CN102621322A - Kit for detecting 3-methyl-quinoxaline-2-carboxylic acid - Google Patents
Kit for detecting 3-methyl-quinoxaline-2-carboxylic acid Download PDFInfo
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Abstract
The invention discloses a kit for detecting 3-methyl-quinoxaline-2-carboxylic acid. The enzyme linked immunosorbent assay (ELISA) kit for detecting 3-methyl-quinoxaline-2-carboxylic acid comprises a monoclonal antibody which has an anti-preservation number of CGMCC No.5778 and is excreted by an olaquindox metabolite monoclonal antibody hybrid tumor 5A2. The kit comprises the monoclonal antibody, and a conjugate of a compound and a barrier protein, which is shown as a formula (I). The kit has the advantages of high sensitivity, high accuracy, high precision, low cost, simplicity for operation, short detection time, suitability for use by various units, simplicity for storage and long shelf life, can be used for quickly detecting a large number of samples at the same time, and can realize on-site high-flux quick detection. The ELISA kit is suitable for determining the residual quantity of olaquindox metabolites in feeds, animal tissues or excrements. The antibody, the kit and a detection method can play a major role in detection of the olaquindox metabolites.
Description
Technical field
The present invention relates to the kit of a kind of 3-of detection Jia based quinoxaline-2-carboxylic acid.
Background technology
3-Jia based quinoxaline-2-carboxylic acid (MQCA) is the metabolin that olaquindox, mequindox and quinocetone produce in animal body.Through discovering that quinoxaline original shape medicine and metabolin exist tangible safety issue, have toxic and side effects such as tangible teratogenesis, carcinogenic, mutagenesis, photosensitive and adrenal cortex infringement, serious harm the health of humans and animals.The MRL of relevant quinoxaline medicine regulation is very strict in the world.European Union's regulation carbadox and olaquindox with and metabolic product in animal food, must not detect and stipulate that these two kinds of veterinary drugs forbid selling.China Ministry of Agriculture announces regulation No. 235, and the olaquindox MRL is olaquindox+3-Jia based quinoxaline-2-carboxylic acid: 4ug/kg (muscle), 50ug/kg (liver), 2mg/kg (feed).
Research mainly contains chromatography (TLC), high performance liquid chromatography (HPLC) and immuno analytical method etc. both at home and abroad at present.The specificity of TLC method is relatively poor, and sensitivity is also relatively poor relatively, and required standard items concentration is higher, and potential contaminative is higher; The required instrument and equipment investment of HPLC method is big, and operative technique requires high, and decontaminating column consumes more, and it is higher to detect cost; The immune affinity column specificity is better, but needs to combine HPLC appearance and fluorophotometer ability detection by quantitative, and detection technique requirement and cost are higher, and the ELISA method Comparatively speaking; Easy and simple to handle, quick, to pollute lessly, sensitivity is also higher; Be fit to the general inspection and the screening of sample in batches, the kit of particularly succeeding in developing brings great convenience to the testing staff; Simultaneously, also provide cost savings, meet at present the developing direction of detection range in the world.The immune colloidal gold chromatography technology does not need any instrument and equipment and reagent, be particularly suitable for detection and large tracts of land generaI investigation that grass-roots unit is pressed for time in enormous quantities, so enzyme immunoassay is widely used in the feed safety analysis.
Summary of the invention
The kit that the purpose of this invention is to provide a kind of 3-of detection Jia based quinoxaline-2-carboxylic acid.
The enzyme linked immunological kit of detection 3-Jia based quinoxaline provided by the invention-2-carboxylic acid comprises the monoclonal antibody that anti-olaquindox metabolin monoclonal antibody hybridoma cell 5A2 secretes.Said anti-olaquindox metabolin monoclonal antibody hybridoma cell 5A2 (being called for short hybridoma 5A2) is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on February 15th, 2012 and (is called for short CGMCC; The address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.5778.
Said kit can be following 1) to 4) in any one:
1) comprises the kit of compound shown in the formula (I) and the conjugate of carrier protein, said monoclonal antibody and enzyme labeling antiantibody; Wherein, said conjugate is as coating antigen;
2) comprise the kit of the enzyme labeling thing of compound shown in the formula (I), said monoclonal antibody and antiantibody; Wherein, said antiantibody is as coating antigen;
3) comprise the enzyme labeling thing of compound shown in the formula (I) and the kit of said monoclonal antibody; Wherein, said monoclonal antibody is as coating antigen;
4) comprise the kit of enzyme labeling thing of conjugate and the said monoclonal antibody of compound shown in the formula (I) and carrier protein; Wherein, said conjugate is as coating antigen.
Said carrier protein can be ovalbumin (OVA) or bovine serum albumin(BSA) (BSA).
In the said conjugate, the coupling ratio of compound and carrier protein specifically can be (11-8) shown in the formula (I): 1.Said coupling ratio refers to mol ratio.Compound and said carrier protein shown in the formula (I) specifically can pass through the active ester method coupling.Said conjugate specifically can be suc as formula shown in (II).
Formula (II).
