CN101275947A - Sulfalene oxazole monoclonal antibody reagent kit and detecting method - Google Patents
Sulfalene oxazole monoclonal antibody reagent kit and detecting method Download PDFInfo
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- CN101275947A CN101275947A CNA2008100974145A CN200810097414A CN101275947A CN 101275947 A CN101275947 A CN 101275947A CN A2008100974145 A CNA2008100974145 A CN A2008100974145A CN 200810097414 A CN200810097414 A CN 200810097414A CN 101275947 A CN101275947 A CN 101275947A
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Abstract
The present invention discloses an enzyme linked immunoreaction reagent kit for testing Sulfamethoxazole and a test method thereof. The enzyme linked immunoreaction reagent kit which is provided by the invention for testing Sulfamethoxazole comprises species-specific monoclonal antibody of Sulfamethoxazole and encapsulated coupling compound of the Sulfamethoxazole and the carrier protein and enzyme marking secondary antibody. The monoclone antibody is obtained from the secretion of hybriddoma cell line (preservation number for CGMCC No.2411). The enzyme linked immunoreaction reagent kit for detecting Sulfamethoxazole can simultaneously and quickly detect large quantities of samples. The main reagent is provided with the form of working liquid and the test method is convenient and practical. The invention has the characteristics of high specificity, high sensitivity, high precision, high accuracy and the like. The invention can exert an important function in the test of Sulfamethoxazole residue in animal food and feed stuff.
Description
Technical field
The present invention relates to a kind of enzyme linked immunological kit and detection method that detects Sulfamethoxazole in the detection of veterinary drugs in food analysis technical field.
Background technology
Sulfamethoxazole is one of widely used sulfamido microbiotic, it has controlled Animal diseases effectively, promotes growth of animal, aspect the animal derived pathogen discharging of reduction improvement public health, brought into play and important effect, obtained good economic benefit and social benefit; But long-term and heavy dose of the use makes sulfa drug accumulate in vivo, causes medicine residual in human body and animal tissue, thereby potential hazard health.Accumulating of sulfa drug can cause the drug-fast generation of sulfa drug in the human body, and has potential carcinogenic, mutagenicity.Therefore, Codex Committee on Food of the United Nations (CAC) and many national regulations, the single content of sulfamido material such as Sulfamethoxazole all must not surpass 100 μ g/kg in food and the feed.
The method that detects the sulfalene oxazole relict amount mainly contains microbial method, instrument detecting method, immuno-chemical method.Wherein the instrument detecting method comprises fluorophotometric method, thin-layered chromatography (TLC), vapor-phase chromatography (GM), gas-matter on-line method (GC/MS), high performance liquid chromatography (HPLC), liquid-matter on-line method (HPLC/MS), capillary electrophoresis (CE) etc., because complicated instrument and equipment and loaded down with trivial details process are not suitable for on-site supervision and great amount of samples examination.
The innovation and creation content
The purpose of this invention is to provide a kind of enzyme linked immunological kit that detects Sulfamethoxazole.
The monoclonal antibody reagent kit of detection Sulfamethoxazole provided by the present invention comprises Sulfamethoxazole specific monoclonal antibody, Sulfamethoxazole and the carrier protein couplet thing and the ELIAS secondary antibody of wrapping quilt.
Wherein, described Sulfamethoxazole specific antibody is a Sulfalene oxazole monoclonal antibody, is preferred Sulfamethoxazole mouse monoclonal antibody, and this monoclonal antibody prepares according to a conventional method; Described carrier protein can be bovine serum albumin(BSA) (BSA), human serum albumins (HSA), ovalbumin (OVA), hemocyanin common carrier albumen such as (KLH), and the conjugate of described Sulfamethoxazole and carrier protein can obtain by Sulfamethoxazole and carrier protein are carried out coupling with glutaraldehyde method or diazotising method; Described marker enzyme can be horseradish peroxidase or alkaline phosphatase, is preferably horseradish peroxidase, horseradish peroxidase can by glutaraldehyde method or sodium periodate method crosslinked two anti-on; Described two anti-anti-mouse or the anti-rabbit antiantibodys of being preferably.
