CN101839918B - Method for detecting spiramycin and special enzyme-linked immunoassay kit thereof - Google Patents
Method for detecting spiramycin and special enzyme-linked immunoassay kit thereof Download PDFInfo
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- CN101839918B CN101839918B CN 200910237822 CN200910237822A CN101839918B CN 101839918 B CN101839918 B CN 101839918B CN 200910237822 CN200910237822 CN 200910237822 CN 200910237822 A CN200910237822 A CN 200910237822A CN 101839918 B CN101839918 B CN 101839918B
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Abstract
The invention discloses a method for detecting spiramycin and a special enzyme-linked immunoassay kit thereof. The enzyme-linked immunoassay kit comprises a spiramycin specific antibody, a coating and an enzyme marker, wherein the spiramycin specific antibody is a monoclonal or polyclonal antibody of spiramycin. The enzyme-linked immunoassay kit has the advantages of simple structure, convenient use, low price, portability, and high-efficiency, accurate, simple and convenient detection method, can be used for on-site monitoring, is suitable for qualitative and quantitative screening of a large number of samples, and is expected to play a significant role in the detection of spiramycin.
Description
Technical field
The present invention relates to a kind of method and special ELISA reagent kit thereof that detects spiramvcin.
Background technology
Spiramvcin belongs to macrolide antibiotics, and staphylococcus, micrococcus scarlatinae, streptococcus pneumonia etc. are had antibacterial action.Be mainly used in anti-livestock and poultry bacterium and mycoplasma infection, also adjuvant capable of using as feed is to promote weightening finish and to improve feed conversion rate.The international food code council, European Union and Japan stipulate that all the maximum residue limit(MRL) value of this medicine is 200 μ g/L; Therefore, in practice, need a kind of degree of accuracy, highly sensitive detection spiramvcin method.
At present, the conventional sense method of spiramycin residues amount mainly contains cylinder plate method, high performance liquid chromatography/mass spectrum/mass spectroscopy, high performance liquid chromatography (HPLC), liquid chromatography/tandem mass spectrum etc. in the animal tissue, but these methods exist that testing process is loaded down with trivial details, instrument and equipment is complicated and to the demanding shortcoming of reviewer's technical ability, be not suitable for the examination of on-site supervision and great amount of samples, apply being restricted.
Summary of the invention
An object of the present invention is to provide a kind of enzyme linked immunological kit that detects spiramvcin.
The enzyme linked immunological kit of detection spiramvcin provided by the present invention comprises spiramvcin specific antibody and coating antigen and enzyme labeling thing; Described coating antigen is conjugate or the antiantibody of spiramvcin haptens and carrier protein; Described enzyme labeling thing is enzyme mark antiantibody or enzyme mark spiramvcin haptens; When described coating antigen was the conjugate of spiramvcin haptens and carrier protein, described enzyme labeling thing was enzyme mark antiantibody; When described coating antigen was antiantibody, described enzyme labeling thing was enzyme mark spiramvcin haptens.
For more convenient on-site supervision and great amount of samples examination, described kit also can comprise spiramvcin standard solution, colour developing liquid, stop buffer, concentrated cleaning solution, concentrate redissolution liquid.
Wherein, described concentrated cleaning solution can be the pH value and is 6.2-6.7, the Tween-20 that contains 0.7-1.0%, the sodium azide of 0.03-0.05 ‰, 0.15-0.2mol/L citrate buffer; It is barbital-sodium chloride damping fluid of 7.1-7.6,0.3mol/L that described concentrated redissolution liquid can be the lowlenthal serum, the pH value that contain 5-8%, and described percentage composition is the weight volumn concentration; When marker enzyme is horseradish peroxidase, described colour developing liquid is made up of colour developing liquid A liquid and colour developing liquid B liquid, colour developing liquid A liquid can be hydrogen peroxide or urea peroxide, and colour developing liquid B liquid can be o-phenylenediamine or tetramethyl benzidine, and stop buffer can be 1-2mol/L sulfuric acid or hydrochloric acid solution; When marker enzyme was alkaline phosphatase, developer can be the nitro phosphate buffer, and stop buffer can be the 2mol/L sodium hydroxide solution.
