CN101046474A - Enzyme-linked immunological kit for detecting quinoxaline medicine residue - Google Patents
Enzyme-linked immunological kit for detecting quinoxaline medicine residue Download PDFInfo
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Abstract
The enzyme-linked immunological kit for detecting 3-methyl quinoxaline-2-carboxylic acid (MQCA) as the quinoxaline medicine residue marker includes specific antibody for detecting MQCA and standard enzyme plate, standard enzyme secondary antibody and other enzyme-linked immunological agent coated with MQCA derivative and carrier protein coupler. The present invention is suitable for detecting mass samples, and has main reagent in liquid form, fast and convenient detecting process, high specificity, high sensitivity, high precision, high accuracy and other features.
Description
Technical field
The present invention relates to the detection of veterinary drugs in food analysis technical field, particularly a kind of Enzymoimmune reagent kit that detects quinoxaline medicine residue.
Background technology
Quinoxaline medicine is the feed medicated premix in China's consumption maximum, have antibiotic, growth promotion, the raising price of deed, improve effect such as livestock products quality, comprise that olaquindox, mequindox, quinocetone and China that China's approved uses do not ratify but has the medicines such as carbadox of a large amount of illegal uses.
Have and studies have shown that quinoxaline prototype medicine and metabolin exist tangible safety issue.For example, olaquindox and residue thereof can produce photosensitive toxicity, adrenal cortex infringement and water-electrolyte metabolism imbalance, chromosome aberration, genetoxic, the disorder of enteron aisle flora and hepatorenal damage etc.; Toxic reactions such as mequindox causes that functional disturbances of gastrointestinal tract, alimentary canal and respiratory mucosa are hemorrhage, body temperature descends, blood clotting is bad, liver and splenomegaly; Carbadox is the strongest kind of this class toxic, and carcinogenesis is arranged.But the phenomenon that quinoxaline medicine is abused in China's aquaculture is very serious, and for example: China allows olaquindox to be used for 35kg pig in the past, but not only is used for the production overall process of pig in the practice, but also uses in a large number on chicken, aquatic livestock and other animals; Mequindox approval be raw material and tablet, regulation is as the curative of swine dysentery and pig, ox bacillary enteritis, but practical ranges is extended.Therefore, the medicament residue that illegally uses this class medicine to be caused has constituted very big threat to the consumer health, and the detection of strengthening this class medicine in the animal food is very necessary.
3-Jia based quinoxaline-2-carboxylic acid (MQCA) is marker and the monitored object that the food additives joint specialist council (JECFA) under the World Food Programme (FAO) and the The World Health Organization (WHO) determines the De quinoxaline medicine residue.China owing to can't obtain MQCA, can only measure the tissue concentration of olaquindox original shape (rather than its residual marker) to the residual research of such medicine (olaquindox) in the past.In view of condition at that time, the minimum quantitative limit of used high-efficiency liquid chromatography method for detecting only about 0.5mg/kg, differs greatly with residual requirement of limiting the quantity of 50 μ g/kg (liver) and 4 μ g/kg (chicken).Therefore, can not reflect the truth that olaquindox is residual, these methods can not be used for the monitoring of olaquindox and residual marker thereof, more do not have to form the immune reagent kit that can monitor quinoxaline medicine residue in enormous quantities.
Summary of the invention
A kind of Enzymoimmune reagent kit that detects quinoxaline medicine residue of providing at the deficiencies in the prior art is provided, and it is highly sensitive, and is convenient and swift, economic and practical.
The object of the present invention is achieved like this:
A kind of Enzymoimmune reagent kit that detects quinoxaline medicine residue, it comprises the specific antibody of detection 3-Jia based quinoxaline-2-carboxylic acid (MQCA), is ELISA Plate, ELIAS secondary antibody, MQCA series concentration standard items, concentrated cleaning solution, developer, stop buffer, the concentrated liquid that redissolves of coating antigen bag quilt with MQCA derivant and carrier protein couplet thing.
The specific antibody of described detection MQCA is to be MQCA monoclonal antibody or the MQCA polyclonal antibody that the immunogen immune animal obtains with MQCA derivant and carrier protein couplet thing; MQCA monoclonal antibody or MQCA polyclonal antibody can be mouse source, rabbit source, Yang Yuan, cavy source and horse source antibody, described MQCA monoclonal antibody is preferably the MQCA mouse monoclonal antibody, described MQCA polyclonal antibody is preferably rabbit polyclonal antibody, and above antibody all can prepare according to a conventional method.
