CN101358966A - Method for detecting colimycin and special ELISA kit thereof - Google Patents
Method for detecting colimycin and special ELISA kit thereof Download PDFInfo
- Publication number
- CN101358966A CN101358966A CNA2008101184435A CN200810118443A CN101358966A CN 101358966 A CN101358966 A CN 101358966A CN A2008101184435 A CNA2008101184435 A CN A2008101184435A CN 200810118443 A CN200810118443 A CN 200810118443A CN 101358966 A CN101358966 A CN 101358966A
- Authority
- CN
- China
- Prior art keywords
- colistin
- kit
- liquid
- antiantibody
- enzyme
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention discloses a method of detecting colistin, and a special enzyme linked immunosorbent kit thereof. The enzyme linked immunosorbent kit comprises the colistin and specific antibodies of the colistin; and the specific antibodies are polyclonal antibodies or monoclonal antibodies of the colistin. The kit adopts the high specificity monoclonal antibodies of the colistin, which guarantees the reliability of detection results, and the experimental results show that the kit has the characteristics of high specificity, high sensitivity, high precision, high accuracy and the like; and the main reagents of the kit all adopt the form of working liquid, the operation is convenient, and the cost is low. The operation of the method which adopts the kit to detect the colistin is simple, the detection time is shortened, the operation have low requirements for the pretreatment of the sample, and the method can be used for simultaneously and rapidly detecting the samples in bulk. Therefore, the method which adopts the enzyme linked immunosorbent kit for detection can play a significant role in the detection of the colistin.
Description
Technical field
The present invention relates to a kind of method and special ELISA reagent kit thereof that detects colistin.
Background technology
(colistin CLS), claims polymyxin e (polymyxin E) again to colistin, is produced by a plurality of colistins (B.polymyxa).It has very strong inhibiting effect to Gram-negative bacteria; can treat the infection that Shigella shigae, Escherichia coli, pseudomonas aeruginosa, Salmonella and proteus vulgaris cause; it can also be used as feed addictive; improve food conversion ratio; promote growth of animals or poultry, the generation of livestock and poultry in the prevention large-scale cultivation.Antibacterial action is strong, residual quantity is low, be difficult for producing chemical sproof characteristics because of colistine sulfate has, and therefore, China, the U.S., European Union and Japan etc. ratify it as feed addictive or veterinary drug many countries and regions.
Along with the application of colistine sulfate in aquaculture is increasing, the residue problem of colistine sulfate is subjected to the attention of various countries gradually in the animal food, European Union has taken the lead in formulating the maximum residue limit(MRL) standard of colistin in the animal edible tissues, and wherein muscle, hepatic and/or renal maximum residue limit(MRL) standard are respectively 150,150 and 200 μ g/kg.The colistin maximum residue limit(MRL) that China Ministry of Agriculture announces regulation in No. 235 file (issue " animal food herbal medicine maximum residue limit(MRL) ") is consistent with European Union.
At present, the analytical approach that is used for detecting the colistin residual quantity mainly is a microbiological assay, but colistin belongs to high molecular (for common antibiotics), high polar material, its poor heat stability causes it should not adopt the GC method to analyze, and its poor selectivity causes using instrumental method can not reach required detection sensitivity and detectability.
Summary of the invention
An object of the present invention is to provide a kind of enzyme linked immunological kit that detects colistin.
The enzyme linked immunological kit of detection colistin provided by the present invention comprises the specific antibody of colistin and colistin; Described specific antibody is the polyclonal antibody or the monoclonal antibody of described colistin.Wherein, the specific antibody of described colistin and colistin can exist with following any form:
1) described colistin and carrier protein are carried out coupling, obtain the conjugate of colistin and carrier protein, as coating antigen, described specific antibody carries out after the enzyme labeling as the enzyme labeling thing with it;
2) described specific antibody is a coating antigen, and described colistin carries out after the enzyme labeling as the enzyme labeling thing.
Described kit can also not only comprise the specific antibody of colistin and colistin but also comprise antiantibody; Described antiantibody is sheep anti mouse antiantibody or goat-anti rabbit antiantibody; Wherein, described colistin and antiantibody exist with following any form:
1) described colistin and carrier protein are carried out coupling, obtain the conjugate of colistin and carrier protein, as coating antigen, described antiantibody carries out after the enzyme labeling as the enzyme labeling thing with it;
2) described antiantibody is a coating antigen, and described colistin carries out after the enzyme labeling as the enzyme labeling thing.
Described colistin polyclonal antibody and described colistin monoclonal antibody all are that the conjugate with colistin 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.
Described colistin monoclonal antibody is to be the antibody that secretion produces to the monoclonal hybridoma strain A-4-4 of colistin medicine of CGMCC No.2541 by deposit number.
Described colistin polyclonal antibody can be mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source antibody.
For more convenient on-site supervision and great amount of samples examination, described kit also comprises colistin standard solution, colour developing liquid, stop buffer, concentrated cleaning solution, concentrates redissolution liquid.
Wherein, described concentrated cleaning solution can be and contains 0.8-1.3% bovine serum albumin, 45-60% glycerine, and the pH value is the carbonate buffer solution of 9.2-9.5,0.05-0.1mol/L; It is 9.1-9.9,0.05-0.1mol/L carbonate buffer solution that described concentrated redissolution liquid can be the human albumin, the pH that contain 0.2-0.5%; Described percentage composition is the quality percentage composition.
