporins medicine provided by the present invention comprises cephalosporins medicine specific antibody working fluid and pre-coated former ELISA Plate of bag and enzyme labeling thing working fluid; Described enzyme labeling thing is the antiantibody of enzyme labeling, enzyme labeling cephalosporins medicine haptens or enzyme labeling cephalosporins medicine specific antibody; Described antiantibody is sheep anti mouse antiantibody or goat-anti rabbit antiantibody.
Described cephalosporins medicine haptens is the Ceftiofur n-caproate haptens that obtains by Ceftiofur and the amino n-caproic acid reaction of 6-.
The marker enzyme of described enzyme labeling thing is horseradish peroxidase or alkaline phosphatase, wherein preferred horseradish peroxidase; The sheep anti mouse antiantibody of enzyme labeling or goat-anti rabbit antiantibody adopt glutaraldehyde method or sodium periodate method that marker enzyme and antiantibody are carried out coupling and obtain; Enzyme labeling cephalosporins medicine haptens adopts mixed acid anhydride or carbodiimide method that marker enzyme and cephalosporins medicine hapten conjugation are obtained; The enzyme labeling specific antibody adopts glutaraldehyde method or sodium periodate method that marker enzyme and specific antibody coupling are obtained, horseradish peroxidase can adopt several different methods of the prior art that itself and antiantibody are carried out coupling, as glutaraldehyde method, sodium periodate method etc., the present invention creates through long-term work the sodium periodate method is improved, make it save time, reduce the concentration rate of horseradish peroxidase (HRP) and antiantibody, saved starting material.
Described cephalosporins medicine specific antibody can be cephalosporins medicine monoclonal antibody or cephalosporins medicine polyclonal antibody; They all are to obtain as immunogene with the conjugate that cephalosporins medicine haptens and carrier protein adopt mixed anhydride method or carbodiimide method to obtain; Described cephalosporins medicine polyclonal antibody can be mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source antibody, described cephalosporins medicine monoclonal antibody is preferably the cephalosporins medicine mouse monoclonal antibody, and described cephalosporins medicine polyclonal antibody is preferably the cephalosporins medicine rabbit polyclonal antibody.
Described cephalosporins medicine monoclonal antibody is preferably the monoclonal antibody of the monoclonal hybridoma strain C-4-3CGMCC No.3111 secretion of cephalosporins medicine.
Described cephalosporins medicine monoclonal hybridoma strain C-4-3CGMCC No.3111 (classification name: the monoclonal antibody hybridoma cell strain of cephalosporins medicine) be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 05 26th, 2009 and (be called for short CGMCC, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101).
Above antibody all can use the conjugate of cephalosporins medicine haptens and carrier protein as immunogen preparing.Described carrier protein can be common carrier albumen such as mouse haemocyanin, thyroprotein (BCG), bovine serum albumin, rabbit anteserum albumen, human albumin, ovalbumin, hemocyanin and fibrinogen; The conjugate of described cephalosporins medicine haptens and carrier protein can obtain by cephalosporins medicine haptens and carrier protein are carried out coupling with mixed anhydride method or carbodiimide method.
For more convenient on-site supervision and great amount of samples examination, described kit also comprises Ceftiofur standard solution, developer, stop buffer, concentrated cleaning solution, concentrates redissolution liquid.
When marker enzyme is horseradish peroxidase, developer is made up of colour developing liquid A liquid and colour developing liquid B liquid, colour developing liquid A liquid is hydrogen peroxide or urea peroxide, and described colour developing liquid B liquid is o-phenylenediamine or tetramethyl benzidine, and stop buffer is sulfuric acid or the hydrochloride buffer of 1～2mol/L; When marker enzyme was alkaline phosphatase, colour developing liquid was for being 1～2mol/L sodium hydroxide solution to nitro phosphate buffer, stop buffer.
Described concentrated cleaning solution is to contain the phosphate buffer of pH value for 7.5-8.2,0.8-1.2% Tween-20,0.01-0.02 ‰ thimerosal antiseptic, 0.1-0.2mol/L, and described number percent is weight percentage.
Described concentrated redissolution liquid is to contain 8-12% casein, the pH value phosphate buffer for 7.3-7.8,0.1-0.3mol/L, and described number percent is weight percentage.