For more convenient on-site supervision and great amount of samples examination, the mentioned reagent box also comprises Sulfamethoxazole standard solution, developer, colour developing dilution, concentrated cleaning solution, stop buffer.
Described developer is made up of colour developing liquid A liquid and colour developing liquid B liquid, and described colour developing liquid A liquid is hydrogen peroxide, and described B liquid is tetramethyl benzidine (TMB), and described colour developing dilution is a sodium acetate solution; Described concentrated cleaning solution is the phosphate buffer of 0.05%-1.2% Tween-20; Described stop buffer is the sulfuric acid solution of 1-2mol/L.
Detection principle of the present invention is adsorbed on the solid phase carrier as coating antigen for the conjugate with Sulfamethoxazole and carrier protein, add sample and Sulfalene oxazole monoclonal antibody, add ELIAS secondary antibody, the colour developing back stops, test sample product light absorption value, sulfalene oxazole relict thing content is negative correlation in this value and the sample, relatively can draw the content of Sulfamethoxazole with typical curve, can draw roughly concentration range according to the depth of color sample on the ELISA Plate.
The enzyme linked immunological kit of detection Sulfamethoxazole of the present invention mainly adopts the residual quantity of Sulfamethoxazole in the qualitative or samples such as detection by quantitative animal tissue, serum, urine sample, milk and feed of indirect competitive ELISA method; To sample pre-treatments require low, sample pretreatment process simple, fast detecting gross sample simultaneously; Adopt the Sulfalene oxazole monoclonal antibody of high specific, main agents all provides with the working fluid form, and detection method is simple and easy to do; Have characteristics such as high specific, high sensitivity, pinpoint accuracy, pin-point accuracy, will in the detection of food and feed sulfalene oxazole relict amount, play a significant role.
Description of drawings
Fig. 1 is the structural representation of the enzyme linked immunological kit of detection Sulfamethoxazole
Fig. 2 is the Sulfamethoxazole typical curve
Embodiment
The preparation of embodiment 1 antigen and antibody
1. the preparation of Sulfamethoxazole-carrier protein couplet thing
(1) with reference to FrnakeM in 1999, the etal. reported method was synthesized the SMZ-HSA holoantigen, and concrete steps are as follows: at first get 20mg SMZ and be added in the 300 μ l dimethyl formamide solutions, slowly be added to 1ml 0.5mol/LH again
2SO
4In, then under magnetic agitation with 1ml1mol/L NaNO
2Slowly be added in the SMZ solution, reaction 10min forms diazo salt.
(2) under the magnetic agitation, get 100mg HSA respectively and 100mg OVA respectively is added in the 4ml phosphate buffer.
(3) the diazotising salt solusion with above-mentioned formation slowly is added under magnetic agitation in HSA solution and the OVA solution, with 1MNaOH pH is maintained 8-10,4 ℃ of reaction 1h;
(4) reactant liquor is placed on the 3d that dialyses in the physiological saline, changes physiology salt solution every day 2 times, to remove unreacted small-molecule substance and SMZ etc.
(5) with orange SMZ-HSA and SMZ-OVA solution separated into two parts, a part is preserved down for ultraviolet and liquid phase in 4 ℃ and is tested usefulness, and another part is preserved down in-20 ℃.