Used marker enzyme is horseradish peroxidase or alkaline phosphatase in the described enzyme labeling; 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 is hydrogen peroxide or urea peroxide, and colour developing liquid B liquid is o-phenylenediamine or tetramethyl benzidine, and stop buffer is 1-2mol/L sulfuric acid or hydrochloric acid solution; When marker enzyme was alkaline phosphatase, developer was the nitro phosphate buffer, and stop buffer is the 1-2mol/L sodium hydroxide solution.
Described concentrated cleaning solution specifically can be and contains that 1.0% bovine serum albumin, 55% (quality percentage composition) glycerine and pH value are 9.3, the carbonate buffer solution of 0.1mol/L; Described concentrated redissolution liquid specifically can be and contains that 0.4% human albumin, pH are 9.6, the 0.1mol/L carbonate buffer solution.
The method that described antiantibody carries out enzyme labeling is to adopt the sodium periodate method that described marker enzyme and described antiantibody are carried out coupling to obtain enzyme and mark antiantibody; In the described sodium periodate method, the molar concentration rate of described marker enzyme and described antiantibody is 2: 1.The sodium periodate method of this improvement of the present invention has been omitted the step of amino on the sealase, has saved the time, has reduced the concentration rate of horseradish peroxidase (HRP) with antiantibody again, has saved starting material.
The colistin molecular weight has only immunoreactivity less than 3000, does not have immunogenicity, can not bring out body and produce immune response, must with the macromolecular carrier albumen coupling after just have immunogenicity.Colistin can by glutaraldehyde method or carbodiimide method be direct and carrier protein couplet, obtains immunogenic or bag by the source.In preparation during immunogene, it is low or too high all unfavorable to immunity that colistin and the ratio that combines of carrier protein are crossed, and the present invention shows that by experiment colistin is (8-10) with the mol ratio that combines of carrier protein: 1 is proper; Described carrier protein specifically can be thyroprotein.In preparation during coating antigen, the mole proportioning of colistin and described carrier protein be 10: 1 proper.
When making was coated with the ELISA Plate of coating antigen, used bag is cushioned liquid can be the carbonate buffer solution of pH value for 9.1-9.9,0.1mol/L; Described confining liquid can be 4.0-5.0,0.1mol/L citrate buffer solution for oralbumin, the skimmed milk power of 0.2-0.5%, the sucrose of 0.05-0.3%, the pH value that contains 0.4-0.6%; Described percentage composition is the quality percentage composition.
Another purpose of the present invention provides a kind of method that detects colistin.
The method of detection colistin provided by the present invention may further comprise the steps:
1) sample pre-treatments
In every 1.0g animal tissue homogenate, add 9-12ml 0.03-0.07M sulfuric acid solution, mixing places 37 ℃ of incubation 20-40min, under 5-10 ℃ with the centrifugal 5-15min of the speed more than the 3000g, get supernatant liquid 3-5ml, add the sodium hydroxide solution of 140-170ml 1.5-2.5M, mixing, adjust pH is to pipette liquid behind the 6-8, add isopyknic above-mentioned arbitrary described redissolution liquid, mixing, sampling is analyzed;
2) utilize above-mentioned arbitrary described enzyme linked immunological kit to detect 1) described in dilution.
By deposit number is that the colistin monoclonal antibody that secretion produces to the monoclonal hybridoma strain A-4-4 of colistin medicine of CGMCC No.2541 belongs to protection scope of the present invention.
Deposit number is that the monoclonal hybridoma strain A-4-4 to the colistin medicine of CGMCC No.2541 also belongs to protection scope of the present invention.
The enzyme linked immunological kit of detection colistin of the present invention mainly adopts the residual quantity of colistin in the qualitative or detection by quantitative sample of indirect competitive ELISA method.Adopt the colistin monoclonal antibody of high specific in this kit, guaranteed the reliability of testing result, experimental result shows that this kit has specificity height, highly sensitive, characteristics such as precision is high, accuracy height; The main agents of this kit all adopts the working fluid form, and is easy to use, with low cost.Detect the method for colistin with kit of the present invention, easy and simple to handle, simplified the step of traditional detection method, shortened the time of detecting, the pre-treatment of sample is required low, fast detecting batch samples simultaneously.Therefore, the method for utilizing enzyme linked immunological kit of the present invention to detect can be carried out the qualitative and quantitative examination of on-site supervision and suitable great amount of samples, will play a significant role in the detection of colistin.
Description of drawings
Fig. 1 is that coating antigen, ELIAS secondary antibody are the canonical plotting of the kit of enzyme labeling thing for the conjugate with colistin haptens and carrier protein.
Embodiment
Employed experimental technique is conventional method if no special instructions among the following embodiment.
Embodiment 1, be that coating antigen, ELIAS secondary antibody are the preparation and the use of the kit of enzyme labeling thing with the conjugate of colistin haptens and carrier protein
One, be that coating antigen, ELIAS secondary antibody are that the detection principle of kit of enzyme labeling thing is as follows with the conjugate of colistin haptens and carrier protein:
When the coating antigen on the ELISA Plate capillary strip is the conjugate of colistin haptens and carrier protein, in the ELISA Plate micropore, add standard solution or sample solution, add the colistin specific antibody again, colistin coupled antigen competition colistin specific antibody on residual colistin and the ELISA Plate in the sample, add enzyme mark antiantibody again, with the colour developing of colour developing liquid, the content of sample light absorption value and colistin is negative correlation, relatively can draw the residual content of colistin in the sample with typical curve.