Described MQCA derivant has following general formula:
Wherein R is the (CH of 4-, 5-, 6-or 7-position
2)
nX, (CH
2)
nOH, (CH
2)
nNH
2, (CH
2)
nSH, n are 0 to 6 natural number, and X is Cl, Br or I.
Described carrier protein is bovine serum albumin(BSA) (BSA), human serum albumins (HSA), ovalbumin (OVA), hemocyanin (KLH), poly-D-lysine common carrier albumen such as (PLL).
Described MQCA derivant and carrier protein couplet thing as coating antigen is by two succinic acids-N-hydroxy-succinamide ester method (BSNHS method) or Toluene-2,4-diisocyanate, and 4-two isocyanide acid systems (TDIC method) chemical coupling forms.Described MQCA derivant that is used to wrap quilt and carrier protein couplet thing be by with MQCA derivant and carrier protein with two succinic acids-N-hydroxy-succinamide ester method (BSNHS method) or Toluene-2,4-diisocyanate, 4-two isocyanide acid systems (TDIC method) coupling obtains.
Marker enzyme in described ELISA Plate, the ELIAS secondary antibody is horseradish peroxidase or alkaline phosphatase, is preferably horseradish peroxidase, and horseradish peroxidase can be crosslinked on antibody by glutaraldehyde method or periodic acid method; Described two anti-sheep anti mouse or the goat-anti rabbit antiantibodys of being preferably.
Described ELISA Plate form by the plastic stent and the plastic strip in the cave with holes that separates separately and with MQCA derivant and carrier protein couplet thing be the coating antigen bag by and the filter membrane vacuum packaging.
Described concentrated cleaning solution is the phosphate buffer that contains the 0.8%-1.2% tween.
Described developer is made up of colour developing liquid A and colour developing liquid B, and colour developing liquid A is hydrogen peroxide or urea peroxide, and colour developing liquid B is o-phenylenediamine (OPD) or tetramethyl benzidine (TMB).
Described concentrated redissolution liquid is for containing the phosphate buffer of 0.1%-0.5% dimethyl sulfoxide (DMSO) (DMF).
Detection principle of the present invention detects the residual of monitoring quinoxaline medicine for indicate residue MQCA by enzyme immune detection quinoxaline medicine, being about to MQCA derivant and carrier protein couplet thing is adsorbed on the solid phase carrier as coating antigen, add sample and MQCA specific antibody, in the testing sample on residual MQCA and the solid phase carrier MQCA derivant of bag quilt compete specific antibody with the carrier protein couplet thing, the sheep anti mouse antiantibody that adds horseradish peroxidase-labeled, the colour developing back stops the working sample light absorption value.MQCA content in light absorption value and the sample is negative correlation, relatively can draw the MQCA content of sample with typical curve, and further infer the residual quantity that quinoxaline medicine in the sample.
The present invention mainly adopts the qualitative or sample Zhong quinoxaline medicines sign residue MQCA content such as detection by quantitative animal tissue, serum, urine sample and feed of indirect competitive enzyme-linked immunosorbent absorption (ELISA) method, thereby the monitoring quinoxaline medicine is residual; Pre-treatment requirement to sample is low, and sample pretreatment process is simple, simultaneously the fast detecting gross sample; Adopt the MQCA monoclonal antibody or the polyclonal antibody of high specific, main agents all provides with the working fluid form, and the method for inspection is convenient and easy; Have characteristics such as high specific, high sensitivity, high precision, pin-point accuracy, will in the detection of food and feed quinoxaline medicine residue, play a significant role.
Description of drawings
Fig. 1 is a structural representation of the present invention
Fig. 2 is ELISA Plate structural representation among the present invention
Embodiment
1, (to work as R is 6-CH in the preparation of MQCA derivant
2During OH the preparation that example is introduced the MQCA derivant in detail)
A) first step: 1 part of 5-methylol benzo furazan is dissolved in 2～6 parts the ethyl acetoacetate, in the presence of 2～3 parts of alkali, continues to stir 6～8 hours, yellow mercury oxide, be 3-methyl-6-methylol-2-carbethoxyl group quinoxaline-1,4-dioxide.Temperature of reaction is-10～50 ℃; The alkali that reaction is used for catalyzer can be organic base and inorganic base, and organic base comprises diethylamine, triethylamine, and trimethylamine, n-propylamine, pyridine, picoline, piperidines etc., inorganic base comprises NaOH, potassium hydroxide, calcium hydroxide, oxyammonia etc.