Described spiramvcin specific antibody is spiramvcin polyclonal antibody or spiramvcin monoclonal antibody; They all are that conjugate with spiramvcin haptens and carrier protein obtains as immunogene; Described carrier protein can be thyroprotein, bovine serum albumin, mouse haemocyanin, human albumin, rabbit anteserum albumen, hemocyanin, fibrinogen or ovalbumin etc.
Spiramvcin is small-molecule substance, has only immunoreactivity, does not have immunogenicity, can not bring out body and produce immune response, must with the macromolecular carrier albumen coupling after just have immunogenicity.Therefore with reaction obtains the spiramvcin haptens with succinic anhydride again after spiramvcin and the oxammonium hydrochloride reaction, given prominence to the characteristic group in the spiramvcin molecular structure like this, made the spiramvcin antibody of preparation very high to the specificity of spiramvcin.Wherein, crossing in conjunction with ratio of spiramvcin haptens and carrier protein is low or too high all unfavorable to immunity, and the mole proportioning of spiramvcin haptens and described carrier protein is 20-23: 1 is proper.
Described spiramvcin polyclonal antibody can be mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source antibody.
Described spiramvcin monoclonal antibody is to be the antibody that secretion produces to the monoclonal antibody hybridoma cell strain D-1-2 of spiramvcin medicine of CGMCC No.3355 by preserving number.
Described enzyme mark antiantibody adopts the sodium periodate method that described marker enzyme and described antiantibody are carried out coupling and obtains; In the described sodium periodate method, the molar concentration rate of described marker enzyme and described antiantibody is 2: 1; Described marker enzyme is alkaline phosphatase or horseradish peroxidase, is preferably horseradish peroxidase.
It is 6.5 that described concentrated cleaning solution is specially the pH value, contain 0.8% Tween-20,0.05 ‰ sodium azide, the citrate buffer of 0.2mol/L; Described concentrated redissolution liquid is to contain that 6% lowlenthal serum, pH are 7.5, barbital-sodium chloride damping fluid of 0.3mol/L; Described percentage composition is the weight volumn concentration; Described substrate colour developing liquid A liquid is urea peroxide, and described substrate colour developing liquid B liquid is tetramethyl benzidine; Described stop buffer is the hydrochloric acid of 2mol/L.
The effect that adds a certain amount of Tween-20 and sodium azide in cleansing solution is: Tween-20 can reduce the non-specific adsorption of antibody in the damping fluid; can also play the certain protection effect to albumen; after adding sodium azide; then Sodium azide suppresses the growth of bacterium in solution, and the stability of solution is played a protective effect.
Another object of the present invention provides a kind of method that detects spiramvcin.
The method of detection spiramvcin provided by the present invention may further comprise the steps:
1) sample pre-treatments:
The pre-treating method of animal's liver tissue, feed sample: in every 1.0g homogenate, add the 5ml extract, vibration 10min, put in 37 ℃ of constant temperature ovens and react 30min, take out back vibration 30s, the above centrifugal 10min of 3000g, mixed feed, chicken/pork liver: get 100 μ l and redissolve to 700 μ l that mixing is used for measuring (diluting 40 times) in the liquid; Concentrate/premix: get 50 μ l and redissolve to 750 μ l that mixing is used for measuring (diluting 80 times) in the liquid; Sampling is analyzed.
Animal muscle tissue's sample-pretreating method: in per 1.0 animal tissue's homogenates, add the 5ml extract, vibration 10min puts in 37 ℃ of constant temperature ovens and reacts 30min, the above centrifugal 10min of 3000g; Chicken/pork: get 100 μ l and redissolve to 700 μ l that mixing is used for measuring (diluting 40 times) in the liquid; Sampling is analyzed.
2) utilize above-mentioned arbitrary described enzyme linked immunological kit to detect 1) described in sample.