Two, be that coating antigen, ELIAS secondary antibody are that the kit of enzyme labeling thing generally can comprise as follows with the conjugate of colistin haptens and carrier protein:
1, is coated with the ELISA Plate of coating antigen (coating antigen is the conjugate of colistin haptens and carrier protein); The concentration of coating antigen can be 0.15-0.25 μ g/ml;
2, enzyme mark antiantibody working fluid: ELIAS secondary antibody is sheep anti mouse antiantibody or goat-anti rabbit antiantibody with horseradish peroxidase-labeled; The dilution of ELIAS secondary antibody is that the human albumin, the pH that contain 0.2-0.5% are 9.1-9.9,0.05-0.1mol/L carbonate buffer solution, and enzyme mark antiantibody working fluid dilutability is 1: 400, and described percentage composition is the quality percentage composition.
3, colistin specific antibody working fluid: can be colistin polyclonal antibody working fluid or colistin monoclonal antibody working fluid; With dilution the colistin specific antibody is diluted 2500 times, obtain the specific antibody working fluid, described dilution is for sodium azide, the pH value that contains 3.0% (quality percentage composition) casein and 0.003% (quality percentage composition) is 5.6, the phosphate buffer of 0.1mol/L.
4, colistin standard items (going up sea cowry base bio tech ltd) 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; The solution of preparation standard items is that human albumin, the pH that contains 0.2-0.5% is 9.1-9.9,0.05-0.1mol/L carbonate buffer solution.
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: 1-2M sulfuric acid or hydrochloric acid; The 7ml/ bottle, 1 bottle;
7, concentrated cleaning solution is for containing 0.8-1.3% bovine serum albumin, 45-60% glycerine, and the pH value is the carbonate buffer solution of 9.2-9.5,0.05-0.1mol/L; The 50ml/ bottle, 1 bottle; Described percentage composition is the quality percentage composition.
8, redissolution liquid is that human albumin, the pH that contains 0.2-0.5% is 9.1-9.9,0.05-0.1mol/L carbonate buffer solution; The 400ml/ bottle, 1 bottle; Described percentage composition is the quality percentage composition.
Three, the conjugate with colistin haptens and carrier protein is that coating antigen, ELIAS secondary antibody are the concrete composition and the preparation thereof of the kit of enzyme labeling thing:
(1) forms
1, is coated with the ELISA Plate of coating antigen (coating antigen is colistin haptens and hemocyanin conjugate); The concentration of coating antigen is 0.2 μ g/ml.
2, enzyme mark antiantibody working fluid: with the sheep anti mouse antiantibody of horseradish peroxidase-labeled; The ELIAS secondary antibody dilution is to contain that promising 0.4% human albumin, pH are 9.6, the 0.1mol/L carbonate buffer solution, and enzyme mark antiantibody working fluid dilutability is 1: 400, and described percentage composition is the quality percentage composition.
3, colistin monoclonal antibody working fluid: the colistin monoclonal antibody is to be that the secretion to the monoclonal hybridoma strain A-4-4 of colistin medicine of CGMCCNo.2541 produces by deposit number; Colistin monoclonal antibody working fluid prepares in accordance with the following methods: with 2500 times of colistin monoclonal antibody dilutions, obtain the monoclonal antibody working fluid with dilution; Described dilution is 5.6 for containing 3.0% (quality percentage composition) casein and 0.003% (quality percentage composition) sodium azide, pH value, the phosphate buffer of 0.1mol/L.
4, colistin standard items (going up sea cowry base bio tech ltd) solution: 6 bottles, 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, and the solution of preparation standard items is to contain that 0.4% human albumin, pH are 9.6, the 0.1mol/L carbonate buffer solution.
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: 2mol/L sulfuric acid, 7ml/ bottle, 1 bottle.
7, concentrated cleaning solution: contain 1.0% bovine serum albumin, 55% glycerine and pH value and be 9.3, the carbonate buffer solution of 0.1mol/L; The 50ml/ bottle, 1 bottle.
8, concentrate to redissolve liquid: contain 0.4% human albumin, pH and be 9.6, the 0.1mol/L carbonate buffer solution; The 400ml/ bottle, 1 bottle.
(2) preparation
1, is coated with the preparation of the ELISA Plate of colistin haptens and hemocyanin conjugate
(1) preparation of coating antigen
With colistin and hemocyanin, adopt carbodiimide method to carry out coupling and obtain envelope antigen.
Take by weighing hemocyanin 36mg, make it fully to be dissolved in the 2mL distilled water I liquid; Take by weighing EDC20mg and NHS15mg, make it fully to be dissolved in and obtain II liquid in the 0.5mL distilled water; Under stirring condition I liquid is added in the II liquid then, room temperature priming reaction 1h gets solution III; Get colistin 5mg and separate with 1.5ml is water-soluble, slowly add in the solution III then, stirring reaction spends the night, and the conjugate that obtains colistin and hemocyanin is a coating antigen; Wherein the mole proportioning of colistin and described hemocyanin is 10: 1.