B) second step: 1 part of 3-methyl-6-methylol-2-carbethoxyl group quinoxaline-1, the 4-dioxide reacted 2～3 hours with 3～6 parts of reductive agents in suitable quantity of water, through chloroform extraction, and the evaporated under reduced pressure organic solvent, get white crystals, be 3-methyl-6-methylol-2-carbethoxyl group quinoxaline.Temperature of reaction is 10～100 ℃.Reductive agent comprises metallic reducing agent such as iron powder, zinc powder, zinc amalgam etc.; Sulphur and sulfide reductive agent such as sodium sulphide, sodium dithionate, sulphite etc.
C) the 3rd step: with 3-methyl-6-methylol-2-carbethoxyl group quinoxaline (pH10～14) heating hydrolysis 1 hour under alkali condition, cessation reaction, add appropriate amount of acid and adjust pH, separate out the flesh pink precipitation, be 3-methyl-6-methylol Oxoquinoxaline-2-carboxylic acid 3～4.Wherein temperature of reaction is between 50～100 ℃, and acid wherein is meant hydrochloric acid, sulfuric acid, and acetic acid and acid mixture thereof etc., alkali is meant NaOH, potassium hydroxide, calcium hydroxide, oxyammonia etc.
2, the preparation of antigen and antibody
(1) immunogene, coating antigen is synthetic
Adopt two succinic acids-N-hydroxy-succinamide ester method (BSNHS method) to carry out coupling and obtain immunogene in MQCA derivant and bovine serum albumin(BSA) (BSA).
MQCA derivant and ovalbumin (OVA) are adopted Toluene-2,4-diisocyanate, and 4-two isocyanide acid systems (TDIC method) carry out coupling and obtain coating antigen.
(2) preparation of MQCA mouse monoclonal antibody
A. the animal immune program adopts BALB/c mouse as immune animal, and as immunogene, immunizing dose is 80-100 μ g/ with MQCA and bovine serum albumin(BSA) conjugate.When head exempts from the Freund's complete adjuvant intermixture of antigen and equivalent is made emulsifying agent, the subcutaneous multi-point injection of nape portion, At intervals of two to three weeks are got the same dose immunogene and are added equivalent incomplete Freund mixing and emulsifying, and booster immunization once, four exempt from the pneumoretroperitoneum booster immunization once, extracting spleen cell after 3 days.
B. immune BALB/c mouse splenocyte is got in Fusion of Cells and cloning, and in 5-10: 1 ratio and SP2/0 myeloma cell are merged, and adopts indirect competitive ELISA to measure cell conditioned medium liquid, screens positive hole.Utilize limiting dilution assay that cloning is carried out in positive hole, up to the hybridoma cell strain that obtains the stably excreting monoclonal antibody.
C. cell cryopreservation and recovery are got the hybridoma that is in exponential phase and are made 1 * 10 with cryopreserving liquid
6-5 * 10
6The cell suspension of individual/mL is sub-packed in frozen pipe, 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. MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying adopt in the body and induce method, and crust match in 8 ages in week (BALB/c) mouse peritoneal is only injected sterilization paraffin oil 0.5mL/, 7-14 days pneumoretroperitoneum injection hybridomas 5 * 10
5-1 * 10
6Individual/as only, to gather ascites after 7-10 days.Carry out the ascites purifying through sad-saturated ammonium sulfate method, bottle packing ,-20 ℃ of preservations.
(3) preparation of MQCA rabbit polyclonal antibody
Adopting new zealand white rabbit as immune animal, is immunogene with MQCA derivant and bovine serum albumin(BSA) conjugate, and immunizing dose is 1mg/kg.b.w..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.Take a blood sample after last immune 7-10 days, measure serum antibody titer, the arteria carotis bloodletting obtains the polyclonal antibody of purifying through ammonium sulfate precipitation.
(4) two anti-preparations
As immune animal, is that immunogene carry out immunity with mouse or rabbit immunoglobulin IgG with sheep, obtains sheep anti mouse or goat-anti rabbit antiantibody.