Wherein used bag is cushioned the preferred pH 9.4-9.7 of liquid in the ELISA Plate preparation, 0.1-0.3mol/L carbonate is towards liquid, used confining liquid is to contain the carbonate buffer solution of the pH value of 8-10% ovalbumin, 0.1-0.3% Tween-20 and 4-7% sucrose for the 0.02-0.03mol/L of 8.9-9.4, and described number percent is weight percentage.
The preparation process of ELISA Plate is among the present invention: be cushioned liquid with bag coating antigen is diluted to 0.2～0.3 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubation 2h or 4 ℃ spend the night, and the coating buffer that inclines is with cleansing solution washing 2 times, each 30 seconds, pat dry, in every hole, add 150～200 μ l confining liquids then, 37 ℃ of incubation 1～2h, inclining, liquid pats dry in the hole, and preserve with the vacuum seal of aluminium film dry back.
Immunogenic building-up process is among the present invention:
The cephalosporins medicine haptens synthesizes (synthetic route such as Fig. 2)
The cephalosporins medicine haptens is the Ceftiofur n-caproate haptens that obtains by Ceftiofur (structural formula such as Fig. 1) and the amino n-caproic acid reaction of 6-, and concrete reactions steps is as follows:
(1) gets Ceftiofur 10g and be dissolved among the 50ml DMSO, add the reaction of 1ml isobutyl chlorocarbonate stirring at room and spend the night.Add the amino n-caproic acid of 6-again and transfer pH value to 8.5, be warming up to 45 ℃, reaction 12h.
(2) reaction product is used the Rotary Evaporators evaporate to dryness,, transferred pH value to 3 with 2mol/L hydrochloric acid again with the dissolution of sodium hydroxide of 100ml 1mol/L, centrifugal.
(3) get precipitation, the hydrochloric acid with 0.1mol/L washs 4～5 times, with the amount of ethyl acetate dissolving, adds 2ml acetone again.
(4) after chromatographic silica gel post purifying, with ethyl acetate and acetone, methyl alcohol=7: 2: 1, wash-out, flow down at nitrogen again and dry up, the pressed powder that obtains is Ceftiofur n-caproate haptens.
Adopt carbodiimide method to carry out coupling Ceftiofur n-caproate haptens and carrier protein and obtain immunogene.
Immunogenic concrete preparation method:
(1) get cephalosporins medicine haptens 16.2mg adding 1mlDMF and dissolve fully, get the EDC of 30.5mg, the water of 1ml dissolves the back fully and adds under stirring condition in the cephalosporins medicine haptens solution, and room temperature reaction 24h obtains A liquid;
2, get among the PBS of 39.6mgBSA adding 5ml pH7.4 0.01mmol/L, the vibration dissolving prepares B liquid;
3, under magnetic agitation, reactant liquor A is dropwise joined among the reactant liquor B, reseal beaker, stirring reaction 6h under the room temperature;
4, reactant liquor is packed in the bag filter, dislysate is changed in 4 ℃ of dialysis three days every day three times, and dialysis finishes, and packing is standby in-20 ℃ of preservations.
The preparation of cephalosporins medicine antibody
1, the preparation of cephalosporins medicine mouse monoclonal antibody
The animal immune program: adopting the Balb/c mouse as immune animal, is immunogene with cephalosporins medicine haptens and carrier protein couplet thing, obtain preferably polyvalent antibody after, the taking-up spleen carries out Fusion of Cells.
Fusion of Cells and cloning: get immune Balb/c mouse boosting cell and SP2/0 myeloma cell and merge, the screening cell line is up to the hybridoma cell strain that obtains the stably excreting monoclonal antibody.
2, the preparation of cephalosporins medicine rabbit polyclonal antibody
Adopting new zealand white rabbit as immune animal, is that immunogene is carried out immunity to new zealand white rabbit with cephalosporins medicine haptens and carrier protein couplet thing, and repeatedly the immunity back is measured serum antibody titer and obtained polyclonal antibody.
The preparation process of antiantibody of the present invention: the sheep anti mouse antiantibody be with sheep as immune animal, be that immunogene is carried out immunity to anosis thalline sheep with mouse source antibody, obtain the sheep anti mouse antiantibody; Goat-anti rabbit antiantibody be with sheep as immune animal, be that immunogene is carried out immunity to anosis thalline sheep with rabbit source antibody, obtain goat-anti rabbit antiantibody.