Said kit also comprises at least a in cleansing solution, sample concentration liquid, substrate colour developing liquid and the stop buffer.
Per 1 liter of cleansing solution can obtain according to following method preparation: 10ml polysorbas20,5g sodium azide and 990ml phosphate buffer are mixed, obtain cleansing solution.Said phosphate buffer can be the phosphate buffer of pH7.2-7.6,0.005M-0.015M, and the concentration that specifically can be can be pH7.4,0.01M) sodium phosphate buffer.
Said sample concentration liquid can be the phosphate buffer that concentration is 0.03mol/L-0.05mol/L, is preferably the PBS damping fluid of pH7.4,0.04mol/L.
Said substrate colour developing liquid comprises colour developing liquid A and colour developing liquid B, can be the colour developing liquid A and colour developing liquid B of independent packaging, also can directly colour developing liquid A be mixed obtaining with colour developing liquid B equal-volume.Said colour developing liquid can be superoxol or urea peroxide solution; Said colour developing liquid B can be o-phenylenediamine (OPD) solution or tetramethyl benzidine (TMB) solution.Said colour developing liquid A specifically can be 2% (g/100ml) urea peroxide WS.Said colour developing liquid B specifically can be 1% (g/100ml) tetramethyl biphenyl amine aqueous solution.
Said stop buffer specifically can be the 0.2M aqueous sulfuric acid.
More than arbitrary said kit all can be used for detecting olaquindox metabolin (like 3-Jia based quinoxaline-2 carboxylic acid Huo quinoxaline-2 carboxylic acid).
More than arbitrary said kit all can be used for detecting whether contain olaquindox metabolin (like 3-Jia based quinoxaline-2 carboxylic acid Huo quinoxaline-2 carboxylic acid) in the sample to be tested.
The present invention adopts the 3-first based quinoxaline-2-carboxylic acid monoclonal antibody of high specific, and the method for inspection is convenient and easy, has specificity height, highly sensitive, characteristics such as degree of accuracy is high, accuracy height.Kit of the present invention and detection method are low to the pre-treatment requirement of sample, and sample pretreatment process is simple, simultaneously the fast detecting batch samples; That kit of the present invention has is highly sensitive, accuracy is high, precision is high, cost is low, simple to operate, detection time short, be fit to various units uses, stores advantage simple, long shelf-life; The fast detecting batch samples can realize on-the-spot high flux fast detecting simultaneously.Enzyme linked immunological kit provided by the invention is applicable to the residual quantity of measuring olaquindox metabolin in feed, animal tissue or the excreta.Antibody of the present invention, kit and detection method will be brought into play significant role in the detection of olaquindox metabolin.
Description of drawings
Fig. 1 is the ultraviolet spectrogram of 3-Jia based quinoxaline-2-carboxylic acid artificial antigen.
Fig. 2 is for adopting the canonical plotting that 3-Jia based quinoxaline-the 2-carboxylic acid is made.
Fig. 3 is the canonical plotting of kit.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is conventional method.Used test material among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.Used PBS damping fluid among the embodiment like no specified otherwise, is the PBS damping fluid of pH7.4,0.01M.Used carbonate buffer solution is the sodium carbonate buffer of pH9.6,0.05mol/L among the embodiment.Bovine serum albumin(BSA) is called for short BSA.Ovalbumin is called for short OVA.
3-Jia based quinoxaline-2-carboxylic acid is available from German Dr.Ehrenstorfer company, and catalog number is 81121, and structural representation is seen formula (III).
Formula (III).
N, dinethylformamide (DMF) is suc as formula shown in (IV).
Formula (IV)
N-hydroxy-succinamide (NHS) is suc as formula shown in (V).
Formula (V)
1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) is suc as formula shown in (VI).
Formula (VI)
Embodiment 1, preparation 3-Jia based quinoxaline-2-carboxylic acid haptens
One, 3-Jia based quinoxaline-haptenic preparation of 2-carboxylic acid
1, takes by weighing 3-Jia based quinoxaline-2-carboxylic acid 10mg, place the 10mL reaction bulb; The adding proper amount of acetone makes it with a small amount of DMF dissolves fully (acetone is as reaction system dissolving 3-Jia based quinoxaline-2-carboxylic acid, and the effect of DMF is to promote to dissolve), adds thionyl chloride 10 μ L (acting as activated carboxyl), heating reflux reaction 1h; Add normal hexane 0.5ml then and blow to constant volume, add normal hexane 0.5ml then and blow to constant volume, add normal hexane 0.5ml then and blow to constant volume, obtain solution I with nitrogen with nitrogen with nitrogen.
2, take by weighing the 20mg GABA; Be dissolved in the KOH WS (as reaction system dissolving GABA) of 1ml 2mol/L; Add 0.5ml pyridine (catalyzer that reacts as 3-Jia based quinoxaline-2-carboxylic acid and GABA), obtain solution II.