3, detect the enzyme linked immunological kit of MQCA
(1) structure of the enzyme linked immunological kit of detection quinoxaline medicine residue
The structure of kit as shown in Figure 1, 2, mainly by box body 1, vacuum-packed 96 holes of filter membrane/40 hole ELISA Plate 2, MQCA series concentration standard items 3, enzyme labeling thing working fluid 4, MQCA antibody working fluid 5, substrate colour developing liquid A liquid 6, substrate colour developing liquid B liquid 7, stop buffer 8, concentrated cleaning solution 9, concentrated liquid 10, the foam carriage 11 of redissolving, be shaped on shrinkage pool on the foam carriage 11, mentioned reagent bottle 3-10 is placed in the shrinkage pool of foam carriage, and foam carriage 11 and ELISA Plate 2 are placed in the box body.Wherein ELISA Plate 2 is made up of plastic stent and detachable plastic strip.
(2) preparation of agents useful for same
A.MQCA standard solution: 6 bottles of MQCA series standard solution, 1-3mL/ bottle, 0 μ g/L, 0.01 μ g/L, 0.25 μ g/L, 2.5 μ g/L, 5 μ g/L, 10 μ g/L.
B. bag is cushioned liquid: pH9.6, the carbonate buffer solution of 0.05mol/L.
C. confining liquid: 3-10% sheep blood serum.
D. concentrated cleaning solution: contain the phosphate buffer (0.1M pH7.4) of 0.5-1% tween, for the 10-20 of normal working concentration doubly, 30-50mL/ bottle, 1 bottle.
E.MQCA antibody working fluid: it is that 0.5-5.0 μ g/mL uses 5-8mL/ bottle, 1 bottle that antibody dilution is become protein concentration.
F. enzyme labeling thing working fluid: the antiantibody dilution of anti-mouse of enzyme labeling or anti-rabbit, 5-8mL/ bottle, 1 bottle; Or the enzyme-labelled antigen dilution, 5-8mL/ bottle, 1 bottle.
G. substrate colour developing liquid A liquid: hydrogen peroxide or urea peroxide, 5-8mL/ bottle, 1 bottle.
H. substrate colour developing liquid B liquid: o-phenylenediamine (OPD) or tetramethyl benzidine (TMB), 5-8mL/ bottle, 1 bottle.
I. stop buffer: 1-2mol/L sulfuric acid or hydrochloric acid, 5-8mL/ bottle, 1 bottle.
J. concentrate to redissolve liquid: contain the phosphate buffer (0.1M pH7.4) of 0.51% tween, for the 10-20 of normal working concentration doubly, 30-50mL/ bottle, 1 bottle.。
(3) preparation of ELISA Plate
The ELISA Plate preparation method of MQCA derivant and ovalbumin conjugate bag quilt
Be cushioned liquid with bag MQCA derivant and ovalbumin conjugate are diluted to 0.1-1 μ g/mL, every hole adds 100 μ L, 37 ℃ of incubation 2h, and 4 ℃ are spent the night, coating buffer inclines, with cleansing solution washing 3 times, each 30 seconds, pat dry, in every hole, add 150-200 μ L confining liquid then, 37 ℃ of incubation 1-2h, liquid in the hole of inclining, preserve with the vacuum seal of aluminium film dry back.
4, residual De quinoxaline medicine in the test sample
(1) sample pre-treatments
A. animal tissue: take by weighing (5 ± 0.05) g animal tissue, place the 50mL centrifuge tube, add 1mol/L sodium hydroxide solution 10mL, the vortex mixing is put in 95～100 ℃ of water-baths hydrolysis 35min.Be cooled to room temperature, add 1mol/L hydrochloric acid 10mL, ethyl acetate 15mL, capping plug, hand vibration 20s, the centrifugal 5min of 4000r/min gets upper solution and changes separating funnel over to; Repeat to extract twice with ethyl acetate, merge upper solution in same separating funnel.Adding citric acid damping fluid 5mL, jolting 30s, standing demix is collected lower floor's solution to 25mL glass test tube with cover; Repeat to extract once with citrate buffer solution, merge lower floor's solution, add hydrochloric acid 2mL, standby.
B. serum: the centrifugal 20min of 3000 commentaries on classics/min, draw supernatant and also discard lower sediment., get 50 μ L and be used for detecting dilution (1 portion of supernatant+1 portion damping fluid) in its 1: 1 with damping fluid.
C. urine sample: the centrifugal 10min of 3500 commentaries on classics/min, abandon lower sediment and draw supernatant.Diluted (1 portion of supernatant+1 portion damping fluid) with damping fluid with its 1: 1 again, get 50 μ L and be used for detecting.
