Detect enzyme linked immunological kit and the application thereof of cephalosporins medicine
Technical field
The present invention relates to the enzyme linked immunosorbent detection technology, be specifically related to a kind of enzyme linked immunological kit for detection of 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 detection analytic approach Sample pretreatment that uses 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 (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 object of the invention is to provides a kind of enzyme linked immunological kit for the cephalosporins medicine detection for 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 ENR-HRP, 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 coated antiantibody and enzyme labeling thing are ENR-HRP on enzyme labeling antiantibody or the ELISA Plate);
(4) Ceftiofur standard solution;
(5) substrate nitrite ion;
(6) stop buffer;
(7) concentrated cleaning solution;
(8) the concentrated liquid that redissolves.
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 ELISA Plate and the enzyme labeling thing working fluid of cephalosporins medicine specific antibody working fluid and pre-coated coating antigen; 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, such as glutaraldehyde method, sodium periodate method etc., the present invention improves the sodium periodate method through long-term labor and creation, 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 And Nomenclature: 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 prepare as immunogene with the conjugate of cephalosporins medicine haptens and carrier protein.Described carrier protein can be the 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, the concentrated liquid that redissolves.
When marker enzyme is horseradish peroxidase, developer is comprised of nitrite ion A liquid and nitrite ion B liquid, nitrite ion A liquid is hydrogen peroxide or urea peroxide, and described nitrite ion 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, nitrite ion was for being 1~2mol/L sodium hydroxide solution to nitro phosphate buffer, stop buffer.
Described concentrated cleaning solution is to contain the pH value to be 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.
Described concentrated redissolution liquid is to contain 8-12% casein, pH value to be the phosphate buffer of 7.3-7.8,0.1-0.3mol/L, and described number percent is weight percentage.
The used preferred pH 9.4-9.7 of coated damping fluid in ELISA Plate preparation wherein, 0.1-0.3mol/L carbonate rushes liquid, used confining liquid is to contain the pH value of 8-10% ovalbumin, 0.1-0.3% Tween-20 and 4-7% sucrose to be the carbonate buffer solution of 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: with coated damping fluid 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, then in every hole, add 150~200 μ l confining liquids, 37 ℃ of incubation 1~2h, inclining, liquid pats dry in the hole, preserves with the vacuum seal of aluminium film after dry.
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 again the amino n-caproic acid of 6-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, with the dissolution of sodium hydroxide of 100ml 1mol/L, transferred pH value to 3 with 2mol/L hydrochloric acid again, centrifugal.
(3) get precipitation, the hydrochloric acid with 0.1mol/L washs 4~5 times, uses a small amount of acetic acid ethyl dissolution, 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, add under stirring condition in the cephalosporins medicine haptens solution after the water of 1ml dissolves fully, room temperature reaction 24h obtains A liquid;
2, get among the PBS of 39.6mgBSA adding 5ml pH7.40.01mmol/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 it is complete to dialyse, and packing saves backup in-20 ℃.
The preparation of cephalosporins medicine antibody
1, the preparation of cephalosporins medicine mouse monoclonal antibody
The animal immune program: adopt the Balb/c mouse as immune animal, take cephalosporins medicine haptens and carrier protein couplet thing as immunogene, 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 until obtain the hybridoma cell strain of stably excreting monoclonal antibody.
2, the preparation of cephalosporins medicine rabbit polyclonal antibody
Adopt new zealand white rabbit as immune animal, as immunogene new zealand white rabbit is carried out immunity take cephalosporins medicine haptens and carrier protein couplet thing, repeatedly measure serum antibody titer after the immunity and obtain polyclonal antibody.
The preparation process of antiantibody of the present invention: the sheep anti mouse antiantibody be with sheep as immune animal, take mouse source antibody as immunogene anosis thalline sheep is carried out immunity, obtain the sheep anti mouse antiantibody; Goat-anti rabbit antiantibody be with sheep as immune animal, take rabbit source antibody as immunogene anosis thalline sheep is carried out immunity, 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 pre-coated cephalosporins medicine coupled antigen on capillary strip, after adding sample solution or standard solution, add again cephalosporins medicine specific antibody solution, coated cephalosporins medicine coupled antigen competition cephalosporins medicine specific antibody on residual cephalosporins medicine and the ELISA Plate in the sample, add the enzyme labeling antiantibody and carry out amplification, develop the color with nitrite ion, 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 rough concentration range of cephalosporins medicine residual quantity in the judgement sample of the comparison of the Ceftiofur standard solution color of series concentration.