3, in ice bath, solution I is dropwise joined in the solution II, stirring reaction 1.5h transfers about pH to 6.0 with 6mol/L HCl then; With twice of dichloromethane extraction (each 5ml), merge the organic phase of twice extraction, with water washing 3 times; Organic phase is revolved inspissation after with anhydrous acid sodium drying to contract; Obtain the 15mg product, be 3-Jia based quinoxaline-2-carboxylic acid haptens, hereinafter to be referred as MQCA-GABA.
Two, 3-Jia based quinoxaline-haptenic sign of 2-carboxylic acid
Product to the step 1 preparation carries out ultimate analysis, and the result is following:
C：61.51；H：5.52；N：15.40；O：17.57
The result shows that the product of step 1 preparation is a compound shown in the formula (I).
Preparation and the sign of embodiment 2,3-Jia based quinoxaline-2-carboxylic acid artificial antigen
One, the immunogenic synthetic and sign of 3-Jia based quinoxaline-2-carboxylic acid
1,3-Jia based quinoxaline-immunogenic preparation of 2-carboxylic acid
(1) compound is dissolved in 2mL N shown in the formula that 10mg embodiment 1 is prepared (I); In N '-dimethylformamide; Add 10mg N-hydroxy-succinamide and 10mg1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, the room temperature lower magnetic force stirs 2h, obtains solution III.
(2) the 30mg bovine serum albumin(BSA) is added in the 2mL PBS damping fluid, fully dissolving is solution IV.
(3) solution III is slowly dropped in the solution IV, go into bag filter after slowly stirring 24h, 4 ℃ of dialysis 72h (water is changed 6 times in the centre) in physiological saline; Then under 4 ℃ of conditions; The centrifugal 30min of 8000rmp gets supernatant, i.e. 3-Jia based quinoxaline-2-carboxylic acid immunogen solution; Be sub-packed in the ampere bottle-20 ℃ of preservations.3-Jia based quinoxaline-2-carboxylic acid immunogene is called for short MQCA-BSA, and 3-Jia based quinoxaline-2-carboxylic acid immunogen solution is called for short MQCA-BSA solution.
(4) with MQCA-BSA solution with after the PBS damping fluid dilution; Measure the spectrophotometric value of 280nm and 260nm; By formula calculate the protein concentration in the dilution, the protein concentration that records is on duty to be the MQCA-BSA concentration in the former MQCA-BSA solution behind its extension rate.Protein concentration (mg/ml)=1.45 * OD
280-0.74 * OD
260MQCA-BSA concentration in the MQCA-BSA solution is 6.2mg/ml.
2, the evaluation of 3-Jia based quinoxaline-2-carboxylic acid artificial antigen
MQCA-BSA solution is diluted (concentration that makes MQCA-BSA is 5mg/mL) with the PBS damping fluid, as the solution first; The PBS damping fluid that will contain 5mg/mL MQCA-GABA is as solution second; The PBS damping fluid that will contain 5mg/mL BSA is as solution third.Respectively solution first, solution second and solution third are carried out ultraviolet (200-380nm) spectral scan, the uv scan result sees Fig. 1.Significant change has taken place in the uv-spectrogram of comparing the solution first with solution third, and compound and BSA success coupling is described.
The maximum absorption wave long value of solution second is 297nm, and the maximum absorption wave long value of solution third is 280nm.Calculate the extinction coefficient (K) of each compound according to formula K=A/CL (A is the absorbance under the maximum absorption wave long value, and C is a solution concentration, and L is the thickness of liquid layer).
Adopt the maximum absorption wave long value of solution second and solution third that the solution first is carried out uv scan respectively; And according to the concentration of this compound of extinction coefficient backwards calculation in the solution first of this compound that has calculated; Obtain the volumetric molar concentration of this compound divided by molecular weight with concentration value; Calculate coupling ratio, the coupling ratio of compound and BSA is 11: 1 shown in the formula (I), and promptly compound shown in 11 formulas (I) combines 1 BSA.
Two, the preparation and the sign of 3-Jia based quinoxaline-2-carboxylic acid coating antigen
1, the preparation of 3-Jia based quinoxaline-2-carboxylic acid coating antigen
Replace bovine serum albumin(BSA) with ovalbumin, other is with 1 of step 1.
3-Jia based quinoxaline-2-carboxylic acid coating antigen is called for short MQCA-OVA, and 3-Jia based quinoxaline-2-carboxylic acid coating antigen solution is called for short MQCA-OVA solution.
MQCA-OVA concentration in the MQCA-OVA solution is 3.2mg/ml.
2, the sign of 3-Jia based quinoxaline-2-carboxylic acid coating antigen
Replace MQCA-BSA with MQCA-OVA, replace BSA with OVA, other is with 2 of step 1.
The coupling ratio of compound and OVA is 8: 1 shown in the formula (I), and promptly compound shown in 8 formulas (I) combines 1 OVA.
Embodiment 3,3-Jia based quinoxaline-2-carboxylic acid MONOCLONAL ANTIBODIES SPECIFIC FOR
Balb/c mouse: purchase in Beijing Vital River Experimental Animals Technology Co., Ltd.;
SP2/0 myeloma cell: available from Sigma-Aldrich company, catalog number is 08060101.