2. the preparation of anti-Sulfalene oxazole monoclonal antibody
(1) immunity: utilize the prepared Sulfamethoxazole-human serum albumins of applicant (HSA) conjugate immunity Balb/C mouse (available from the PLA Academy of Military Science Experimental Animal Center), the program of being excused from an examination is to select 4 of female Balb/c mouse in age in 6-8 week for use, body weight 18-22g, get immunizing antigen 100mg (calculating) with protein content, add the emulsification of equal-volume complete Freund's adjuvant, with syringe suction stirring repeatedly in the ampere bottle, in water, be not drips of solution (oil-in-water state) until the emulsification soup.Subcutaneous multi-point injection carries out exempting from Balb/C mouse carotid back, every mouse 0.5ml; After two weeks, the antigen amount reduces by half and mixes with incomplete Freund at interval, and method is the same, carries out the immunity second time, after two weeks, carries out immunity for the third time, and method together last time.Three exempt from back 10d immunized mice tail vein blood, measure the immune mouse serum antibody titer by indirect ELISA method.Select antibody titer the higher person to be used for Fusion of Cells, merge the first two day to contain 30 μ g immunizing antigen aquas for the mouse intrasplenic injection to carry out booster immunization.
(2) monoclonal antibody preparation: after the immunized mice eye socket was got blood, the vertebra dislocation was put to death, and sterilization is placed in the disinfecting surface ware on the superclean bench, the careful spleen that takes out through immunity enlargement, and with DMEM nutrient culture media flushing 2 times, each 3ml.Have 5.6 * 10 through counting cells
8Individual, in 37 ℃ of water-baths, SP2/0 cell (being provided by life institute of Peking University monoclonal antibody chamber) and splenocyte are mixed in the 50ml centrifuge tube in 10: 1 ratios, stir evenly gently, the centrifugal 5min of 1000rpm abandons supernatant, makes cell be mixed into pasty state at the bottom of the attack pipe.Centrifuge tube is placed 37 ℃ of water-baths, suction pipe is drawn the 0.8ml 50%PEG1500 (available from Sigma company) of 37 ℃ of preheatings, with the bottom that suction pipe inserts centrifuge tube, left hand is coil slowly, and right-hand side stirs the limit and adds, add in the 1min, to merge liquid and slowly suck in the suction pipe, keep 0.5min, slowly emit then, emit fully in the 1min, and in centrifuge tube, leave standstill 1min.After leaving standstill, immediately along the incomplete nutrient culture media of tube wall Dropwise 5 ml IDMEM (prescription: DMEM (packed) 13.5g, Benzylpenicillin sodium salt (injection) 100,000 units, streptomycin sulphate (injection) 100,000 units, NaHCO
33.7g, be diluted to 1000ml with redistilled water, and to regulate pH with 0.1mol/L NaOH or HCl be 7.4) and stop fusion reaction, the limit edged slowly stirs, and preceding 0.5min adds 2ml, and back 0.5min adding 3ml adds 5ml then in 1min.Add the IDMEM nutrient culture media to 40ml, the centrifugal 5min of 1000rpm abandons supernatant.Suction pipe is drawn 1ml HAT (prescription: 20ml hyclone, 1mlHAT, 79ml DMEM is made into 100ml) select nutrient culture media after, insert the centrifuge tube bottom, the limit is stirred the limit gently and is added, make cell suspension, can not blow, add HAT to 40ml then, and mixing cell gently, every hole 0.1ml adding has been completed in 96 orifice plates of feeder cells, spreads 4 blocks of plates altogether.Change the HAT nutrient solution next day of merging the back.Merge after 3-4 days, as seen cell clone is in a small amount arranged, after merging 10 days, use HT nutrient culture media (prescription: 20ml hyclone, 1ml HT instead, 80ml DMEM is made into 100ml), greatly about about 12-16 days, hybridoma cell clone grows at the bottom of the hole 1/4, adopts indirect ELISA to detect specific antibody in the nutrient solution supernatant, statistics has the hole count of hybridoma growth and positive hybridoma cell growth, calculates fusion rate and positive rate.Adopt Sulfamethoxazole-ovalbumin as screening antigen, utilize the ELISA method to filter out the positive hole of the anti-Sulfamethoxazole of secretion.The positive hole that screens at once with the ratio of limiting dilution assay in 10,5,2 cells in every hole, is put into the culture plate of completing feeder cells in advance with cell and cultivated.Observe cloning cell number and state, and in time measure every hole supernatant positive, until finding positive monoclonal cell strain, finishing screen is selected the monoclonal hybridoma system of the anti-Sulfamethoxazole of secretion, this clone is numbered A-7-1, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), deposit number: CGMCC No.2411 on March 21st, 2008.