3) analyzing and testing result.
Be that the spiramvcin monoclonal antibody that secretion produces to the monoclonal antibody hybridoma cell strain D-1-2 of spiramvcin medicine of CGMCC No.3355 also belongs to protection scope of the present invention by preserving number.
Preserving number is that the monoclonal antibody hybridoma cell D-1-2 to the spiramvcin medicine of CGMCC No.3355 also belongs to protection scope of the present invention.
The enzyme linked immunological kit that the present invention detects spiramvcin mainly adopts the residual quantity of spiramvcin in the qualitative or quantitative test 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 spiramvcin 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, the screening that simple in structure, easy to use, low price, carrying convenience, detection method be efficient, accurate, easy, be suitable for on-the-spot batch samples.Kit of the present invention will play a significant role in the detection of spiramvcin.
Description of drawings
Fig. 1 is the canonical plotting of the kit of coating antigen for the conjugate with spiramvcin haptens and carrier protein.
Embodiment
Employed experimental technique is conventional method if no special instructions among the following embodiment.
Embodiment 1, be preparation and the detection of the kit of coating antigen with the conjugate of spiramvcin haptens and carrier protein.
One, be that the detection principle of kit of coating antigen is as follows with the conjugate of spiramvcin haptens and carrier protein:
When on capillary strip, wrapping by the spiramvcin coupled antigen in advance, after adding sample solution or standard solution, add ELIAS secondary antibody immediately, add spiramvcin specific antibody solution again, the spiramvcin coupled antigen competition spiramvcin specific antibody of bag quilt on residual spiramvcin medicine and the ELISA Plate in the sample, the enzyme labeling antiantibody that adds carries out amplification, with the colour developing of colour developing liquid, sample light absorption value and spiramvcin content of medicines are negative correlation, relatively can draw the residual quantity of spiramvcin in the sample with typical curve.Simultaneously according to the depth of color on the ELISA Plate, with the comparison of the spiramvcin standard solution color of the series concentration concentration range of spiramycin residues amount in the judgement sample roughly.
Two, be that the kit of coating antigen generally can comprise following composition with the conjugate of spiramvcin haptens and carrier protein:
1, bag is by the ELISA Plate of spiramvcin coupled antigen; The concentration of coating antigen can be 0.10-0.20 μ g/ml;
2, enzyme mark antiantibody working fluid: with sheep anti mouse antiantibody or the goat-anti rabbit antiantibody of horseradish peroxidase-labeled; The dilution of ELIAS secondary antibody is to contain 2-4% rabbit anteserum albumen, pH value to be the phosphate buffer of 7.2-8.1,0.1-0.3mol/L; Described number percent is percent weight in volume; Enzyme mark antiantibody working fluid dilutability is 1: 300.
3, spiramvcin specific antibody working fluid: can be spiramvcin monoclonal antibody working fluid or polyclonal antibody working fluid; Antibody diluent is that the pH value is 7.8-8.3, contains the 5-10% ovalbumin, and the phosphate buffer of 0.1-0.2mol/L, described number percent are percent weight in volume, and antibody working fluid dilutability is 1: 3000;
4, the spiramvcin standard solution is 6 bottles, and concentration is respectively 0 μ g/L, 0.2 μ g/L, 0.6 μ g/L, 1.8 μ g/L, 5.4 μ g/L, 16.2 μ g/L; The solution of preparation standard items is that the lowlenthal serum, the pH value that contain 5-8% are barbital-sodium chloride damping fluid of 7.1-7.6,0.3mol/L, and described number percent is percent weight in volume.
5, substrate colour developing liquid is made up of A liquid and B liquid, and substrate colour developing liquid A liquid is urea peroxide or hydrogen peroxide, 7ml/ bottle, 1 bottle; Substrate colour developing liquid B liquid is tetramethyl benzidine or o-phenylenediamine; The 7ml/ bottle, 1 bottle;
6, stop buffer is 1-2mol/L sulfuric acid or hydrochloric acid; The 7ml/ bottle, 1 bottle;
7, concentrated cleaning solution can be the pH value and is 6.2-6.7, the Tween-20 that contains 0.7-1.0%, the sodium azide of 0.03-0.05 ‰, 0.15-0.2mol/L citrate buffer, and described percentage composition is the weight volumn concentration.