One, animal immune
MQCA-BSA solution immunity Balb/c mouse with embodiment 2 preparations; Every mouse single immunization 100 μ gMQCA-BSA, immunity is 4 times altogether, each two weeks at interval; The immunization ways of first three time is the subcutaneous multi-point injection of nape portion, and back three times immunization ways is an intraperitoneal injection.
Two, Fusion of Cells and cloning
1, the 4th immunity be after 3 days, and extracting spleen cell merges in 5: 1 (quantitative proportion) ratios and SP2/0 myeloma cell, adopts indirect competitive ELISA to measure cell conditioned medium liquid, screens positive hole.
2, utilize limiting dilution assay that cloning is carried out in positive hole, obtain to secrete the hybridoma cell strain of 3-Jia based quinoxaline-2-carboxylic acid monoclonal antibody.With the anti-olaquindox metabolin of strain of hybridoma strain called after monoclonal antibody hybridoma cell 5A2 (being called for short hybridoma 5A2); Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on February 15th, 2012 and (be called for short CGMCC; The address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.5778.
Three, cell cryopreservation and recovery
With cryopreserving liquid hybridoma 5A2 is processed 1 * 10
6The cell suspension of individual/ml is in the medium-term and long-term preservation of liquid nitrogen.During recovery, take out frozen pipe, put into 37 ℃ of water-bath middling speeds immediately and melt, move in the culture flask behind the centrifugal removal cryopreserving liquid and cultivate.
Four, MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying
1, increment cultivation
The preparation method of cell culture medium (7.4): in the RPMI-1640 nutrient culture media, add calf serum and soda mint, the final concentration of calf serum is 20% (quality percentage composition), and the final concentration of soda mint is 0.2% (quality percentage composition).
5A2 places cell culture medium with hybridoma, cultivates 2 days for 37 ℃, with sad-saturated ammonium sulfate method the nutrient solution that obtains is carried out purifying, obtains monoclonal anti liquid solution (20 ℃ of preservations).
Protein concentration in the monoclonal antibody (mg/ml)=1.45 * OD
280-0.74 * OD
260
Adopt above formula to calculate the protein concentration in the monoclonal antibody, be 24.1mg/ml.
2, ascites preparation
Balb/c mouse peritoneal injection sterilization paraffinum liquidum (0.4mL/ only).7 days pneumoretroperitoneum injection hybridoma 5A2 (5 * 10
5Individual/only).Gather ascites after 7 days, carry out purifying, the ascites behind the purifying-20 ℃ preservation with sad-saturated ammonium sulfate method.
Five, the evaluation of monoclonal antibody
The monoclonal anti liquid solution that 1 of step 4 obtains is identified respectively as follows:
1, adopt ELISA monoclonal antibody hypotype detection kit (Sigma Company products, catalog number are 19285) to detect the hypotype of monoclonal antibody, the immunoglobulin subclass of monoclonal antibody is IgG1.
2, utilize noncompetitive ELISA method to measure the affinity of monoclonal antibody
(1) with MQCA-OVA as the coating antigen coated elisa plate
Adopt the MQCA-OVA solution (diluting) of embodiment 2 preparations to encapsulate 100 μ L/ holes with carbonate buffer solution; Following MQCA-OVA is set respectively encapsulates concentration: 1 μ g/mL, 0.5 μ g/mL, 0.25 μ g/mL, 0.125 μ g/mL.
Hatched 16 hours for (2) 4 ℃.
(3) seal and wash plate.
(4) every hole adds the dilution (diluting with the PBS damping fluid) of 100 μ L monoclonal anti liquid solutions; Protein concentration in the dilution is respectively 1.25,0.625,0.3125,1.5625 * 10
-1, 7.8 * 10
-2, 3.9 * 10
-2, 1.95 * 10
-2, 9.75 * 10
-3, 4.88 * 10
-3, 2.44 * 10
-3, 1.22 * 10
-3, 6.1 * 10
-4Mg/L; Every kind of dilution is provided with three multiple holes.
(5) incubated at room 2h washes plate.
(6) every hole adds the sheep anti-mouse igg of 100 μ L horseradish peroxidase-labeled, incubated at room 2h.
(7) wash plate.
(8) add TMB colour developing liquid, lucifuge colour developing 15min.
(9) every hole adds 100 μ L 2mol/L sulfuric acid stopped reactions; Read OD
450Value.
Natural logarithm value with the protein concentration in the monoclonal antibody (mol/L) is a horizontal ordinate, is that ordinate is made curve with its corresponding absorbance.
Each antigen coated concentration obtains 1 S type curve, obtains 4 S type curves altogether.Find out the top of S curve, corresponding OD
450Value is set at ODMAX.Find out the corresponding AC of each bar curve 50%ODMAX respectively.Adopt 1 μ g/mL to encapsulate concentration, the AC that 50%ODMAX is corresponding is 4.2 * 10
-12Mol/L.Adopt 0.5 μ g/mL to encapsulate concentration, the AC that 50%ODMAX is corresponding is 19.4 * 10
-12Mol/L.Adopt 0.25 μ g/mL to encapsulate concentration, the AC that 50%ODMAX is corresponding is 185.7 * 10
-12Mol/L.Adopt 0.125 μ g/mL to encapsulate concentration, the AC that 50%ODMAX is corresponding is 415.8 * 10
-12Mol/L.