Ceftiofur standard solution in the kit of the present invention: 6 bottles of standard solutions, 0 μ g/L, 0.5 μ g/L, 1.5 μ g/L, 4.5 μ g/L, 13.5 μ g/L, 40.5 μ g/L.
Detection principle of the present invention is:
When on capillary strip, wrapping in advance by cephalo-type drug coupling antigen, after adding sample solution or standard solution, add cephalosporins medicine specific antibody solution again, the cephalosporins medicine coupled antigen competition cephalosporins medicine specific antibody of bag quilt on residual cephalosporins medicine and the ELISA Plate in the sample, add the enzyme labeling antiantibody and carry out amplification, with the colour developing of colour developing liquid, the content of sample light absorption value and cephalosporins medicine is negative correlation, relatively can draw the residual quantity of cephalosporins medicine in the sample with typical curve.Simultaneously according to the depth of color on the ELISA Plate, with the comparison of the Ceftiofur standard solution color of the series concentration concentration range of cephalosporins medicine residual quantity in the judgement sample roughly.
When on capillary strip, wrapping in advance by cephalo-type drug specificity antibody, after adding sample solution or standard solution, add enzyme labeling cephalosporins medicine haptens solution again, cephalosporins medicine and enzyme-labelled antigen residual in the sample are competed the cephalosporins medicine specific antibody that is coated on the ELISA Plate, with the colour developing of colour developing liquid, the content of sample light absorption value and cephalosporins medicine is negative correlation, relatively can draw the residual content of cephalosporins medicine in the sample with typical curve.Simultaneously according to the depth of color on the ELISA Plate, with the comparison of the Ceftiofur standard solution color of the series concentration concentration range of cephalosporins medicine residual quantity in the judgement sample roughly.
When on capillary strip, wrapping in advance by cephalo-type drug coupling antigen, after adding sample solution or standard solution, add enzyme labeling cephalosporins medicine specific antibody solution again, the cephalosporins medicine coupled antigen competition cephalosporins medicine specific antibody of bag quilt on residual cephalosporins medicine and the ELISA Plate in the sample, with the colour developing of colour developing liquid, the content of sample light absorption value and cephalosporins medicine is negative correlation, relatively can draw the residual content of cephalosporins medicine in the sample with typical curve.Simultaneously according to the depth of color on the ELISA Plate, with the comparison of the Ceftiofur standard solution color of the series concentration concentration range of cephalosporins medicine residual quantity in the judgement sample roughly.
When on capillary strip, wrapping in advance by antiantibody, after adding the cephalosporins medicine antibody incubation, after adding sample solution or standard solution, add enzyme labeling cephalosporins medicine coupled antigen solution again, cephalosporins medicine and enzyme labeling cephalosporins medicine coupled antigen residual in the sample are competed anti-cephalosporins medicine specific antibody, with colour developing liquid colour developing, the content of sample light absorption value and cephalosporins medicine is negative correlation, relatively can draw the residual content of cephalosporins medicine in the sample with typical curve.Simultaneously according to the depth of color on the ELISA Plate, with the comparison of the Ceftiofur standard solution color of the series concentration concentration range of cephalosporins medicine residual quantity in the judgement sample roughly.
The present invention also provides a kind of method of using above-mentioned enzyme linked immunological kit monitoring cephalosporins medicine, and it comprises step:
(1) sample pre-treatments;
(2) detect with kit;
(3) analyzing and testing result.
The pre-treatment of sample mainly is for acquisition cephalosporins medicine solution from sample, thereby is used for follow-up detection.Be the pre-treating method of common several samples below:
1, chicken, pork, fish, shrimp sample pre-treating method
Take by weighing the equal pledge of 2.0 ± 0.05g to 50ml polystyrene centrifuge tube, add 8ml and extract working fluid, vibration 5min, more than the 3000g, centrifugal 5min; Get the 1ml supernatant to the clean glass test tube of 10ml, flow down in 50～60 ℃ of water-bath nitrogen and dry up; Add the 1ml normal hexane,, add 1ml redissolution liquid again with vortex instrument whirling motion 30s dissolving dried residue, with vortex instrument whirling motion 30s, the above centrifugal 5min of 3000g; Remove upper strata normal hexane phase, take off layer water 50 μ l and be used for analyzing.