(2) be coated with the preparation of the ELISA Plate of coating antigen
Be cushioned liquid with bag coating antigen is diluted to 0.15-0.25 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubation 2h, and 4 ℃ are spent the night again, coating buffer inclines, with cleansing solution washing 2 times, each 30 seconds, pat dry, in every hole, add 150-200 μ l confining liquid then, 37 ℃ of incubation 1-2h, liquid pats dry in the hole of inclining, and preserve with the vacuum seal of aluminium film dry back.
It is that the pH value is 9.9, the carbonate buffer solution of 0.1mol/L that bag is cushioned liquid.
Confining liquid is to contain 0.4% oralbumin, 0.3% skimmed milk power, 0.2% sucrose, pH value to be the 4.20.1mol/L citrate buffer solution; Described percentage composition is the quality percentage composition.
2, colistin MONOCLONAL ANTIBODIES SPECIFIC FOR
(1) immunogenic preparation
Adopt glutaraldehyde method to carry out coupling colistin and thyroprotein and obtain immunogene.
Get colistin 5mg and separate with 0.5ml is water-soluble, adding concentration under stirring condition is the glutaraldehyde 0.1ml of 2.5% (quality percentage composition), and room temperature priming reaction 18h adds thyroprotein 50mg again, and stirring reaction spends the night, and obtains the colistin immunogene; Colistin is 9: 1 with the mol ratio that combines of thyroprotein.
(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, in 7: 1 ratio (quantitative proportion) merge with 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 colistin monoclonal antibody, with the monoclonal hybridoma strain A-4-4 of this cell line called after to the colistin medicine, this cell line has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center and (has been called for short CGMCC on 06 04th, 2008, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number is CGMCC No.2541.
C. cell cryopreservation and recovery
The monoclonal hybridoma strain of colistin is made 1 * 10 with cryopreserving liquid
9The 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
Can prepare monoclonal antibody with following two kinds of methods:
Method 1: the Balb/c mouse peritoneal is only injected sterilization paraffin oil 0.5ml/, the monoclonal hybridoma strain 5 * 10 of 7 days pneumoretroperitoneum injection colistins
7Individual/as only, to gather ascites after 7 days.Carry out the ascites purifying with sad-saturated ammonium sulfate method, obtain monoclonal antibody ,-20 ℃ of preservations.
Method 2: increment cultivation: it is 7.4,0.2% sodium bicarbonate, 1640+20% calf serum nutrient culture media that hybridoma CGMCC No.2541 is placed pH, under 37 ℃ of conditions, cultivate, with sad-saturated ammonium sulfate method the nutrient solution that obtains is carried out purifying, obtain monoclonal antibody ,-20 ℃ of preservations.
3, Polyclonal Antibody Preparation
Adopt new zealand white rabbit as immune animal, with colistin antigen and bovine serum albumin(BSA) conjugate is immunogene, 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:
The sheep anti mouse antiantibody is available from Beijing Bo Aosen company, article No. bse-0296G.
Goat-anti rabbit antiantibody is available from Beijing Bo Aosen company, article No. bse-0295G.
(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 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, 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.
Four, the detection of colistin in the sample
Kit of the present invention can be used to detect animal tissue (as pig muscle, chicken liver, fish, shrimp etc.).
1, sample pre-treatments
Get 1g animal tissue homogenate, add 10ml 0.05M sulfuric acid solution, with vortex instrument whirling motion 5min, place 37 ℃ of incubation 30min, the above 10 ℃ of centrifugal 10min of 3000g get upper strata liquid 4ml, add 160ml 2M sodium hydroxide solution, with vortex instrument whirling motion 30s, it is neutral transferring pH, pipettes liquid 500 μ l, add 500 μ l redissolution liquid, with vortex instrument whirling motion 30s, fully mixing is taken a sample and is analyzed.The pre-treatment of sample mainly is for the object in the more accurate extraction sample, thereby is used for follow-up detection.
2, detect with kit
In the ELISA Plate micropore that is coated with the colistin coupled antigen, add colistin standard solution or sample solution 50 μ l, add colistin specific antibody working fluid 50 μ l,, react 30min in 25 ℃ of lucifuge environment with cover plate film shrouding.Liquid in the hole is dried, every hole adds 250 μ l cleansing solutions, pour out liquid in the hole after 10 seconds, so repetitive operation is washed plate 5 times altogether, pat dry with thieving paper, add colistin enzyme mark antiantibody working fluid 100 μ l again, with cover plate film shrouding, react 30min in 25 ℃ of lucifuge environment, liquid in the hole is dried the repeated washing step, every hole adds substrate colour developing liquid A liquid urea peroxide and each 50 μ l of substrate colour developing liquid B liquid tetramethyl benzidine (TMB), the mixing that vibrates gently with cover plate film shrouding, reacts 30min in 25 ℃ of lucifuge environment, every hole adds 2mol/L stop buffer sulfuric 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
Multiply by 100% with the absorbance mean value (B) of the standard solution of each concentration that is obtained 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, B
0It is the mean light absorbency value of 0 μ g/L standard solution.
With colistin standard items concentration (μ g/L) value is X-axis, and the percentage absorbance is a 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 colistin 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 hour.