With one group in twos of 4 concentration, calculate the affinity costant of monoclonal antibody according to formula
Ka＝(n-1)/2(n[Ab]t
1-[Ab]t
2)
In the formula, two multiples that encapsulate concentration during n is every group, [Ab] t
1, [Ab] t
2Be respectively two ACs (mol/L) that 50%ODMAX is corresponding in every group.For example: 1 μ g/mL encapsulates concentration, 50%OD
450Corresponding AC is 4.2 * 10
-12Mol/L, 0.5 μ g/mL encapsulates concentration, 50%OD
450Corresponding AC is 19.4 * 10
-12Mol/L, Ka=(2-1)/2 (2 * 19.4 * 10
-12-4.2 * 10
-12)=14.4 * 10
9M
-1And the like, obtain all the other 5 Ka values, be respectively 1.4 * 10
9M
-1, 0.7 * 10
9M
-1, 2.0 * 10
9M
-1, 0.9 * 10
9M
-1, 1.0 * 10
9W
-1, the affinity costant that calculates monoclonal antibody of averaging is 3.4 * 10
9M
-1
3, monoclonal antibody Sensitivity calculation
(1) adopt the MQCA-OVA solution (diluting) of embodiment 2 preparations to encapsulate 100 μ L/ holes with carbonate buffer solution; The concentration that encapsulates of MQCA-OVA is 1.0 μ g/mL.
Hatched 16 hours for (2) 4 ℃.
(3) seal and wash plate.
(4) every hole adds 50 μ L 3-Jia based quinoxaline-2-carboxylic acid standard solutions and (is made up of 3-Jia based quinoxaline-2-carboxylic acid and PBS damping fluid; The concentration of 3-Jia based quinoxaline-2-carboxylic acid is respectively 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; With the hole that only adds the PBS damping fluid as control wells), each concentration is provided with 3 multiple holes.
(5) every hole adds the monoclonal anti liquid solution that 1 of 50 μ L step 4 obtain.
(6) incubated at room 2h washes plate.
(7) every hole adds the sheep anti-mouse igg of 100 μ L horseradish peroxidase-labeled, incubated at room 2h.
(8) wash plate.
(9) add TMB colour developing liquid, lucifuge colour developing 15min.
(10) every hole adds 100 μ L 2mol/L sulfuric acid stopped reactions; Read OD
450Value.
The light absorption value (mean values in three multiple holes) that the standard solution that adopts each concentration is obtained multiply by 100 as ordinate again divided by the light absorption value of control wells; Natural logarithm value with the 3-Jia based quinoxaline-2-carboxylic acid concentration (μ g/L) in each standard solution is horizontal ordinate curve plotting figure, sees Fig. 2.
Map 2 obtains Y value and equals 50% o'clock corresponding 3-Jia based quinoxaline-2-carboxylic acid concentration and be the IC50 value.The sensitivity (IC50 value) that monoclonal antibody detects 3-Jia based quinoxaline-2-carboxylic acid is 3.1ng/mL.
Embodiment 4,3-Jia based quinoxaline-2-carboxylic acid Polyclonal Antibody Preparation
New zealand white rabbit: purchase in Beijing Vital River Experimental Animals Technology Co., Ltd..
New zealand white rabbit is carried out immunity (immunization ways is the subcutaneous multi-point injection of nape portion) with the 3-Jia based quinoxaline-2-carboxylic acid immunogene (MQCA-BSA) of preparation among the embodiment 2.Every each immune 1.5mg of rabbit (in the BSA amount); Per three all immunity once; During immunity for the first time 3-Jia based quinoxaline-2-carboxylic acid immunogene and Freund's complete adjuvant are mixed and made into emulsifying agent and carry out immunity; For the second time 3-Jia based quinoxaline-2-carboxylic acid immunogene and incomplete Freund are mixed and made into emulsifying agent and carry out immunity to the 6th immunity; The 7th immunity only carried out immunity with 3-Jia based quinoxaline-2-carboxylic acid immunogene, and the 7th immunity be the heart blood sampling after 10 days, obtains the polyclonal antibody of purifying with ammonium sulfate precipitation.
The preparation of the enzyme linked immunological kit of embodiment 5, detection 3-Jia based quinoxaline-2-carboxylic acid
One, the composition of enzyme linked immunological kit
Enzyme linked immunological kit is made up of following substances:
1, cleansing solution: per 1 liter of cleansing solution obtains according to following method preparation: 10ml polysorbas20,5g sodium azide and 990ml PBS damping fluid damping fluid are mixed, obtain cleansing solution.