2, milk sample pre-treating method
Get 100 μ l fresh milks in the 2ml centrifuge tube, add 900 μ l redissolution working fluid, mixing is got 50 μ l and is used for analyzing.
When detecting with kit among the present invention: when coating antigen is the cephalosporins medicine coupled antigen, adding standard solution or sample solution add antibody again in the ELISA Plate micropore, washing pats dry behind the incubation, add enzyme mark antiantibody again, washing pats dry behind the incubation, colour developing, termination are measured absorbance with microplate reader; When coating antigen was the cephalosporins medicine coupled antigen, adding standard solution or sample solution added enzymic-labelled antibody again in the ELISA Plate micropore, and washing pats dry behind the incubation, and colour developing, termination are measured absorbance with microplate reader; When coating antigen was the cephalosporins medicine specific antibody, adding standard solution or sample solution added enzyme labeling cephalosporins medicine haptens again in the ELISA Plate micropore, and washing pats dry behind the incubation, and colour developing, termination are measured absorbance with microplate reader; When coating antigen is antiantibody, in the ELISA Plate micropore, add cephalosporins medicine antibody, washing pats dry behind the incubation, add enzyme mark cephalosporins medicine haptens after adding standard solution or sample solution again, washing pats dry behind the incubation, and colour developing, termination are measured absorbance with microplate reader.
The testing result analytic process is among the present invention: the absorbance mean value (B) of standard solution of using each concentration that is obtained is divided by the absorbance (B of first standard solution (0 standard)
0) multiply by 100% again, i.e. percentage absorbance.Computing formula is:
Percentage absorbance (%)=(B/B
0) * 100%
Semilog value with the concentration (μ g/L) of Ceftiofur standard solution is an X-axis, and the percentage absorbance is a Y-axis, the drawing standard curve map.With the percentage absorbance of same way calculation sample solution, the concentration of corresponding each sample then can be read the residual quantity of cephalosporins medicine the sample from typical curve.
The analysis of testing result also can be adopted regression equation method among the present invention, calculates sample solution concentration.
The analysis of testing result can also utilize computer professional software among the present invention, the be more convenient for express-analysis of a large amount of samples of this method, and whole testing process only needed to finish in 75 minutes.
The enzyme linked immunological kit that the present invention detects cephalosporins medicine mainly adopts the residual quantity of cephalosporins medicine in the qualitative or detection by quantitative sample of indirect competitive ELISA method; Pre-treatment requirement to sample is low, and sample pretreatment process is simple, simultaneously the fast detecting batch samples; Adopt the cephalosporins medicine monoclonal antibody of high specific, main agents provides with the form of working fluid, and the method for inspection is convenient and easy, has specificity height, highly sensitive, characteristics such as degree of accuracy is high, accuracy height.Enzyme linked immunological kit of the present invention, simple in structure, easy to use, low price, carrying convenience, detection method be efficient, accurate, easy, be suitable for the qualitative, quantitative of batch samples screening.Kit of the present invention will play a significant role in the detection of cephalosporins medicine.
Description of drawings
Fig. 1: Ceftiofur medicines structure figure
Fig. 2: cephalosporins medicine haptens synthetic route chart
Fig. 3: the conjugate with cephalosporins medicine haptens and carrier protein is the canonical plotting of the kit of coating antigen.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment only are used to illustrate the present invention, and be not used for limiting the scope of the invention.
The preparation of embodiment 1 reagent constituents
1. antigen is synthetic
A, immunogene are synthesized
The cephalosporins medicine haptens synthesizes (synthetic route such as Fig. 2)
Be the Ceftiofur n-caproate haptens that obtains by Ceftiofur (structural formula such as Fig. 1) and the amino n-caproic acid reaction of 6-, concrete reactions steps is as follows:
(1) gets Ceftiofur 10g and be dissolved among the 50ml DMSO, add the reaction of 1ml isobutyl chlorocarbonate stirring at room and spend the night, add the amino n-caproic acid of 6-again and transfer pH value to 8.5, be warmed up to 45 ℃ of reactions 12 hours.
(2) reaction product is used the Rotary Evaporators evaporate to dryness,, transferred pH value to 3 with 2mol/L hydrochloric acid again with the dissolution of sodium hydroxide of 100ml 1mol/L, centrifugal.
(3) get precipitation, the hydrochloric acid with 0.1mol/L washs 4～5 times, with the amount of ethyl acetate dissolving, adds 2ml acetone again.