8, concentrate redissolving liquid, to can be the lowlenthal serum, the pH value that contain 5-8% be barbital-sodium chloride damping fluid of 7.1-7.6,0.3mol/L, and described percentage composition is the weight volumn concentration;
Three, this experiment is that the kit of coating antigen is specifically composed as follows with the conjugate of spiramvcin haptens and carrier protein:
1, bag is by the ELISA Plate of spiramvcin coupled antigen; The concentration of coating antigen can be 0.15 μ g/ml;
2, the dilution of ELIAS secondary antibody is to contain that 4% rabbit anteserum albumen, pH value are 7.9, the phosphate buffer of 0.2mol/L; Described percentage composition is the quality percentage composition; Enzyme mark antiantibody working fluid dilutability is 1: 300.
3, spiramvcin monoclonal antibody working fluid: the spiramvcin monoclonal antibody is to be that the secretion to the monoclonal antibody hybridoma cell strain D-1-2 of spiramvcin medicine of CGMCC No.3355 produces by preserving number; Spiramvcin monoclonal antibody working fluid prepares in accordance with the following methods: with dilution the spiramvcin monoclonal antibody is diluted 3000 times, obtain the monoclonal antibody working fluid, described dilution is that the pH value is 8.0, contains 10% fourth of the twelve Earthly Branches albumin, 0.1mol/L phosphate buffer, described percentage composition is the weight volumn concentration.
4, spiramvcin standard solution: 6 bottles, concentration is respectively 0 μ g/L, 0.2 μ g/L, 0.6 μ g/L, 1.8 μ g/L, 5.4 μ g/L, 16.2 μ g/L; The solution of preparation standard items is to contain that 7% lowlenthal serum, pH value are 7.3, barbital-sodium chloride damping fluid of 0.3mol/L, and described percentage composition is the weight volumn concentration.
5, substrate colour developing liquid: be made up of A liquid and B liquid, substrate colour developing liquid A liquid is urea peroxide, 7ml/ bottle, 1 bottle; Substrate colour developing liquid B liquid is tetramethyl benzidine, 7ml/ bottle, 1 bottle.
6, stop buffer is 2mol/L hydrochloric acid; The 7ml/ bottle, 1 bottle;
7, concentrated cleaning solution: the pH value is 6.5, contain 0.8% Tween-20,0.05 ‰ sodium azide, 0.2mol/L citrate buffer; Described percentage composition is the weight volumn concentration; The 40ml/ bottle, 1 bottle;
8, concentrate to redissolve liquid: contain 6% lowlenthal serum, pH value and be 7.5, barbital-sodium chloride damping fluid of 0.3mol/L; Described percentage composition is w/v; The 200ml/ bottle, 1 bottle.
Wherein, it is as follows to be coated with the preparation method of sheep anti mouse antiantibody working fluid of ELISA Plate, spiramvcin antibody working fluid, horseradish peroxidase-labeled of spiramvcin haptens and ovalbumin conjugate:
Used bag is cushioned liquid and confining liquid is as follows:
Used bag is cushioned liquid can be 4.5-6.0,0.1-0.2mol/L acetic acid-ammonium acetate buffer for the pH value; Used confining liquid can be the phosphate buffer of 6.7-7.2,0.1-0.2mol/L for bovine serum albumin(BSA), the gelatin of 0.6-1.0%, the glycerine of 0.5-1%, the pH value that contains 4-7%, and described number percent is the weight volumn concentration.
1, the preparation of ELISA Plate:
The preparation of a, coating antigen: spiramvcin haptens and ovalbumin coupling are obtained coating antigen, and concrete steps are as follows: wherein the mole proportioning of spiramvcin haptens and described ovalbumin is 22: 1.