This clone is carried out chromosome counting, the result shows: SP2/0 myeloma cell's chromosome number is 36 pairs, the chromosome number of Balb/C mouse spleen cell is 20 pairs, and this hybridoma chromosome number is between the 96-102 bar, as seen the chromosome number of the cell strain of monoclonal antibody of this test gained is similar substantially to the chromosome number sum of two parent's cells, proves that this cell is the product of two parent's Fusion of Cells.With this clone injection Balb/C mouse peritoneal, produce monoclonal antibody.Employing is available from the mouse monoclonal antibody typing Immunotype of Sigma company
TMKit carries out hypotype to the resulting monoclonal antibody of the present invention and identifies that the result is mouse IgG
1Subclass.
3, the purifying of anti-Sulfalene oxazole monoclonal antibody
Concrete steps are as follows: the HAc-NaAc damping fluid of 4ml 0.06mol/L pH4.8 is divided into two parts, and major part is mixed with 1ml ascites, and stirs gently; Other has a spot of HAc-Aac damping fluid and sad (needing sad 33 μ L altogether) to be mixed into emulsion.Sad emulsion slowly is added dropwise in the ascites solution, and the limit edged stirs gently, adds the back and continues to stir 30min, and a large amount of white precipitates is arranged this moment.The centrifugal 30min of 12000rpm stays supernatant to discard precipitation under the room temperature, drips the saturated ammonium sulfate solution of 4 ℃ of pH7.4 of equal-volume, the limit edged stirs, and transferring pH with 1.0mol/LNaOH solution is 7.4, and solution is separated out a large amount of precipitations, after 4 ℃ of solution leave standstill 2h, 4 ℃ of centrifugal 30min of 12000rpm.Remove supernatant, resolution of precipitate in 0.01mol/L pH7.4PBS, to PBS dialysis desalination 4 times, is dripped 0.02%NaN in the final antibody solution
3, divide to be installed on-20 ℃ of preservations.The mensuration protein concentration is 8.25mg/ml.Use MbaTrap
TMGThe affinity column antibody purification, (PB, 0.02mol/LpH7.0) after the suitable dilution, last sample is used for the good MbaTarP of level pad balance to ascites through level pad
TMGAffinity column behind the thorough flush away foreign protein of level pad, changes eluent (Gly-HCI, 0.01mol/L, pH2.7) antibody is washed, the antibody that washes is used neutralization buffer (Tris-HCI, 1mol/L immediately, PH9.0) neutralization returns to about 7.0 pH, in order to avoid lose activity.
4, the preparation of Sulfamethoxazole rabbit polyclonal antibody
3 of new zealand white rabbits, average weight 2.5kg, single cage is raised.Head exempts from: get an amount of synthetic immunogene, and with the Freund's complete adjuvant mixing of sterile saline dilution back, fully emulsified with asepsis injector with equivalent.Every rabbit injections of antigens amount is 1mg/mL, through back intracutaneous multi-point injection; Two are replaced by incomplete Freund with Freund's complete adjuvant when exempting to exempt from three, the subcutaneous multi-point injection of nape portion, and three exempted from the back the 7th day, and rabbit ear edge vein is adopted the 1mL whole blood, gets serum, measures serum titer with indirect ELISA.Tire reach requirement after, carry out booster immunization, the 7th day arteria carotis blood sampling behind the booster immunization.Obtain the polyclonal antibody of purifying through the saturated ammonium sulphate method.