When pre-coated cephalosporins medicine specific antibody on capillary strip, after adding sample solution or standard solution, add again enzyme labeling cephalosporins medicine haptens solution, cephalosporins medicine and enzyme-labelled antigen residual in the sample are competed the cephalosporins medicine specific antibody that is coated on the ELISA Plate, develop the color with nitrite ion, 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 rough concentration range of cephalosporins medicine residual quantity in the judgement sample of the comparison of the Ceftiofur standard solution color of series concentration.
When pre-coated cephalosporins medicine coupled antigen on capillary strip, after adding sample solution or standard solution, add again enzyme labeling cephalosporins medicine specific antibody solution, coated cephalosporins medicine coupled antigen competition cephalosporins medicine specific antibody on residual cephalosporins medicine and the ELISA Plate in the sample, develop the color with nitrite ion, 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 rough concentration range of cephalosporins medicine residual quantity in the judgement sample of the comparison of the Ceftiofur standard solution color of series concentration.
When pre-coated antiantibody on capillary strip, after adding the cephalosporins medicine antibody incubation, after adding sample solution or standard solution, add again enzyme labeling cephalosporins medicine coupled antigen solution, cephalosporins medicine and enzyme labeling cephalosporins medicine coupled antigen residual in the sample are competed anti-cephalosporins medicine specific antibody, with nitrite ion 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 rough concentration range of cephalosporins medicine residual quantity in the judgement sample of the comparison of the Ceftiofur standard solution color of series concentration.
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.
Determination mainly is for acquisition cephalosporins medicine solution from sample, thereby is used for follow-up detection.The below is common several determination methods:
1, chicken, pork, fish, shrimp Sample pretreatment 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, with vortex instrument whirling motion 30s dissolving dried residue, add again 1ml redissolution liquid, 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 pretreatment 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, add standard solution in the ELISA Plate micropore or sample solution adds antibody again, washing pats dry behind the incubation, add again enzyme mark antiantibody, washing pats dry behind the incubation, colour developing, termination are measured absorbance with microplate reader; When coating antigen is the cephalosporins medicine coupled antigen, add standard solution in the ELISA Plate micropore or sample solution adds enzymic-labelled antibody again, washing pats dry behind the incubation, colour developing, stops, and measures absorbance with microplate reader; When coating antigen is the cephalosporins medicine specific antibody, add standard solution in the ELISA Plate micropore or sample solution adds enzyme labeling cephalosporins medicine haptens again, washing pats dry behind the incubation, colour developing, stops, and measures 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 again standard solution or sample solution, washing pats dry behind the incubation, and colour developing, termination are measured absorbance with microplate reader.
The Analysis of test results process is among the present invention: use the absorbance mean value (B) of standard solution of each concentration that obtains divided by the absorbance (B of first standard solution (0 standard)
0) multiply by again 100%, i.e. percentage absorbance.Computing formula is:
Percentage absorbance (%)=(B/B
0) * 100%
Take the semilog value of the concentration (μ g/L) of Ceftiofur standard solution as X-axis, the percentage absorbance is 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 from typical curve the residual quantity of cephalosporins medicine the sample.
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 quantitative test sample of indirect competitive ELISA method; Low to the determination requirement, 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, have the specificity height, highly sensitive, degree of accuracy is high, the accuracy high.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 canonical plotting take the conjugate of cephalosporins medicine haptens and carrier protein as 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 for explanation the present invention, and be not used for limiting the scope of the invention.
The preparation of embodiment 1 kit components
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 again the amino n-caproic acid of 6-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, with the dissolution of sodium hydroxide of 100ml 1mol/L, transferred pH value to 3 with 2mol/L hydrochloric acid again, centrifugal.