Haptenic preparation
Take by weighing the 20mg spiramvcin and be dissolved in the 2.5ml absolute ethyl alcohol, mix with the oxammonium hydrochloride 5mg that is dissolved in the 1ml distilled water, under condition of ice bath, react 2.5h, during splash into the about 1ml of NaOH solution of 0.05mol/L.Splash into the about 1ml of acetate buffer of 0.2mol/L, pH4 after the reaction, and add the about 4mg of trash ice, white precipitate occurs, under 4 ℃, leave standstill 1d, the centrifugal 10min of back 10000rpm/min room temperature, abandoning supernatant.White precipitate adds the 6mg succinic anhydride, room temperature reaction 2h after dissolving with the 2.5ml dimethyl formamide.Add triethylamine 100 μ l subsequently, continue reaction 1h, obtain the spiramvcin haptens.
The coating antigen preparation:
Get the completely reacted acylate 1.3ml of step (1), be cooled to 10 ℃, add isobutyl chlorocarbonate 5 μ l, 10 ℃ of stirring reaction 30min can obtain reactant liquor A.Take by weighing OVA36mg, make it fully to be dissolved in the 2mL 50mmol/L sodium carbonate liquor B liquid, reactant liquor A dropwise slowly is added drop-wise among the reactant liquor B.10 ℃ of reaction 4h, 4 ℃ are spent the night then.3d changes dislysate 3 times every day with the 0.01mol/LPBS dialysis, obtains coating antigen.Packing, standby in-20 ℃ of preservations.
The preparation of b, ELISA Plate
Be cushioned liquid with bag the spiramvcin coupled antigen is diluted to 0.15 μ g/ml, every hole adds 100 μ l, 4 ℃ are spent the night, and the coating buffer that inclines is with the washing of the concentrated cleaning solution after the dilution 2 times, each 30 seconds, pat dry, and then add 150 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.
2, spiramvcin MONOCLONAL ANTIBODIES SPECIFIC FOR
(1) immunogene is synthetic
Spiramvcin is small-molecule substance, has only immunoreactivity, does not have immunogenicity, can not bring out body and produce immune response, must with the macromolecular carrier albumen coupling after just have immunogenicity.
1) take by weighing the 20mg spiramvcin and be dissolved in the 2.5ml absolute ethyl alcohol, mix with the oxammonium hydrochloride 5mg that is dissolved in the 1ml distilled water, under condition of ice bath, react 2.5h, during splash into the about 1ml of NaOH solution of 0.05mol/L.Splash into the about 1ml of acetate buffer of 0.2mol/L, pH4 after the reaction, and add the about 4mg of trash ice, white precipitate occurs, under 4 ℃, leave standstill 1d, the centrifugal 10min of back 10000rpm/min room temperature, abandoning supernatant.White precipitate adds the 6mg succinic anhydride, room temperature reaction 2h after dissolving with the 2.5ml dimethyl formamide.Add triethylamine 100 μ l subsequently, continue reaction 1h, obtain the spiramvcin haptens.
2) get the completely reacted acylate 1.3ml of step (1), be cooled to 10 ℃, add isobutyl chlorocarbonate 5 μ l, 10 ℃ of stirring reaction 30min can obtain reactant liquor A.Take by weighing hemocyanin 36mg, make it fully to be dissolved in the 2mL50mmol/L sodium carbonate liquor B liquid, reactant liquor A dropwise slowly is added drop-wise among the reactant liquor B.10 ℃ of reaction 4h, 4 ℃ are spent the night then.3d changes dislysate 3 times every day with the 0.01mol/LPBS dialysis, obtains the spiramvcin immunogene.Packing, standby in-20 ℃ of preservations.
(2) preparation monoclonal antibody
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 in 7: 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, up to the hybridoma cell strain that obtains energy stably excreting spiramvcin monoclonal antibody, with this monoclonal antibody hybridoma cell strain called after D-1-2 to the spiramvcin medicine, this cell line is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on October 28th, 2009 and (is called for short CGMCC, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number is CGMCC No.3355.