5, two preparations that resist
As immune animal, is that immunogene carry out immunity with mouse or rabbit igg with sheep, obtains sheep anti mouse or goat-anti rabbit antiantibody
Embodiment 2 detects the Sulfalene oxazole monoclonal antibody reagent kit assembling
1. the structure of Sulfalene oxazole monoclonal antibody reagent kit
The structure of kit mainly comprises as shown in Figure 1: 1 box body, the vacuum-packed 96 hole ELISA Plate of 2 aluminium films, 3 standard Sulfamethoxazole antigens, 4 enzyme conjugates working fluids, 5 Sulfalene oxazole monoclonal antibody working fluids, 6 colour developing liquid A liquid, 7 colour developing liquid B liquid, 8 colour developing liquid dilutions, 9 concentrated cleaning solutions, 10 stop buffers, 11 foam carriages have shrinkage pool on the foam carriage, the mentioned reagent bottle all is placed in the shrinkage pool, and foam carriage and ELISA Plate are placed in the box body.Wherein ELISA Plate is made up of plastic stent and detachable plastic strip.
2. the preparation of agents useful for same
A standard Sulfamethoxazole antigen: 6 bottles of Sulfamethoxazole standard solution, its concentration range is at 1-1000 μ g/L.
B coating buffer: PH9.6, the carbonate buffer solution of 0.05mol/L.
C confining liquid: 10% calf serum.
The D concentrated cleaning solution: (0.01M PH7.4), is 15 times of normal working concentration to the phosphate buffer of 0.05%-1.2% tween, 1 bottle.
E Sulfalene oxazole monoclonal antibody working fluid: antibody is carried out 10
4Doubly dilution is used, and promptly contains the Sulfamethoxazole mouse resource monoclonal antibody of 0.3-30mg/L, 6-10ml/ bottle, 1 bottle.
F enzyme conjugates working fluid: the antiantibody dilution of the anti-mouse of enzyme labeling, 6-10ml/ bottle, 1 bottle.
G substrate colour developing liquid A liquid: hydrogen peroxide, 6-10ml/ bottle, 1 bottle.
H substrate colour developing B liquid: tetramethyl benzidine (TMB) 1ml/ bottle, 1 bottle.
I colour developing liquid dilution: 0.2mol/L sodium acetate solution, 10ml/ bottle, 1 bottle.
J stop buffer: 1-2M sulfuric acid solution, 6-10ml/ bottle, 1 bottle.
3. the preparation of ELISA Plate
The ELISA Plate of Sulfamethoxazole and ovalbumin conjugate bag quilt, the preparation method is as follows:
Be cushioned liquid with bag Sulfamethoxazole and ovalbumin conjugate are diluted to 1 μ g/ml, every hole 100 μ l, 4 ℃ are spent the night, and remove coating buffer, and it is inferior to give a baby a bath on the third day after its birth with cleansing solution, each 3min, pat dry, add 200 μ l confining liquids in every then hole, 37 ℃ of incubation 2h, remove liquid in the hole, preserve with the vacuum seal of aluminium film dry back.
4. the preparation of ELIAS secondary antibody
Sodium periodate method with improvement is come the mark horseradish peroxidase.
Residual Sulfamethoxazole in embodiment 3 test sample
1. sample pre-treatments
(1) pre-service of pig urine
To get supernatant liquid evaporate to dryness or in nitrogen stream, dry up phosphate buffer (the PBS) (prescription: NaCl 8g, KCl 0.2g, Na of usefulness 1ml 0.01M pH7.4 for the centrifugal 10min of examination urine 3000rpm
2HPO
412H
2O 2.9g, KH
2PO
40.2g) dissolution residual substance.
(2) pre-service of milk sample
Milk sample is through the centrifugal 10min of 3500rpm, get 3ml and add the 6ml ethyl acetate 10min that vibrates up and down, the centrifugal 10min of room temperature 3000rpm gets 2ml supernatant liquid evaporate to dryness or dry up phosphate buffer (PBS) dissolution residual substance of usefulness 1ml 0.01M pH7.4 in nitrogen stream.