(2) detection method
In 96 hole ELISA Plate micropores of MQCA derivant and ovalbumin conjugate bag quilt, add series standard solution or sample solution (each 2 hole) 50 μ L, add MQCA mouse monoclonal antibody working fluid 50 μ L then, with cover plate film shrouding, react 30min in 37 ℃ of constant temperature ovens.Pour out liquid in the hole, every hole adds 250 μ L cleansing solutions, pours out liquid in the hole after 30 seconds, pats dry with thieving paper, and so repetitive operation is washed plate 5 times altogether.The sheep anti mouse antiantibody working fluid 100 μ L that add horseradish peroxidase-labeled with cover plate film shrouding, react 1h in 37 ℃ of constant temperature ovens.Take out ELISA Plate, wash plate as described above 5 times.Every hole adds substrate colour developing liquid A liquid 50 μ L, adds substrate colour developing liquid B liquid 50 μ L again, the mixing that vibrates gently, 37 ℃ of constant temperature oven lucifuge colour developing 15-30min.Every hole adds stop buffer 50 μ L, and the mixing that vibrates is gently measured every hole absorbance (OD value) with microplate reader.
The used reagent of present embodiment is all prepared according to the reagent compound method in the step 3.
(3) interpretation of result
Each the concentration standard solution that is obtained and the mean value (B) of sample absorbance are divided by the absorbance (B of first standard (0 standard)
0) multiply by 100 again, i.e. percentage absorbance.
Percentage absorbance (%)=B/B
0* 100
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.Natural logarithm value with MQCA concentration (μ g/L) is an X-axis, and the percentage absorbance is a Y-axis, the drawing standard curve map.The concentration of corresponding each sample (μ g/L) can be read from typical curve.Also can use regression equation method, calculate sample solution concentration.Utilize computer professional software, the express-analysis of a large amount of samples of being more convenient for.
According to the depth of the color sample on the ELISA Plate, but with the concentration range of the comparison judgement sample of the standard solution color of series concentration.
5, kit sensitivity, specificity, precision, accuracy and storage life test
(1) kit sensitivity test
50% inhibition concentration (is IC
50, refer to the drug concentration of 50% place correspondence of the absorbance of 0 μ g/L standard solution) and be usually used in estimating the index of competitive ELISA kit sensitivity.Measure 50% inhibition concentration of 20 typical curves respectively, determine this kit standard curve I C
50Should be in the scope of 0.5-1 μ g/L.Measure the blank sample of 20 parts of chicken, chicken gizzard, pork, pork liver, serum and urine samples and 0 standard items of 20 parts of MQCA respectively.The result judges that this kit is limited to 0.02 μ g/L to the 0 standard items lowest detection of MQCA.To the lowest detectable limit of chicken, chicken gizzard, pork, pork liver, serum and urine sample all less than 0.05 μ g/L.
(2) kit specificity test
Selection is Yu quinoxaline medicine and similar several drugs are measured cross reacting rate.Typical curve by various medicines obtains its 50% inhibition concentration respectively.Calculate the cross reacting rate of kit with following formula to other drug.Cross reacting rate is littler, shows that the specificity of kit is higher.
Cross reacting rate (%)=cause 50% suppresses the concentration of MQCA/cause 50% analog concentration * 100 that suppress
It is as shown in the table for kit specificity test findings, the cross reacting rate that the result shows this kit Dui quinoxaline medicine olaquindox, mequindox, quinocetone, carbadox all＜1%, to the cross reacting rate of sulfadimidine, chloromycetin, penicillin all＜0.01%, this kit is described at MQCA specificity height, can guarantees reliability animal-derived food and feed Zhong quinoxaline medicine residue testing result.
(3) kit precision test
A. standard repeatability
From three batches of ELISA Plate, every plate is extracted 20 micropores out, measures same concentration standard solution absorbency value (OD value), and replication 10 times calculates the coefficient of variation.Coefficient of variation scope is at 4.2%-10.1%.
B. sample repeatability
Get the MQCA standard items of variable concentrations, add in the sample and go, get each three of the kits of three different batches respectively, each concentration repeats 5 times.Calculate in the plate interassay coefficient of variation respectively.The Variation Lines number average of liver is lower than 10% as a result, and the Variation Lines number average of chicken is lower than 9.6%, and the Variation Lines number average of serum, urine sample is lower than 8%, and the Variation Lines number average of feed is lower than 8.5%.
(4) accuracy of kit
Get the MQCA standard items of 5 concentration, sample added recovery test, each concentration establish 4 parallel, calculate recovery rate respectively.The interpolation recovery of pork, pork liver, chicken, chicken gizzard, serum, urine sample is all in the 70%-115% scope.