C. cell cryopreservation and recovery
The monoclonal hybridoma strain of spiramvcin is made the cell suspension of 1 * 109/ml with cryopreserving liquid, preserve in that liquid nitrogen is medium-term and long-term.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/, and 5 * 107/of the monoclonal hybridoma strains of 7 days pneumoretroperitoneum injection spiramvcins were gathered ascites after 7 days.Carry out the ascites purifying with sad-saturated ammonium sulfate method, obtain monoclonal antibody ,-20 ℃ of preservations.
3, Polyclonal Antibody Preparation
Adopt new zealand white rabbit as immune animal, be immunogene with spiramvcin antigen and hemocyanin conjugate, immunizing dose is 1.5mg/kg, Freund's complete adjuvant with immunogene and equivalent when head exempts from is mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, getting the same dose immunogene 3-4 week 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.
4, the preparation process of the antiantibody of horseradish peroxidase-labeled:
(1) preparation of antiantibody:
The preparation of sheep anti mouse antiantibody: as immune animal, be that immunogene to pathogen-free domestic sheep carry out immunity with mouse source antibody with sheep, obtain the sheep anti mouse antiantibody.
The preparation of goat-anti rabbit antiantibody: as immune animal, be that immunogene to pathogen-free domestic sheep carry out immunity with rabbit source antibody with sheep, obtain goat-anti rabbit antiantibody.
(2) preparation of the antiantibody of horseradish peroxidase-labeled
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, Huo Hua horseradish peroxidase molecule has served as the bridge that connects each molecule like this, reduced the enzymatic activity of enzyme labeling thing, made in the conjugate of preparation and be mixed with many condensates.
The present invention utilizes the sodium periodate method of improvement to carry out the enzyme mark of antibody, and it has saved amino closed process, because can produce self amino amino reality that connects seldom.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.
Three, the detection of spiramvcin in the sample
1, sample pre-treatments
The pre-treating method of animal's liver tissue, feed sample: in every 1.0g homogenate, add the 5ml extract, vibration 10min, put in 37 ℃ of constant temperature ovens and react 30min, take out back vibration 30s, the above centrifugal 10min of 3000g, mixed feed, chicken/pork liver: get 100 μ l and redissolve to 700 μ l that mixing is used for measuring in the liquid; Concentrate/premix: get 50 μ l and redissolve to 750 μ l that mixing is used for measuring in the liquid; Sampling is analyzed.
Animal muscle tissue's sample-pretreating method: in per 1.0 animal tissue's homogenates, add the 5ml extract, vibration 10min puts in 37 ℃ of constant temperature ovens and reacts 30min, the above centrifugal 10min of 3000g; Chicken/pork: get 100 μ l and redissolve to 700 μ l that mixing is used for measuring in the liquid; Sampling is analyzed.
2, detect with kit
In the ELISA Plate micropore that is coated with spiramvcin haptens and ovalbumin conjugate, add spiramvcin standard solution or sample solution 50 μ l/ holes, the sheep anti mouse antiantibody working fluid 50 μ l/ holes of horseradish peroxidase-labeled immediately, and then adding spiramvcin monoclonal antibody working fluid 50 μ l/ holes, with cover plate mould shrouding, react 45min in 25 ℃ of lucifuge environment, pour out liquid in the hole, every hole adds 250 μ l cleansing solutions, pour out liquid in the hole behind each interval 10s, pat dry with thieving paper, so repetitive operation is washed plate 5 times altogether.Every hole adds substrate colour developing liquid A liquid urea peroxide, substrate colour developing liquid B liquid tetramethyl benzidine (TMB), mixing gently vibrates, react 15min in 25 ℃ of lucifuge environment, every hole adds 2mol/L stop buffer sulfuric acid, 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
Multiply by 100% with the absorbance mean value (B) of the standard solution of each concentration that obtains again divided by the absorbance (B0) of first standard solution (0 standard), obtain the percentage absorbance.