(3) pre-service of tissue sample
With tissue sample homogenate to be checked, get 3g tissue sample to be checked and add the 6ml ethyl acetate 10min that vibrates up and down, the centrifugal 10min of room temperature 3000rpm gets 2ml supernatant liquid evaporate to dryness or dry up phosphate buffer (PBS) dissolution residual substance of usefulness 1ml 0.01M pH7.4 in nitrogen stream.
(4) pre-service of egg sample
Draw 3ml yolk and add the 6ml ethyl acetate 10min that vibrates up and down, the centrifugal 10min of room temperature 3000rpm gets 2ml supernatant liquid evaporate to dryness or dries up in nitrogen stream, with phosphate buffer (PBS) dissolution residual substance of 1ml 0.01M pH7.4.
(5) feed The pretreatment
Take by weighing feed 1g, add methanol-water solution, Extraction by Ultrasound 30min gets the liquid nitrogen air-blowing of 3ml upper strata and does, with phosphate buffer (PBS) dissolution residual substance of 1ml 0.01MpH7.4.
2. detection method;
(1) the sample 50 μ l of preincubate 30min or the variable concentrations standard antigen 50 μ l of hatching 30min add Sulfalene oxazole monoclonal antibody working fluid 50 μ l then in 96 each hole of hole ELISA Plate, hatch 30min for 37 ℃, wash three times, pat dry.
(2) add ELIAS secondary antibody: every hole adds 5000 * sheep anti mouse ELIAS secondary antibody, 100 μ l, hatches 30min for 37 ℃, washs five times, pats dry.
(3) add substrate: add substrate solution, every hole 100 μ l, room temperature is placed 5min.
(4) cessation reaction: add stop buffer 50 μ l, mixing gently vibrates.
(5) reading: microplate reader is measured the absorption photometric value (OD value) in every hole.
3. interpretation of result
(1) calculates each standard model, treat the OD450 value of test sample and reference sample.
(2) concentration with standard model is X-axis, is the Y-axis mapping with the inhibiting rate, and this figure is a linear graph,
Wherein, inhibiting rate=(with reference to OD450-OD450 to be measured)/reference opening * 100%
(3) utilize typical curve to calculate antigen amount in the testing sample
As shown in Figure 2.The concentration of corresponding each sample can be read from typical curve, also can use regression equation method, calculates sample solution concentration, utilizes computer professional software, is more conducive to the express-analysis of batch samples.
According to the depth of the color sample on the ELISA Plate,, can judge the sample concentration approximate range with the comparison of the standard solution color of series concentration.
The sensitivity of embodiment 4 kits, specificity, the mensuration of precision and storage life experiment
(1) mensuration of sensitivity
The definition of lowest detectable limit has multiple, and 10% expression lowest detectable limit of inhibiting rate is adopted in this test.According to test findings, determine that lowest detection is limited to 10ng/mL.
(2) specific mensuration
Select 7 kinds of sulfonamides,, measure its typical curve, and calculate inhibition concentration, calculate cross reacting rate the sulfa drug replacement SMZ standard solution of variable concentrations.Formula: the SMZ concentration that cross reacting rate=50% suppresses/50% competition substrate concentration * 100%.
The result shows among the figure, kit to other in 7 the cross reacting rate of sulfa drug all be lower than 0.1%, illustrate that this kit is very high at the Sulfamethoxazole specificity, can guarantee reliability to sulfalene oxazole relict testing result in animal foodstuff and the feed.
(3) precision test
Precision can be described as repeatability again, and the reflection assay method is to the repetition degree of repeatedly measuring the gained result of a certain specific sample, and this is the basic parameter of quality assessment, can represent with the coefficient of variation.
Plate within variance coefficient: from numerous samples, randomly draw 6 samples and detect, 8 repetitions of each sample according to the kit detecting pattern.