2, encapsulate the ELISA Plate of MQCA-OVA
With the MQCA-OVA solution dilution (concentration that makes MQCA-OVA be 0.5 μ g/mL) of carbonate buffer solution, be coating buffer with embodiment 2 preparations; Coating buffer is added 96 hole polystyrene ELISA Plates (48 holes also can), every hole 100 μ L, 37 ℃ of incubation 2h; The coating buffer that inclines, with cleansing solution washing 3 times, each 30s claps and does; In every hole, add 200 μ L confining liquids then, 37 ℃ of incubation 2h, liquid in the hole of inclining, preserve with the vacuum seal of aluminium film dry back.
Per 1 liter of said confining liquid is prepared according to following method: 5ml horse serum, 1g sodium azide and 30g casein are dissolved with phosphate buffer (pH7.2,0.02M) and be settled to 1000ml, obtain confining liquid.
3, sample concentration liquid: PBS damping fluid (pH7.4,0.04M).
Sample concentration liquid is diluted with water to 20 times of volumes, is sample diluting liquid.
4, antibody working fluid: the monoclonal anti liquid solution that 1 of the step 4 of embodiment 3 is obtained dilutes with sample diluting liquid, and making protein concentration is 6.5ng/mL.
5, ELIAS secondary antibody working fluid
The sheep anti-mouse antibody of horseradish peroxidase (HRP) mark is available from U.S. Sigma-Aldrich company, and catalog number is A7058, by specification preparation ELIAS secondary antibody working fluid.
6, standard solution
Standard items are 3-Jia based quinoxaline-2-carboxylic acid.
With sample diluting liquid dilution dissolving 3-Jia based quinoxaline-2-carboxylic acid, obtain each standard solution.3-Jia based quinoxaline-2-carboxylic acid concentration is respectively 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 in each standard solution.
Sample diluting liquid is the standard solution (0 standard) of 0 μ g/L as 3-Jia based quinoxaline-2-carboxylic acid concentration.
7, substrate colour developing liquid
Mixed by A liquid and B liquid equal-volume, A liquid is 2% (g/100ml) urea peroxide WS, and B liquid is 1% (g/100ml) tetramethyl biphenyl amine aqueous solution.
8, stop buffer: 0.2M aqueous sulfuric acid.
Two, kit test method
Adopt the kit of step 1 preparation to detect as follows:
(1) sample pre-treatments
When detecting sample and being pork or pork liver: accurately take by weighing tissue sample behind the homogeneous in centrifuge tube; Add sample extracting solution (sample diluting liquid that contains 0.2% polysorbas20); Fully whirling motion disperses to tissue, and centrifugal 10min more than the 4000g gets 50 μ L supernatants as sample to be tested solution.
When the detection sample is urine sample: the centrifugal 10min of 3000g, draw supernatant and sample diluting liquid by 1: 1 volume mixture, get 50 μ L mixed liquors as sample to be tested solution.
(2) kit test method
1, the making of typical curve
In the ELISA Plate that encapsulates MQCA-OVA, add standard solution (50 μ L/ holes; Each standard solution is provided with three multiple holes); Add antibody working fluid (50 μ L/ hole) again,, react 60min in 37 ℃ of constant temperature ovens with cover plate film shrouding; Pour out liquid in the hole; Wash 4 times (step of each washing is: every hole adds 250 μ L cleansing solutions, pours out liquid in the hole behind the 30s), clap with thieving paper and do.Add ELIAS secondary antibody working fluid (100 μ L/ hole), react 30min in 37 ℃ of constant temperature ovens, pour out liquid in the hole; Wash 4 times (step is the same), every hole adds substrate colour developing liquid 100 μ L, and mixing gently vibrates; 37 ℃ of constant temperature oven lucifuge colour developing 15min, every hole adds stop buffer 50 μ L, and mixing gently vibrates; Measure every hole absorbance (OD630 and OD450 dual wavelength, the 450th, predominant wavelength, the 630th, reference wavelength) with ELIASA.
The absorbance mean value (B) of the standard solution of each concentration of usefulness multiply by 100% again divided by the absorbance (B0) of first standard solution (0 standard), obtains the percentage absorbance.Utilization Originpro 7.0 softwares are analyzed the data result, are the X axle with the natural logarithm value of standard items concentration (μ g/L), and the percentage absorbance simulates typical curve for the Y axle.Typical curve is seen Fig. 3.
2,3-Jia based quinoxaline-2-carboxylic acid concentration's mensuration in the sample
In the ELISA Plate that encapsulates MQCA-OVA, add sample to be tested solution or its dilution (50 μ L/ holes; Three multiple holes are set), add antibody working fluid (50 μ L/ hole) again, with cover plate film shrouding, react 60min in 37 ℃ of constant temperature ovens, pour out liquid in the hole, wash 4 times (step is the same), clap with thieving paper and do.Add ELIAS secondary antibody working fluid (100 μ L/ hole), react 30min in 37 ℃ of constant temperature ovens, wash 4 times (step is the same); Every hole adds substrate colour developing liquid 100 μ L, the mixing that vibrates gently, 37 ℃ of constant temperature oven lucifuge colour developing 15min; Every hole adds stop buffer 50 μ L, and the mixing that vibrates is gently measured every hole absorbance (OD630 and OD450 dual wavelength with ELIASA; The 450th, predominant wavelength, the 630th, reference wavelength).