(4) after chromatographic silica gel post purifying, with ethyl acetate and acetone, methyl alcohol=7: 2: 1, wash-out, flow down at nitrogen again and dry up, the pressed powder that obtains is Ceftiofur n-caproate haptens.
Adopt carbodiimide method to carry out coupling cephalosporins medicine haptens and bovine serum albumin(BSA) and obtain immunogene.
Immunogenic preparation process:
(1) get cephalosporins medicine haptens 16.2mg adding 1mlDMF and dissolve fully, get the EDC of 30.5mg, after the water of 1ml dissolves fully, add under stirring condition in the cephalosporins medicine haptens solution, room temperature reaction 24h obtains A liquid;
2, get among the PBS of 39.6mgBSA adding 5ml pH7.4 0.01mmol/L, the vibration dissolving prepares B liquid;
3, under magnetic agitation, reactant liquor A is dropwise joined among the reactant liquor B, reseal beaker, stirring reaction 6h under the room temperature;
4, reactant liquor is packed in the bag filter, dislysate is changed in 4 ℃ of dialysis three days every day three times, and dialysis finishes, and packing is standby in-20 ℃ of preservations.
B. the preparation of coating antigen cephalosporins medicine coupled antigen
Cephalosporins medicine haptens and ovalbumin coupling are obtained coating antigen.
The preparation process of coating antigen:
1, take by weighing cephalosporins medicine haptens 11.9mg and be dissolved among the 1mlDMF, stirring fully gets A liquid after the dissolving;
2, get 50mgOVA and add 2ml pH7.5 0.1mol/L phosphate buffer, vibration dissolve B liquid;
3, put on the constant temperature blender with magnetic force, behind A drop adding B liquid, in mixed liquor, slowly add 2.5% glutaraldehyde 50ul immediately, continue to stir stirring reaction 1h under the room temperature;
4, reactant liquor is changed dislysate every day three times with pH7.5 0.1mol/l phosphate buffer dialysis three days.Dialysis finishes, and packing is standby in-20 ℃ of preservations.
MONOCLONAL ANTIBODIES SPECIFIC FOR
A. animal immune
Immunogene is injected in the Balb/c mouse body, and immunizing dose is 100 μ g/, makes it produce polyclonal antibody serum.
B. Fusion of Cells and cloning
After the mice serum measurement result is higher, get its splenocyte, merge, adopt indirect competitive ELISA to measure cell conditioned medium liquid, screen positive hole in 9: 1 ratios and SP2/0 myeloma cell.Utilize limiting dilution assay that cloning is carried out in positive hole, up to the hybridoma cell strain that obtains secrete monoclonal antibody.
Obtain the monoclonal hybridoma strain C-4-3CGMCC No.3111 of cephalosporins medicine through screening.The monoclonal hybridoma strain of cephalosporins medicine can be endless generation cephalosporins medicine specific antibody, and this antibody specificity is at cephalosporins medicine, sensitivity can reach 0.5 μ g/L.
C. cell cryopreservation and recovery
The monoclonal hybridoma strain of cephalosporins medicine is made 1 * 10 with cryopreserving liquid
6The cell suspension of individual/ml is in the medium-term and long-term preservation of liquid nitrogen.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
D. the production of monoclonal antibody and purifying
The Balb/c mouse peritoneal is only injected sterilization paraffin oil 0.5ml/, the monoclonal hybridoma strain 5 * 10 of 7 days pneumoretroperitoneum injection cephalosporins medicines
7Individual/as only, to gather ascites after 7 days.Carry out the ascites purifying with sad-saturated ammonium sulfate method ,-20 ℃ of preservations.
2. Polyclonal Antibody Preparation
Adopt new zealand white rabbit as immune animal, with cephalosporins medicine haptens and bovine serum albumin(BSA) conjugate is immunogene, immunizing dose is 1.5mg/kg, when head exempts from the Fu Shi of immunogene and equivalent helped fully and be mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, getting the same dose immunogene 3～4 weeks adds equivalent incomplete Freund mixing and emulsifying at interval, and booster immunization is once, immunity is 5 times altogether, does not add adjuvant for the last time.Serum antibody titer is measured in last immunity blood sampling after 10 days, and heart is taken a blood sample, and obtains the polyclonal antibody of purifying with ammonium sulfate precipitation.