Five, kit sensitivity, precision, accuracy and storage life detect
(1) standard items precision test:
From embodiment 1, respectively extract 10 kits in the kit of the different batches (01 batch, 03 batch, 03 batch) 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 4.5 μ g/L colistin standard solutions, calculate the coefficient of variation.Result such as table 1, the Variation Lines number average of standard items absorbance meet precision and are less than or equal to 20% regulation between 4.4%-11.3%.
Table 1, the repeatable test of standard (CV%)
Two, sample precision and accuracy test
(1) sample precision detects:
Add colistin in the pork that does not contain colistin, pork liver, chicken, chicken gizzard sample, making its final concentration is 20 μ g/kg (L), carries out sample pre-treatments according to the method for embodiment 1.From embodiment 1, respectively extract 3 kits in the kit of the different batches (01 batch, 02 batch, 03 batch) of different time sections preparation in the step 3, experimentize, each experiment repeats 5 times, calculates the coefficient of variation respectively, and the result is (numerical value in each table is 5 mean values that repeat) shown in table 2-5.The result shows the Variation Lines number average of pork, pork liver, chicken, chicken gizzard sample between 6.6%-14.6%, 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.
Table 2, the repeatable test of chicken muscle sample
Table 3, the repeatable test of pig muscle sample
Table 4, the repeatable test of chicken gizzard sample
Table 5, the repeatable test of pig liver sample
2, sample accuracy test
In the pork that does not contain colistin, pork liver, chicken, chicken gizzard tissue, add colistin respectively, make the final concentration of colistin be respectively 40 μ g/kg, 80 μ g/kg, handle according to the sample pre-treating method described in the embodiment 1 then; Detect colistin in the tissue 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) respectively.The result is as shown in table 6, and the result shows that colistin adds accuracy with 40 μ g/kg, three concentration of 80 μ g/kg between 61.5%-96.3% to chicken, chicken gizzard, pork, pork liver sample.
The accuracy of table 6, kit
(3) cross reacting rate test:
Select to have a kind of drug monitoring cross reacting rate of similar structures and similar functions with Bacillus adhaerens.Typical curve by various medicines obtains its 50% inhibition concentration respectively.With kit in the following formula calculation procedure three to the colistin cross reacting rate.Kit is big more for the colistin cross reacting rate, and this kit is just good more to the specificity of the detection of colistin so.Replication 3 times, results averaged.
Cross reacting rate (%)=(cause 50% concentration that suppresses colistin/cause the 50% colistin analog concentration that suppresses) * 100%
The specificity of table 7, kit
| Medicine name | Cross reacting rate (%) |
| |
100％ |
| Bacitracin Zinc | ＜1 |
The result is as shown in table 7, and experiment shows that kit of the present invention is good to the specificity of colistin, and kit promptly of the present invention can detect colistin.
(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, colistin 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.Can draw kit from above result can preserve more than 6 months at least at 2-8 ℃.
(5) sensitivity of kit
Get the negative pork tissue samples that does not contain colistin and carry out 20 detections respectively with kit in embodiment 1 step 3, the mean value of measurement result adds the lowest detectable limit of 3 times of standard deviations as kit.
Table 8, negative pork sample measurement result statistical form μ g/kg
As shown in Table 8, the kit lowest detection that the present invention developed is limited to 8.21 μ g/kg.
Embodiment 2, the kit that is used to detect colistin can also have following several:
One, coating antigen is a specific antibody, and the enzyme labeling thing is the haptenic kit of enzyme mark colistin
(1) principle of work of this kit is:
When on capillary strip, wrapping in advance, behind adding sample solution or the standard solution, add enzyme labeling colistin haptens solution again by the colistin specific antibody.Colistin in the sample or colistin standard items and enzyme-labelled antigen competition are coated on the colistin specific antibody on the ELISA Plate, with the colour developing of colour developing liquid, the content of colistin becomes negative correlation in sample light absorption value and the sample, relatively can draw the content of colistin in the sample with typical curve.Simultaneously also can be according to the shade on the ELISA Plate, by with the more rough judgement sample of the colistin standard solution color of series concentration in the concentration range of colistin.
(2) consisting of of this kit:
(1) be coated with the ELISA Plate of coating antigen: coating antigen is the colistin monoclonal antibody, is to be that the monoclonal hybridoma strain A-4-4 secretion to the colistin medicine of CGMCC No.2541 produces by deposit number; The concentration of coating antigen can be 0.15-0.25 μ g/ml.
(2) enzyme labeling thing: enzyme mark colistin working fluid; Marker enzyme is a horseradish peroxidase.
(3) colistin standard items (going up sea cowry base bio tech ltd) 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; The solution of preparation standard items is that human albumin, the pH that contains 0.2-0.5% is 9.1-9.9,0.05-0.1mol/L carbonate buffer solution.
(4) substrate colour developing liquid: be made up of colour developing liquid A liquid and colour developing liquid B liquid, colour developing liquid A liquid is hydrogen peroxide, and colour developing liquid B liquid is o-phenylenediamine.
(5) stop buffer is the 1-2mol/L sulfuric acid solution.
(6) concentrated cleaning solution is for containing 0.8-1.3% bovine serum albumin, 45-60% glycerine, and the pH value is the carbonate buffer solution of 9.2-9.5,0.05-0.1mol/L; The 50ml/ bottle, 1 bottle; Described percentage composition is the quality percentage composition.