The result judges: use each sample to be tested solution absorbency mean value (B) to multiply by 100% again divided by the absorbance (B0) of first standard solution (0 standard), obtain the percentage absorbance.The percentage absorbance of corresponding each sample to be tested solution then can be read the concentration value of 3-Jia based quinoxaline-2-carboxylic acid from typical curve, multiply by the extension rate of respective sample again, converses the content of 3-Jia based quinoxaline-2-carboxylic acid in the sample to be tested solution.
Three, kit detects effect assessment
(1) accuracy and precision test
In the pork that does not contain 3-Jia based quinoxaline-2-carboxylic acid, pork liver and pig urine samples, add 3-Jia based quinoxaline-2-carboxylic acid standard items, make the 3-Jia based quinoxaline-final concentration of 2-carboxylic acid standard items in sample be respectively 4 μ g/kg, 10 μ g/kg, 4 μ g/kg; Sample after adding is carried out pre-treatment according to method described in () of step 2 respectively, obtain test sample solution.
From the kit of three different batches, respectively extracting 3 kits detects; Described in (two) of detection method such as step 2; Each experiment repetition 5 times; According to the content of 3-Jia based quinoxaline-2-carboxylic acid in the cubage detection sample of 3-Jia based quinoxaline-2-carboxylic acid in the sample to be tested solution, the result sees table 1.
Table 1 is used the content (μ g/kg) that each kit detects 3-Jia based quinoxaline-2-carboxylic acid in the sample that draws
| Kit 1 | Kit 2 | Kit 3 | |
| The 3-Jia based quinoxaline-final concentration of 2-carboxylic acid in pork is 4 μ g/kg | 3.34 | 3.63 | 3.48 |
| The 3-Jia based quinoxaline-final concentration of 2-carboxylic acid in pork liver is 10 μ g/kg | 9.31 | 7.93 | 8.52 |
| The final concentration of 3-Jia based quinoxaline-2-carboxylic acid in pig urine is 4 μ g/kg | 3.53 | 3.72 | 3.35 |
The difference calculate recovery rate and the coefficient of variation, the result sees table 2.
Content * 100% of the actual 3-Jia based quinoxaline-2-carboxylic acid that adds in the content ÷ milk of 3-Jia based quinoxaline-2-carboxylic acid that the recovery=application kit detection computations goes out.
The coefficient of variation (CV)=mensuration result's the standard deviation and the number percent of its mean value.
The computing method of plate within variance coefficient: certain sample (being generally medium level) replication number of times gained result's the coefficient of variation in plate within variance coefficient=same same block of plate of once measuring.
The computing method of variation within batch coefficient: the variation within batch coefficient=coefficient of variation of each parallel samples in once measuring together.
The computing method of interassay coefficient of variation: interassay coefficient of variation=same sample is got its mean value in different batches mensuration result's the coefficient of variation.
Table 2 accuracy and Precision test result
The result shows: the interpolation recovery of all samples is 79.3%～93.1%, and the variation within batch coefficient is 4.3%～8.3%, and interassay coefficient of variation is 12.3%～14.7%.
(2) kit storage life
The kit preservation condition is 2-8 ℃, and through 12 months mensuration, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, 3-Jia based quinoxaline-2-carboxylic acid added the practical measurement value all within normal range.Consider in transportation and the use, have improper preservation condition and occur that kit the condition held of 37 ℃ of preservations 8 days, is carried out accelerated deterioration and tests, and the result shows that each item 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 8 days, measure the result and show that also kit each item index is normal fully.Can draw kit from above result can preserve more than 12 months at 2-8 ℃ at least.
(3) cross reacting rate test
In the ELISA Plate that encapsulates MQCA-OVA, adding the analogue standard solution (is made up of analogue and PBS damping fluid; The concentration of analogue is respectively 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; With the hole that only adds the PBS damping fluid as control wells; 50 μ L/ holes; Each concentration is provided with 3 multiple holes), add antibody working fluid (50 μ L/ hole) again, with cover plate film shrouding, react 60min in 37 ℃ of constant temperature ovens, pour out liquid in the hole, wash 4 times (step is the same), clap with thieving paper and do.Add ELIAS secondary antibody working fluid (100 μ L/ hole), react 30min in 37 ℃ of constant temperature ovens, wash 4 times (step is the same); Every hole adds substrate colour developing liquid 100 μ L, the mixing that vibrates gently, 37 ℃ of constant temperature oven lucifuge colour developing 15min; Every hole adds stop buffer 50 μ L, and the mixing that vibrates is gently measured every hole absorbance (OD630 and OD450 dual wavelength with ELIASA; The 450th, predominant wavelength, the 630th, reference wavelength).
The light absorption value (mean values in three multiple holes) that the analogue that adopts each concentration is obtained multiply by 100 as ordinate again divided by the light absorption value of control wells, is horizontal ordinate curve plotting figure with the natural logarithm value of the similar substrate concentration in each standard solution (μ g/L).Control curve figure obtains Y value and equals corresponding similar substrate concentration (μ g/L), the i.e. IC of analogue at 50% o'clock
50Value.