B is the mean light absorbency value of standard solution or sample solution in the formula, and B0 is the mean light absorbency value of 0 μ g/L standard solution.
Being worth with spiramvcin standard items concentration (μ g/L) is X-axis, and the percentage absorbance is Y-axis, drawing standard curve map (Fig. 1).The use the same method percentage absorbance of calculation sample solution, the concentration of corresponding each sample, the residual quantity that then can read spiramvcin 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 1.0 hours.
Embodiment 2, kit sensitivity, accuracy and storage life test
(1) standard items precision detects:
From embodiment 1, respectively extract 10 kits in the kit of the different batches of different time sections preparation in the step 3, from the elisa plate of each kit, respectively extract 20 micropores out, measure the absorbance (OD value) of 1.8 μ g/L spiramvcin standard solutions, calculate the coefficient of variation.Experimental result is as shown in table 1, shows, the coefficient of variation of standard items absorbance met precision and is less than or equal to 20% regulation between 3.5%-13.2% during all detected.
Table 1, the repeatable tests of standard items (CV%)
(2) sample precision and accuracy test
1, sample precision detects:
Adding final concentration in the blank muscle that does not contain spiramvcin, liver, the mixed feed sample respectively is the spiramvcin of 40 μ g/kg, to add final concentration in the concentrating of spiramvcin/premix be the spiramvcin of 80 μ g/kg to not containing, and carries out sample pre-treatments according to method described in the embodiment 1 respectively again.Respectively extract 3 kits in three batches the kit (01 batch, 03 batch, 06 batch) of the different time sections preparation from embodiment 1 described in the step 3, carry out test experience, each experiment repeats 5 times, calculate the coefficient of variation respectively, the result is (numerical value in each table is 5 mean values that repeat) shown in table 2-4.The result shows that muscle, liver, mixed feed, the concentrated/coefficient of variation of premix sample all between 4.3%-18.1%, have met " Ministry of Agriculture's file " farming doctor [2005] No. 17 annex 2 kits and put on record with reference to the 4th precision standard in the judgment criteria.
Table 2, the repeatable test of muscle sample
Table 3, the repeatable test of liver samples
Table 4, the repeatable test of mixed feed sample
Table 5, the repeatable test of concentrated/premix sample
2, sample accuracy test
In the muscle that does not contain spiramvcin, liver, mixed feed tissue, add spiramvcin respectively, make the final concentration of spiramvcin be respectively 40 μ g/kg, 80 μ g/kg, in the concentrated/premix that does not contain spiramvcin, add spiramvcin, make the final concentration of spiramvcin be respectively 80 μ g/kg, 160 μ g/kg, handle according to the sample pre-treating method described in the embodiment 1 then; Detect spiramvcin in tissue or the feed with the kit described in the step 3 among the embodiment 1 again, each concentration do 4 parallel, accuracy in computation (accuracy=measured value/interpolation value * 100%) respectively.The result is as shown in table 6 below.
The result shows that muscle, liver, mixed feed, concentrated/premixed feed sample add the recovery between 60.2%-98.6%.
The accuracy testing result of table 6, kit
(3) cross reacting rate test:
Select to have with spiramvcin 7 kinds of drug monitoring cross reacting rates of similar structures and similar functions.Typical curve by various medicines obtains its 50% inhibition concentration respectively.With kit in the following formula calculation procedure three to the cross reacting rate of other medicines.Kit is more big for the spiramvcin cross reacting rate, and then its specificity to this drug test is just more good.Replication 3 times, results averaged.
Cross reacting rate (%)=(cause 50% concentration that suppresses spiramvcin/cause the 50% spiramvcin analog concentration that suppresses) * 100%
The specificity of table 7, kit
The result is as shown in table 7, shows that kit of the present invention is good to the specificity of spiramvcin, and namely kit of the present invention can detect spiramvcin.
(4) kit storage life test
The kit preservation condition is 2-8 ℃, and through 6 months mensuration, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, spiramvcin added the practical measurement value all within normal range in the step 3.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.Therefore, kit of the present invention can be preserved more than 6 months at least at 2-8 ℃.