(5) kit storage life test
The kit preservation condition is 2-8 ℃, and through 6 months mensuration, the maximum absorbance value of kit (0 standard), 50% inhibition concentration, MQCA added the practical measurement value all in normal range.In the process of considering transportation and using, have the situation that exceeds preservation condition and occur, kit was placed 7 days under the condition of 37 ℃ of preservations, quicken failure test, the result shows that the every index of this kit meets the requirements fully; Kit is positioned over-20 ℃ freezing 10 days, measurement result shows that also the every index of kit is normal fully.Can find out that from above result this kit can preserve more than 6 months at least at 2-8 ℃.
Claims (10)
1, a kind of enzyme linked immunological kit that detects the quinoxaline medicine residue thing is characterized in that it comprises the specific antibody of detection 3-Jia based quinoxaline-2-carboxylic acid (MQCA), is ELISA Plate, ELIAS secondary antibody, MQCA series concentration standard items, concentrated cleaning solution, developer, stop buffer, the concentrated liquid that redissolves of coating antigen bag quilt with MQCA derivant and carrier protein couplet thing.
2, according to enzyme linked immunological kit as claimed in claim 1, the specific antibody that it is characterized in that described detection MQCA is to be MQCA monoclonal antibody or the MQCA polyclonal antibody that the immunogen immune animal obtains with MQCA derivant and carrier protein couplet thing.
3, enzyme linked immunological kit according to claim 1 and 2 is characterized in that described MQCA derivant has following general formula:
Wherein R is the (CH of 4-, 5-, 6-or 7-position
2)
nX, (CH
2)
nOH, (CH
2)
nNH
2, (CH
2)
nSH, n are 0 to 6 natural number, and X is Cl, Br or I.
4, enzyme linked immunological kit according to claim 1 and 2 is characterized in that described carrier protein is bovine serum albumin(BSA), human serum albumins, ovalbumin, hemocyanin, poly-D-lysine.
5, enzyme linked immunological kit according to claim 1, it is characterized in that described MQCA derivant and carrier protein couplet thing as coating antigen is by two succinic acids-N-hydroxy-succinamide ester method (BSNHS method) or Toluene-2,4-diisocyanate, 4-two isocyanide acid systems (TDIC method) chemical coupling forms.
6, enzyme linked immunological kit according to claim 1 is characterized in that the marker enzyme in described ELISA Plate, the ELIAS secondary antibody is horseradish peroxidase or alkaline phosphatase, and two anti-are anti-mouse or anti-rabbit antiantibody.
7, enzyme linked immunological kit according to claim 1, it is characterized in that described ELISA Plate is made up of the plastic stent and the plastic strip in the cave with holes that separates separately and with MQCA derivant and carrier protein couplet thing be the coating antigen bag by and the filter membrane vacuum packaging.
8, enzyme linked immunological kit according to claim 1 is characterized in that described concentrated cleaning solution is the phosphate buffer that contains the 0.8%-1.2% tween.
9, enzyme linked immunological kit according to claim 1 is characterized in that described developer is made up of colour developing liquid A and colour developing liquid B, and colour developing liquid A is hydrogen peroxide or urea peroxide, and the liquid B that develops the color is o-phenylenediamine (OPD) or tetramethyl benzidine (TMB).
10, enzyme linked immunological kit according to claim 1 is characterized in that described concentrated redissolution liquid is for containing the phosphate buffer of 0.1%-0.5% dimethyl sulfoxide (DMSO) (DMF).
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| CN101144816B (en) * | 2007-10-31 | 2011-05-11 | 江南大学 | 3-methyl-quinoline-2-carboxylic acid immunomagnetic bead detection method |
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| CN105061339A (en) * | 2015-07-02 | 2015-11-18 | 中国兽医药品监察所 | Semiantigen, artificial antigen thereof, and application of semiantigen in detection of olaquindox residual marker |
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| CN101144816B (en) * | 2007-10-31 | 2011-05-11 | 江南大学 | 3-methyl-quinoline-2-carboxylic acid immunomagnetic bead detection method |
| CN102617493A (en) * | 2012-02-22 | 2012-08-01 | 中国农业大学 | Mequindox artificial antigens and antibodies prepared by same |
| CN102617493B (en) * | 2012-02-22 | 2014-06-04 | 中国农业大学 | Mequindox artificial antigens and antibodies prepared by same |
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