With the cross reacting rate of computes kit to other analogue.The result sees table 3.
The specificity of table 3 kit
| The purchase approach | Cross reacting rate | |
| MQCA | Dr.Ehrenstorfer company, catalog number is 81121 | 100％ |
| QCA | The Huifeng, Wuhan reaches, article No. 879652 | 43.2％ |
| Olaquindox | Dr.Ehrenstorfer GmbH company, article No. C 15716500 | ＜0.1％ |
| Quinocetone | Dr.Ehrenstorfer GmbH company, article No. C 16709000 | ＜0.1％ |
| Mequindox | Sigma Aldrich company, catalog number is M0189 | ＜0.1％ |
Claims (6)
1. an enzyme linked immunological kit that detects 3-first based quinoxaline-2-carboxylic acid comprises the monoclonal antibody that anti-olaquindox metabolin monoclonal antibody hybridoma cell 5A2 secretes; The preserving number of said anti-olaquindox metabolin monoclonal antibody hybridoma cell 5A2 is CGMCC No.5778.
2. kit as claimed in claim 1 is characterized in that: said kit is following 1) to 4) in any one:
1) comprises the kit of compound shown in the formula (I) and the conjugate of carrier protein, said monoclonal antibody and enzyme labeling antiantibody; Wherein, said conjugate is as coating antigen;
2) comprise the kit of the enzyme labeling thing of compound shown in the formula (I), said monoclonal antibody and antiantibody; Wherein, said antiantibody is as coating antigen;
3) comprise the enzyme labeling thing of compound shown in the formula (I) and the kit of said monoclonal antibody; Wherein, said monoclonal antibody is as coating antigen;
4) comprise the kit of enzyme labeling thing of conjugate and the said monoclonal antibody of compound shown in the formula (I) and carrier protein; Wherein, said conjugate is as coating antigen;
3. kit as claimed in claim 2 is characterized in that: said carrier protein is ovalbumin or bovine serum albumin(BSA).
4. like arbitrary described kit in the claim 1 to 3, it is characterized in that: said kit also comprises at least a in cleansing solution, sample concentration liquid, substrate colour developing liquid and the stop buffer.
5. the application of arbitrary said kit in detecting the olaquindox metabolin in the claim 1 to 4.
6. whether arbitrary said kit contains the application in the olaquindox metabolin at the detection sample to be tested in the claim 1 to 4.
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| CN103342683A (en) * | 2013-07-30 | 2013-10-09 | 中国农业大学 | Quinocetone hapten as well as preparation method and application thereof |
| CN108426996A (en) * | 2017-02-15 | 2018-08-21 | 江苏美正生物科技有限公司 | A kind of quick detection kit and its preparation method and application that 3- Jia based quinoxalines -2- is carboxylic acid remained |
| CN109180519A (en) * | 2018-06-22 | 2019-01-11 | 华南农业大学 | A kind of olaquindox metabolite antigen, antibody and enzyme-linked immunologic detecting kit and detection method |
| CN110927376A (en) * | 2019-10-08 | 2020-03-27 | 杭州佰昕科技有限公司 | Magnetic immunochemiluminescence detection kit for olaquindox and application thereof |
| CN110927377A (en) * | 2019-10-08 | 2020-03-27 | 杭州佰昕科技有限公司 | Magnetic immunochemiluminescence detection kit for olaquindox and application thereof |
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| CN103342683A (en) * | 2013-07-30 | 2013-10-09 | 中国农业大学 | Quinocetone hapten as well as preparation method and application thereof |
| CN108426996A (en) * | 2017-02-15 | 2018-08-21 | 江苏美正生物科技有限公司 | A kind of quick detection kit and its preparation method and application that 3- Jia based quinoxalines -2- is carboxylic acid remained |
| CN108426996B (en) * | 2017-02-15 | 2020-09-15 | 江苏美正生物科技有限公司 | Rapid detection kit for 3-methyl quinoxaline-2-carboxylic acid residues and preparation method and application thereof |
| CN109180519A (en) * | 2018-06-22 | 2019-01-11 | 华南农业大学 | A kind of olaquindox metabolite antigen, antibody and enzyme-linked immunologic detecting kit and detection method |
| CN109180519B (en) * | 2018-06-22 | 2021-08-03 | 华南农业大学 | Olaquindox metabolite antigen, antibody, enzyme-linked immunosorbent assay kit and detection method |
| CN110927376A (en) * | 2019-10-08 | 2020-03-27 | 杭州佰昕科技有限公司 | Magnetic immunochemiluminescence detection kit for olaquindox and application thereof |
| CN110927377A (en) * | 2019-10-08 | 2020-03-27 | 杭州佰昕科技有限公司 | Magnetic immunochemiluminescence detection kit for olaquindox and application thereof |
| CN110927376B (en) * | 2019-10-08 | 2024-04-30 | 浙江工商大学 | Magnetic immunochemistry luminescence detection kit of olaquindox and application thereof |
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| CN102621322B (en) | 2014-04-23 |
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