(7) concentrating redissolution liquid is that human albumin, the pH that contains 0.2-0.5% is 9.1-9.9,0.05-0.1mol/L carbonate buffer solution; The 400ml/ bottle, 1 bottle; Described percentage composition is the quality percentage composition.
Two, coating antigen is that colistin and carrier protein couplet thing, enzyme labeling thing are the kit of enzyme mark colistin specific antibody
(1) principle of work
When on capillary strip, wrapping in advance, behind adding sample solution or the standard solution, add enzyme mark colistin specific antibody solution again by colistin and carrier protein couplet thing.The colistin competition colistin specific antibody of bag quilt on colistin in the sample or colistin standard items and the ELISA Plate, with the colour developing of colour developing liquid, the content of colistin becomes negative correlation in sample light absorption value and the sample, relatively can draw the content of colistin in the sample with typical curve.Simultaneously also can be according to the shade on the ELISA Plate, by with the more rough judgement sample of series concentration colistin standard solution color in the concentration range of colistin.
(2) composition of this kit
(1) be coated with the ELISA Plate of coating antigen: coating antigen is colistin and hemocyanin conjugate.
(2) enzyme labeling thing: enzyme mark specific antibody working fluid, marker enzyme is an alkaline phosphatase; Specific antibody is a monoclonal antibody, is the monoclonal hybridoma strain A-4-4 secretion generation to the colistin medicine of CGMCC No.2541 by deposit number.
(3) colistin standard items (going up sea cowry base bio tech ltd) 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; The solution of preparation standard items is that human albumin, the pH that contains 0.2-0.5% is 9.1-9.9,0.05-0.1mol/L carbonate buffer solution.
(4) developer is nitro phosphate buffer (a 4-nitrophenols phosphate buffer).
(5) stop buffer is 1～2mol/L sodium hydroxide solution.
(6) concentrated cleaning solution is for containing 0.8-1.3% bovine serum albumin, 45-60% glycerine, and the pH value is the carbonate buffer solution of 9.2-9.5,0.05-0.1mol/L; The 50ml/ bottle, 1 bottle; Described percentage composition is the quality percentage composition.
(7) concentrating redissolution liquid is that human albumin, the pH that contains 0.2-0.5% is 9.1-9.9,0.05-0.1mol/L carbonate buffer solution; The 400ml/ bottle, 1 bottle; Described percentage composition is the quality percentage composition.
Three, coating antigen is an antiantibody, and the enzyme labeling thing is an enzyme mark colistin
(1) principle of work
When on capillary strip, wrapping in advance by antiantibody, after adding colistin specific antibody is hatched, add sample solution or standard solution, add enzyme mark colistin solution again.Colistin in the sample or colistin standard items and enzyme mark colistin haptens competition colistin specific antibody, with the colour developing of colour developing liquid, the content of colistin becomes negative correlation in sample absorbance and the sample, relatively can draw the content of colistin in the sample with typical curve.Simultaneously also can be according to the shade on the ELISA Plate, by with the more rough judgement sample of series concentration colistin standard solution color in the concentration range of colistin.
(2) kit is composed as follows:
(1) be coated with the ELISA Plate of coating antigen: coating antigen is sheep anti mouse antiantibody or goat-anti rabbit antiantibody; The concentration of coating antigen can be 0.15-0.25 μ g/ml.
(2) enzyme labeling thing: the colistin of horseradish peroxidase-labeled;
(3) specific antibody working fluid: monoclonal antibody: by deposit number is the monoclonal hybridoma strain A-4-4 secretion generation to the colistin medicine of CGMCC No.2541.
(4) colistin standard items (going up sea cowry base bio tech ltd) 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; The solution of preparation standard items is that human albumin, the pH that contains 0.2-0.5% is 9.1-9.9,0.05-0.1mol/L carbonate buffer solution.
(5) substrate colour developing liquid: be made up of A liquid and B liquid, 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, 7ml/ bottle, 1 bottle.
(6) concentrated cleaning solution is for containing 0.8-1.3% bovine serum albumin, 45-60% glycerine, and the pH value is the carbonate buffer solution of 9.2-9.5,0.05-0.1mol/L; The 50ml/ bottle, 1 bottle; Described percentage composition is the quality percentage composition.
(7) concentrating redissolution liquid is that human albumin, the pH that contains 0.2-0.5% is 9.1-9.9,0.05-0.1mol/L carbonate buffer solution; The 400ml/ bottle, 1 bottle; Described percentage composition is the quality percentage composition.
(8) stop buffer: stop buffer is 1～2mol/L sulfuric acid or hydrochloric acid solution.
Claims (10)
1, a kind of enzyme linked immunological kit that detects colistin comprises the specific antibody of colistin and colistin; Described specific antibody is the polyclonal antibody or the monoclonal antibody of described colistin.
2, kit according to claim 1 is characterized in that: the specific antibody of described colistin and colistin exists with following any form:
1) described colistin and carrier protein are carried out coupling, obtain the conjugate of colistin and carrier protein, as coating antigen, described specific antibody carries out after the enzyme labeling as the enzyme labeling thing with it;
2) described specific antibody is a coating antigen, and described colistin carries out after the enzyme labeling as the enzyme labeling thing.