(3) get precipitation, the hydrochloric acid with 0.1mol/L washs 4~5 times, uses a small amount of acetic acid ethyl dissolution, 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 pH7.40.01mmol/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 it is complete to dialyse, and packing saves backup in-20 ℃.
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 pH7.50.1mol/L phosphate buffer, B liquid is dissolved to get in vibration;
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 pH7.50.1mol/l phosphate buffer dialysis three days.It is complete to dialyse, and packing saves backup in-20 ℃.
The preparation of monoclonal antibody
A. animal immune
Immunogene is injected in the Balb/c Mice Body, and immunizing dose is 100 μ g/, makes it produce polyclonal antibody.
B. Fusion of Cells and cloning
After the mice serum measurement result is higher, get its splenocyte, merge in 9: 1 ratios and SP2/0 myeloma cell, adopt indirect competitive ELISA to measure cell conditioned medium liquid, screen positive hole.Utilize limiting dilution assay that cloning is carried out in positive hole, until obtain the hybridoma cell strain of 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 for 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 cryopreservation tube during recovery, put into immediately 37 ℃ of water-bath middling speeds 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. the preparation of polyclonal antibody
Adopt new zealand white rabbit as immune animal, take cephalosporins medicine haptens and bovine serum albumin(BSA) conjugate as 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 section is got the same dose immunogene and add equivalent incomplete Freunds adjuvant mixing and emulsifying in 3~4 week of interval, and booster immunization once, immunity is 5 times altogether, does not add for the last time adjuvant.Serum antibody titer is measured in last immunity afterwards blood sampling in 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, as immunogene the pathogen-free domestic sheep is carried out immunity take mouse source antibody with sheep, obtain the sheep anti mouse antiantibody; The preparation of goat-anti rabbit antiantibody: as immune animal, as immunogene the pathogen-free domestic sheep is carried out immunity take rabbit source antibody with sheep, obtain goat-anti rabbit antiantibody.
4. the preparation of ELISA Plate
With coated damping fluid 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 is preserved with the vacuum seal of aluminium film after dry.
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 of being combined with antiantibody under the effect of strong oxidation, the horseradish peroxidase molecule of activation 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
Easier than traditional method, 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) ELISA Plate of coated cephalosporins medicine coupled 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) the substrate nitrite ion is comprised of A liquid and B liquid, and substrate nitrite ion A liquid is urea peroxide, substrate nitrite ion B liquid tetramethyl benzidine;
(6) stop buffer is 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) concentrated 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, with vortex instrument whirling motion 30s dissolving dried residue, add again 1ml redissolution liquid, 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 again cephalosporins medicine monoclonal antibody working fluid 50 μ l, 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, repeat operation and wash plate 5 times, pat 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 nitrite ion A liquid urea peroxide, substrate nitrite ion 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. Analysis of test results
With the absorbance mean value (B) of the standard solution of each concentration that the obtains absorbance (B divided by first standard solution (0 standard)
0) multiply by again 100%, obtain the percentage absorbance.Take the semilog value of Ceftiofur standard items concentration (μ g/L) as X-axis, the percentage absorbance is 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 from typical curve cephalosporins medicine.
Experimental example 1 standard items precision test:
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%.
The test of experimental example 2 sample preci-sion and accuracies
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 respectively mensuration to milk, gets respectively each five of the kits of three different batches, and each concentration repeats 5 times, calculate respectively the coefficient of variation, 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, Ceftiofur 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 respectively its 50% inhibition concentration.Calculate kit to the cross reacting rate of other medicines with following formula.Cross reacting rate is larger, and this kit is just better 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 procedure, and kit was placed 6 days under 37 ℃ of preservation conditions, carries out the accelerated deterioration experiment, and the result shows that this kit indices meets the requirements fully.Consider that the freezing situation of kit occurs, kit was put into-20 ℃ of refrigerator freezings 5 days, measurement result shows that also the kit indices is fully normal.Can draw kit from above result can preserve more than 6 months at least at 2~8 ℃.