3. the preparation process of sheep anti mouse antiantibody: as immune animal, is that immunogene to pathogen-free domestic sheep carry out immunity with mouse source antibody with sheep, obtains the sheep anti mouse antiantibody; The preparation of goat-anti rabbit antiantibody: as immune animal, is that immunogene to pathogen-free domestic sheep carry out immunity with rabbit source antibody with sheep, obtains goat-anti rabbit antiantibody.
4. the preparation of ELISA Plate
Be cushioned liquid with bag cephalosporins medicine coupled antigen, antibody or antiantibody are diluted to 0.2 μ g/ml, every hole adds 100 μ l, 4 ℃ are spent the night, and the coating buffer that inclines washs 2 times with the concentrated cleaning solution that dilutes 20 times, each 30 seconds, pat dry, and then add 200 μ l confining liquids in every hole, 37 ℃ of incubation 2h, the liquid in the hole that inclines, preserve with the vacuum seal of aluminium film dry back.
5. the preparation of enzyme labeling sheep anti mouse antiantibody
Adopt the sodium periodate method after improveing to carry out coupling antiantibody and horseradish peroxidase (HRP).The molar concentration rate of enzyme and antiantibody is 4: 1 in traditional sodium periodate method requirement reflection system; Because horseradish peroxidase produces many sites that combine with antiantibody under the effect of strong oxidation, Huo Hua horseradish peroxidase molecule has served as the bridge that connects each molecule like this, reduced the enzymatic activity of enzyme labeling thing, make in the conjugate of preparation and be mixed with many condensates, in order to address this problem, we improve traditional method, that is:
1) saved amino closed process, because can produce self amino amino reality that connects seldom.
2) reduced horseradish peroxidase: the volumetric molar concentration ratio to 2 of antiantibody: 1, the method after the improvement is easier than traditional method, and the loss of the activity of enzyme is reduced.
Embodiment 2 detects the establishment of the enzyme linked immunological kit of cephalosporins medicine
Set up the enzyme linked immunological kit that detects cephalosporins medicine, make it comprise following component:
(1) bag is by the ELISA Plate of cephalo-type drug coupling antigen;
(2) the sheep anti mouse antiantibody of usefulness horseradish peroxidase-labeled;
(3) cephalosporins medicine monoclonal antibody working fluid;
(4) the Ceftiofur standard solution is 6 bottles, and concentration is respectively 0 μ g/L, 0.5 μ g/L, 1.5 μ g/L, 4.5 μ g/L, 13.5 μ g/L, 40.5 μ g/L;
(5) substrate colour developing liquid is made up of A liquid and B liquid, and substrate colour developing liquid A liquid is urea peroxide, substrate colour developing liquid B liquid tetramethyl benzidine;
(6) stop buffer is a 2mol/L hydrochloric acid;
(7) concentrated cleaning solution is that the pH value is the phosphate buffer of 7.5-8.2,0.8-1.2% Tween-20,0.01-0.02 ‰ thimerosal antiseptic, 0.1-0.2mol/L, and described number percent is weight percentage.
(8) concentrate to redissolve liquid be contain the 8-12% casein, pH value is the phosphate buffer of 7.3-7.8,0.1-0.3mol/L, described number percent is weight percentage.
The detection of cephalosporins medicine in embodiment 3 samples
1. sample pre-treatments
A) animal tissue (chicken, pork, fish, shrimp etc.)
Take by weighing the equal pledge of 2.0 ± 0.05g to 50ml polystyrene centrifuge tube, add 8ml and extract working fluid, vibration 5min, more than the 3000g, centrifugal 5min; Get the 1ml supernatant to the clean glass test tube of 10ml, flow down in 50～60 ℃ of water-bath nitrogen and dry up; Add the 1ml normal hexane,, add 1ml redissolution liquid again with vortex instrument whirling motion 30s dissolving dried residue, with vortex instrument whirling motion 30s, the above centrifugal 5min of 3000g; Remove upper strata normal hexane phase, take off layer water 50 μ l and be used for analyzing.
B) milk sample
Get 100 μ l fresh milks in the 2ml centrifuge tube, add 900 μ l redissolution working fluid, mixing is got 50 μ l and is used for analyzing.