3, kit according to claim 1 is characterized in that: described kit also comprises antiantibody; Described antiantibody is sheep anti mouse antiantibody or goat-anti rabbit antiantibody;
Described colistin and antiantibody exist with following any form:
1) described colistin and carrier protein are carried out coupling, obtain the conjugate of colistin and carrier protein, as coating antigen, described antiantibody carries out after the enzyme labeling as the enzyme labeling thing with it;
2) described antiantibody is a coating antigen, and described colistin carries out after the enzyme labeling as the enzyme labeling thing.
4, according to claim 1,2 or 3 described kits, it is characterized in that: described monoclonal antibody is to be the antibody that secretion produces to the monoclonal hybridoma strain A-4-4 of colistin medicine of CGMCC No.2541 by deposit number.
5, according to arbitrary described kit among the claim 1-4, it is characterized in that: described kit also comprises colistin standard solution, colour developing liquid, stop buffer, concentrated cleaning solution, concentrates redissolution liquid; Described concentrated cleaning solution is for containing 0.8-1.3% bovine serum albumin, 45-60% glycerine, and the pH value is the carbonate buffer solution of 9.2-9.5,0.05-0.1mol/L; Described concentrated redissolution liquid is that the human albumin, the pH that contain 0.2-0.5% are 9.1-9.9,0.05-0.1mol/L carbonate buffer solution; Described percentage composition is the quality percentage composition.
6, according to arbitrary described kit among the claim 1-5, it is characterized in that: used marker enzyme is horseradish peroxidase or alkaline phosphatase in the described enzyme labeling; 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 is hydrogen peroxide or urea peroxide, and colour developing liquid B liquid is o-phenylenediamine or tetramethyl benzidine, and stop buffer is 1-2mol/L sulfuric acid or hydrochloric acid solution; When marker enzyme was alkaline phosphatase, developer was the nitro phosphate buffer, and stop buffer is the 1-2mol/L sodium hydroxide solution.
7, according to claim 5 or 6 described kits, it is characterized in that: described concentrated cleaning solution is to contain that 1.0% bovine serum albumin, 55% glycerine and pH value are 9.3, the carbonate buffer solution of 0.1mol/L; Described concentrated redissolution liquid is to contain that 0.4% human albumin, pH are 9.6, the 0.1mol/L carbonate buffer solution.
8, according to arbitrary described kit among the claim 1-7, it is characterized in that: the method that described antiantibody carries out enzyme labeling is to adopt the sodium periodate method that described marker enzyme and described antiantibody are carried out coupling to obtain enzyme and mark antiantibody; In the described sodium periodate method, the molar concentration rate of described marker enzyme and described antiantibody is 2: 1.
9, a kind of method that detects colistin may further comprise the steps:
1) sample pre-treatments
In every 1.0g animal tissue homogenate, add 9-12ml 0.03-0.07M sulfuric acid solution, mixing, place 37 ℃ of incubation 20-40min, under 5-10 ℃,, get supernatant liquid 3-5ml, add 140-170ml 1.5-2.5M sodium hydroxide solution with the centrifugal 5-15min of the speed more than the 3000g, mixing, adjust pH is 6-8, pipettes liquid, adds arbitrary described redissolution liquid among isopyknic claim 5-7, mixing, sampling is analyzed;
2) utilize that arbitrary described enzyme linked immunological kit detects 1 among the claim 1-8) described in dilution.
10, be the colistin monoclonal antibody that secretion produces to the monoclonal hybridoma strain A-4-4 of colistin medicine of CGMCC No.2541 by deposit number, or deposit number is the monoclonal hybridoma strain A-4-4 to the colistin medicine of CGMCC No.2541.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101184435A CN101358966B (en) | 2008-08-22 | 2008-08-22 | Method for detecting colimycin and special ELISA kit thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101184435A CN101358966B (en) | 2008-08-22 | 2008-08-22 | Method for detecting colimycin and special ELISA kit thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101358966A true CN101358966A (en) | 2009-02-04 |
| CN101358966B CN101358966B (en) | 2012-11-28 |
Family
ID=40331470
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008101184435A Expired - Fee Related CN101358966B (en) | 2008-08-22 | 2008-08-22 | Method for detecting colimycin and special ELISA kit thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101358966B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102368072A (en) * | 2011-06-30 | 2012-03-07 | 同昕生物技术（北京）有限公司 | Chemiluminescent enzyme-linked immunoassay kit for detecting concentration of cyclosporine A drug |
| GB2530776A (en) * | 2014-10-01 | 2016-04-06 | Randox Lab Ltd | Detection of polymyxins |
| CN107014991A (en) * | 2017-03-29 | 2017-08-04 | 广东工业大学 | A kind of peptide polyclonal antibody of growth hormone release inhibiting hormone 28 and preparation method thereof |
| CN109142760A (en) * | 2018-07-04 | 2019-01-04 | 中国农业大学 | A kind of kit quick and precisely detecting colistin in feed |