The coefficient of variation between plate: 5 blocks of plates are selected in this test altogether, 3 repetitions of every block of plate.Randomly draw 2 samples and carry out the detection of the coefficient of variation between plate.
(4) storage life experiment
The kit preservation condition is 2-8 ℃, and through 6 months mensuration, the maximum absorbance value of kit, Sulfamethoxazole added practical measurement value, 50% concentration all within normal range.Do accelerated aging test simultaneously, kit is placed on following 6 days of 37 ℃ of states, measurement result shows that the every index of kit meets the requirements fully, again kit is put into-20 ℃ freezing 6 days, measurement result shows that also the every index of kit is normal fully.Can obtain kit from above result can preserve more than 6 months at least at 2-8 ℃.
Although content of the present invention is to describe in conjunction with present embodiment, can not think limitation of the scope of the invention, scope of the present invention is limited by appended claims.In addition, those skilled in the art carries out various changes or modification to the present invention in the appended claims restricted portion, and these changes or modified forms drop on protection scope of the present invention equally.
Claims (4)
1. an enzyme linked immunological kit that detects Sulfamethoxazole comprises that Sulfamethoxazole specific antibody, bag are by the solid phase carrier of antigen, Sulfamethoxazole standard solution, enzyme conjugates working fluid, developer, developer dilution, concentrated cleaning solution, stop buffer; Described developer is by colour developing liquid A liquid and colour developing liquid B liquid, and described colour developing liquid A liquid is hydrogen peroxide, and described B liquid is tetramethyl benzidine (TMB); Described colour developing dilution is a sodium acetate solution; Described cleansing solution is the phosphate buffer of 0.05%-1.2% Tween-20; Described stop buffer is the sulfuric acid solution of 1-2M; Used bag is cushioned the carbonate buffer solution that liquid is PH9.6 in the described envelope antigen process, and used confining liquid is 10% calf serum.
2. enzyme linked immunological kit according to claim 1 is characterized in that: described Sulfamethoxazole specific antibody is a Sulfalene oxazole monoclonal antibody.
3. enzyme linked immunological kit according to claim 1 and 2 is characterized in that: described two anti-are anti-mouse or anti-rabbit antiantibody.
4. according to the described enzyme linked immunological kit of claim 1,2 or 3, it is characterized in that: described enzyme is to come the mark horseradish peroxidase by the periodic acid nanofarad of improvement in conjunction with working fluid.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102079788B (en) * | 2009-11-27 | 2013-02-27 | 北京维德维康生物技术有限公司 | Method for detecting sulfanilamide medicine and special enzyme-linked immunoassay reagent kit thereof |
| CN103018454A (en) * | 2011-09-21 | 2013-04-03 | 北京勤邦生物技术有限公司 | Sulfanilamide drug chemiluminescence enzyme-linked immunodetection kit |
| CN101571541B (en) * | 2009-06-01 | 2013-10-30 | 北京望尔生物技术有限公司 | Enzyme-linked immunosorbent inspect kit for inspecting sulfa drugs and method thereof |
-
2008
- 2008-05-26 CN CNA2008100974145A patent/CN101275947A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101571541B (en) * | 2009-06-01 | 2013-10-30 | 北京望尔生物技术有限公司 | Enzyme-linked immunosorbent inspect kit for inspecting sulfa drugs and method thereof |
| CN102079788B (en) * | 2009-11-27 | 2013-02-27 | 北京维德维康生物技术有限公司 | Method for detecting sulfanilamide medicine and special enzyme-linked immunoassay reagent kit thereof |
| CN103018454A (en) * | 2011-09-21 | 2013-04-03 | 北京勤邦生物技术有限公司 | Sulfanilamide drug chemiluminescence enzyme-linked immunodetection kit |
| CN103018454B (en) * | 2011-09-21 | 2016-03-30 | 北京勤邦生物技术有限公司 | A kind of chemical luminescence ELISA detection kit of sulfa drugs |
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