2. detect with kit
In the ELISA Plate micropore that is coated with the cephalosporins medicine coupled antigen, add Ceftiofur standard solution or sample solution 50 μ l, add cephalosporins medicine monoclonal antibody working fluid 50 μ l again, with cover plate mould shrouding, react 30min in the 4-8 ℃ of constant temperature oven, pour out liquid in the hole, every hole adds 250 μ l cleansing solutions, pours out liquid in the hole behind the 30s, so repetitive operation is washed plate 5 times, pats dry with thieving paper.The sheep anti mouse antiantibody working fluid 100 μ l that add horseradish peroxidase-labeled, react 30min in 25 ℃ of constant temperature ovens, pour out liquid in the hole, repeat to wash the plate step, every hole adds substrate colour developing liquid A liquid urea peroxide, substrate colour developing liquid B liquid tetramethyl benzidine (TMB), the mixing that vibrates gently, 25 ℃ of constant temperature oven lucifuge colour developing 15min, every hole adds 2mol/L stop buffer hydrochloric acid 50 μ l, the mixing that vibrates gently at the 450nm place, is measured every hole absorbance (OD value) with the microplate reader wavelength set.
3. testing result analysis
With the absorbance mean value (B) of the standard solution of each concentration that is obtained absorbance (B divided by first standard solution (0 standard)
0) multiply by 100% again, obtain the percentage absorbance.Semilog value with Ceftiofur standard items concentration (μ g/L) is an X-axis, and the percentage absorbance is a Y-axis, the drawing standard curve map, as shown in Figure 3.With the percentage absorbance of same way calculation sample solution, the concentration of corresponding each sample, the residual quantity that then can read cephalosporins medicine from typical curve.
The test of experimental example 1 standard items precision:
Respectively extract a collection of ELISA Plate out respectively from the ELISA Plate of three different time period preparations, every batch is extracted 10 kits, and every plate is extracted 20 micropores out, measures the absorbance of 4.5 μ g/L standard solution, calculates the coefficient of variation.
The repeatable test of table 1 standard (CV%)
Can draw by above-mentioned test findings, every batch of each 10 standard items coefficient of variation of kit meet precision and are less than or equal to 25% regulation between 3.4%-9.6%.
Experimental example 2 sample precision and accuracy tests
1. sample precision test:
With the Ceftiofur of 10 μ g/L concentration to chicken, pork, fish, shrimp, the Ceftiofur of 20 μ g/L concentration adds mensuration respectively to milk, gets each five of the kits of three different batches respectively, and each concentration repeats 5 times, calculate the coefficient of variation respectively, the results are shown in Table 2～6.
The repeatable test of table 2 chicken sample
The repeatable test of table 3 pork sample
The repeatable test of table 4 fish sample
The repeatable test of table 5 shrimp sample
The repeatable test of table 6 milk sample
The result shows the Variation Lines number average of chicken, pork, fish, shrimp, milk sample between 3.1%-12.7%, has met " Ministry of Agriculture's file " farming doctor [2005] No. 17 annex 2 kits and has put on record with reference to the 4th precision standard in the judgment criteria.
B. sample accuracy test
Respectively chicken, pork, fish, shrimp sample are added recovery test with 10 μ g/kg, 20 μ g/kg Ceftiofurs, with 20 μ g/kg, 40 μ g/kg Ceftiofurs the milk sample is added recovery test, each concentration do 4 parallel, accuracy in computation respectively.
The accuracy of table 7 kit
The result shows that chicken, pork, fish, shrimp sample add the recovery between 68.7%-95.2%, and the interpolation recovery of milk sample is between 87.2%-107.5%.
The test of experimental example 3 cross reacting rates:
Selection has 3 kinds of cephalosporins medicines of similar structures and similar functions and measures cross reacting rates, and the typical curve by various medicines obtains its 50% inhibition concentration respectively.Calculate the cross reacting rate of kit with following formula to other medicines.Cross reacting rate is big more, and this kit is just good more to the specificity of the detection of cephalo-type so.
Cross reacting rate (%)=(cause 50% concentration that suppresses Ceftiofur/cause the 50% analog concentration that suppresses) * 100%
The specificity of table 8 kit
Experimental example 4
The kit preservation condition is 2～8 ℃, and through 6 months mensuration, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, cephalosporins medicine added the practical measurement value all within normal range.Consideration has improper preservation condition and occurs in transportation and use, and kit was placed 6 days under 37 ℃ of preservation conditions, carries out the accelerated deterioration experiment, and the result shows that the every index of this kit meets the requirements fully.Consider that the freezing situation of kit takes place, kit was put into-20 ℃ of refrigerators freezing 5 days, measurement result shows that also the every index of kit is normal fully.Can draw kit from above result can preserve more than 6 months at least at 2～8 ℃.