| CN109374907A (en) * | 2018-10-10 | 2019-02-22 | 北京纳百生物科技有限公司 | A kind of colistin gold-immunochromatographyreagent reagent for assay box and its application |
| CN111665307A (en) * | 2020-07-14 | 2020-09-15 | 南京品生医疗科技有限公司 | Kit for detecting concentrations of polymyxin B1and polymyxin B2 in serum |
| CN111735959A (en) * | 2019-03-25 | 2020-10-02 | 重庆乾泰生物医药有限公司 | Enzyme linked immunosorbent assay kit for detecting PF1022A |
-
2008
- 2008-08-22 CN CN2008101184435A patent/CN101358966B/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102368072A (en) * | 2011-06-30 | 2012-03-07 | 同昕生物技术（北京）有限公司 | Chemiluminescent enzyme-linked immunoassay kit for detecting concentration of cyclosporine A drug |
| GB2530776A (en) * | 2014-10-01 | 2016-04-06 | Randox Lab Ltd | Detection of polymyxins |
| US10620225B2 (en) | 2014-10-01 | 2020-04-14 | Randox Laboratories Limited | Detection of polymyxins |
| CN107014991A (en) * | 2017-03-29 | 2017-08-04 | 广东工业大学 | A kind of peptide polyclonal antibody of growth hormone release inhibiting hormone 28 and preparation method thereof |
| CN109142760A (en) * | 2018-07-04 | 2019-01-04 | 中国农业大学 | A kind of kit quick and precisely detecting colistin in feed |
| CN109142760B (en) * | 2018-07-04 | 2021-02-02 | 中国农业大学 | Kit for rapidly and accurately detecting colistin in feed |
| CN109374907A (en) * | 2018-10-10 | 2019-02-22 | 北京纳百生物科技有限公司 | A kind of colistin gold-immunochromatographyreagent reagent for assay box and its application |
| CN111735959A (en) * | 2019-03-25 | 2020-10-02 | 重庆乾泰生物医药有限公司 | Enzyme linked immunosorbent assay kit for detecting PF1022A |
| CN111665307A (en) * | 2020-07-14 | 2020-09-15 | 南京品生医疗科技有限公司 | Kit for detecting concentrations of polymyxin B1and polymyxin B2 in serum |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101358966B (en) | 2012-11-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101256188B (en) | ELISA kit for detecting lincomycin medicine as well as usage thereof | |
| CN101358966B (en) | Method for detecting colimycin and special ELISA kit thereof | |
| CN101013129B (en) | Enzyme linked immunosorbent reagent casing for detecting furacilin metabolite and uses thereof | |
| CN102955031B (en) | Enzyme-linked immunosorbent assay kit for detecting aflatoxin B1-containing medicine and application for same | |
| CN101571541B (en) | Enzyme-linked immunosorbent inspect kit for inspecting sulfa drugs and method thereof | |
| CN101839918B (en) | Method for detecting spiramycin and special enzyme-linked immunoassay kit thereof | |
| CN101413943B (en) | Method for detecting melamine and specific enzyme-linked immunologic reagent kit | |
| CN102967709B (en) | Detect enzyme linked immunological kit and the application thereof of zearalenone medicine | |
| CN102080066B (en) | Method for detecting T-2 toxin and special reagent kit thereof | |
| CN101358967B (en) | Method for detecting chlorpromazine and special ELISA kit thereof | |
| CN101571539A (en) | Elisa kit for detecting cephalo-type medicine and application thereof | |
| CN101013130A (en) | Enzyme linked immunosorbent reagent casing for detecting furantoin metabolite and uses thereof | |
| CN101776685B (en) | Enzyme linked immunosorbent assay kit for detecting trimethoprim medicament and application thereof | |
| CN101315378B (en) | Method and special ELISA reagent kit for detecting nitryl imidazole medicament | |
| CN101571540B (en) | Enzyme-linked immunosorbent kit for inspecting porcine immunoglobulin G and application thereof | |
| CN101021536A (en) | Enzyme-linked immunological kit for detecting neomycin drug and method | |
| CN101329339B (en) | Method for testing cattle immune globulin G and special-purpose enzyme-linked immunologic reagent kit thereof | |
| CN102080067B (en) | Method for detecting deoxynivalenol and special reagent kit thereof | |
| CN101021535B (en) | Enzyme-linked immunalogical kit for detecting gentamicin medicine and method | |
| CN101299046B (en) | Method for detecting erythromycin series compounds and special-purpose enzyme-linked immunologic reagent kit thereof | |
| CN101013131B (en) | Furantoin metabolite enzyme linked immunosorbent analytical reagent casing and uses thereof | |
| CN101782579B (en) | Enzyme-linked immunoassay kit for detecting tiamulin medicaments, and application thereof | |
| CN101349696A (en) | Enzyme-linked immunologic reagent and method for detecting alficetin | |
| CN103018451A (en) | Enzyme-linked immunoassay kit for chloramphenicol detection, and applications thereof | |
| CN101315377B (en) | Method for detecting monensin and its special ELISA reagent kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Free format text: FORMER OWNER: BEIJING WANGER BIOTECHNOLOGY CO., LTD. Effective date: 20120605 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20120605 Address after: 102206 Beijing city Changping Huilongguan international information industry base, four High Street No. 8 Applicant after: Beijing Wanger Biotechnology Co.,Ltd. Address before: 102206 Beijing city Changping Huilongguan international information industry base, four High Street No. 8 Applicant before: Beijing Wanger Kangtai Biotechnology Co.,Ltd. Co-applicant before: Beijing Wanger Biotechnology Co.,Ltd. |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20121128 Termination date: 20210822 |