(5) sensitivity of kit
With made kit in embodiment 1 step 3 zero standard product are carried out 20 times respectively and detect, the mean value of measurement result adds that 3 times of standard deviations are as the sensitivity of kit.
Table 7, sensitivity determination be statistical form μ g/kg as a result
As shown in Table 9, the kit lowest detection developed of the present invention is limited to 0.2 μ g/kg.
Claims (7)
1. an enzyme linked immunological kit that detects spiramvcin comprises spiramvcin specific antibody and coating antigen and enzyme labeling thing; Described coating antigen is conjugate or the antiantibody of spiramvcin haptens and carrier protein; Described enzyme labeling thing is enzyme mark antiantibody or enzyme mark spiramvcin haptens; When described coating antigen was the conjugate of spiramvcin haptens and carrier protein, described enzyme labeling thing was enzyme mark antiantibody; When described coating antigen was antiantibody, described enzyme labeling thing was enzyme mark spiramvcin haptens; Described spiramvcin specific antibody is the monoclonal antibody of spiramvcin, and it is the monoclonal antibody hybridoma cell strain D-1-2 secretion to the spiramvcin medicine of CGMCC No.3355 by preserving number.
2. enzyme linked immunological kit according to claim 1 is characterized in that: described spiramvcin haptens be will spiramvcin and the oxammonium hydrochloride reaction after the product that obtains obtain with the succinic anhydride reaction again.
3. enzyme linked immunological kit according to claim 1 and 2 is characterized in that: described spiramvcin specific antibody is that the conjugate with described spiramvcin haptens and carrier protein obtains as immunogene; Described carrier protein is thyroprotein, bovine serum albumin, mouse haemocyanin, human albumin, rabbit anteserum albumen, hemocyanin, fibrinogen or ovalbumin.
4. enzyme linked immunological kit according to claim 1 and 2 is characterized in that: described enzyme mark antiantibody adopts the sodium periodate method that marker enzyme and described antiantibody are carried out coupling and obtains; In the described sodium periodate method, the molar concentration rate of described marker enzyme and described antiantibody is 2: 1; Described marker enzyme is alkaline phosphatase or horseradish peroxidase.
5. method that detects spiramvcin may further comprise the steps:
1) sample pre-treatments:
The pre-treating method of animal's liver tissue, feed sample: in every 1.0g homogenate, add the 5ml extract, vibration 10min, put in 37 ℃ of constant temperature ovens and react 30min, take out back vibration 30s, the above centrifugal 10min of 3000g, mixed feed, chicken/pork liver: get 100 μ l and redissolve to 700 μ l that mixing is used for measuring in the liquid; Concentrate/premix: get 50 μ l and redissolve to 750 μ l that mixing is used for measuring in the liquid; Sampling is analyzed;
Animal muscle tissue's sample-pretreating method: in the every 1.0g animal tissue homogenate, add the 5ml extract, vibration 10min puts in 37 ℃ of constant temperature ovens and reacts 30min, the above centrifugal 10min of 3000g; Chicken/pork: get 100 μ l and redissolve to 700 μ l that mixing is used for measuring in the liquid; Sampling is analyzed;
Described redissolution liquid is that the lowlenthal serum, the pH value that contain 5-8% are barbital-sodium chloride damping fluid of 7.1-7.6,0.3mol/L, and described percentage composition is the weight volumn concentration;
2) utilize that arbitrary described enzyme linked immunological kit detects 1 among the claim 1-4) described in sample.
6. be the spiramvcin monoclonal antibody to the monoclonal antibody hybridoma cell strain D-1-2 of spiramvcin medicine secretion of CGMCC No.3355 by preserving number.
7. preserving number is the monoclonal antibody hybridoma cell strain D-1-2 to the spiramvcin medicine of CGMCC No.3355.
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| CN102721808A (en) * | 2011-03-29 | 2012-10-10 | 库尔医疗技术(北京)有限公司 | Spiramycin assay kit and method for making the same |
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