Claims (10)
1, a kind of enzyme linked immunological kit that detects cephalosporins medicine is characterized in that it contains:
(1) is coated with the ELISA Plate of coating antigen;
(2) enzyme labeling thing;
(3) Ceftiofur standard solution;
(4) substrate colour developing liquid;
(5) stop buffer;
(6) concentrated cleaning solution;
(7) concentrate redissolution liquid,
Described coating antigen is cephalosporins medicine antigen, antibody or antiantibody, and described enzyme labeling thing is enzyme labeling cephalosporins medicine antigen, enzyme labeling cephalosporins medicine antibody or enzyme labeling antiantibody,
When envelope antigen on the ELISA Plate and enzyme labeling thing are also to contain cephalosporins medicine specific antibody working fluid when bag is enzyme-labelled antigen by antiantibody and enzyme labeling thing on enzyme labeling antiantibody or the ELISA Plate.
2, kit as claimed in claim 1, it is characterized in that described cephalosporins medicine antigen is to be obtained by cephalosporins medicine haptens and carrier protein couplet, described cephalosporins medicine antibody is to be obtained by described antigen preparation, and described cephalosporins medicine haptens is the Ceftiofur n-caproate haptens that obtains by Ceftiofur and the amino n-caproic acid reaction of 6-.
3, kit as claimed in claim 1 or 2 is characterized in that described cephalosporins medicine antibody is monoclonal antibody.
4, kit as claimed in claim 3 is characterized in that described cephalosporins medicine antibody is produced by hybridoma cell strain C-4-3CGMCC No.3111 secretion.
5, kit as claimed in claim 2 is characterized in that described carrier protein is mouse haemocyanin, thyroprotein, bovine serum albumin, rabbit anteserum albumen, human albumin, ovalbumin, hemocyanin or fibrinogen.
6, as claims 1 or 2 described kits, it is characterized in that it is pH9.4-9.7 that used bag is cushioned liquid, 0.1-0.3mol/L carbonate is towards liquid, used confining liquid is to contain the carbonate buffer solution of the pH value of 8-10% ovalbumin, 0.1-0.3% Tween-20 and 4-7% sucrose for the 0.02-0.03mol/L of 8.9-9.4, and described number percent is weight percentage.
7, kit as claimed in claim 1 or 2, the marker enzyme that it is characterized in that described enzyme labeling thing is that horseradish peroxidase or bacterium are extracted alkaline phosphatase, when marker enzyme is horseradish peroxidase, substrate colour developing liquid A liquid is hydrogen peroxide or urea peroxide, substrate colour developing liquid B liquid is o-phenylenediamine or tetramethyl benzidine, and stop buffer is sulfuric acid or the hydrochloride buffer of 1～2mol/L; When the marker enzyme marker enzyme is a bacterium when extracting alkaline phosphatase, substrate colour developing liquid is for being 1～2mol/L NaOH to nitro phosphate buffer, stop buffer.
8, kit as claimed in claim 1 or 2 is characterized in that: concentrated cleaning solution is to contain the phosphate buffer of pH value for 7.5-8.2,0.8-1.2% Tween-20,0.01-0.02 ‰ thimerosal antiseptic, 0.1-0.2mol/L; The liquid that concentrate to redissolve be contain the 8-12% casein, pH value is the phosphate buffer of 7.3-7.8,0.1-0.3mol/L, the concentration of Ceftiofur standard solution is respectively 0 μ g/L, 0.5 μ g/L, 1.5 μ g/L, 4.5 μ g/L, 13.5 μ g/L, 40.5 μ g/L, described number percent is weight percentage.
9, the application of each described kit of claim 1～8 in detecting the cephalo-type medicament residue.
10, the residual method of a kind of test sample cephalosporins medicine comprises step:
(1) sample pre-treatments;
(2) detect with each described kit of claim 1-8;
(3) analyzing and testing result.
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| IL59723A (en) * | 1980-03-27 | 1983-05-15 | Teva Pharma | Determination of antibacterial agents |
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