CN103146652B - Anti-olaquindox monoclonal antibody and its hybridoma cell line, preparation methods of antibody and cell line, and kit for detecting olaquindox in forage - Google Patents
Anti-olaquindox monoclonal antibody and its hybridoma cell line, preparation methods of antibody and cell line, and kit for detecting olaquindox in forage Download PDFInfo
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Abstract
The invention discloses a high-potency, high-sensitivity and high-specificity anti-olaquindox monoclonal antibody, and also discloses a hybridoma cell line having a preservation number of CGMCC No.6260 and capable of producing the anti-olaquindox monoclonal antibody, preparation methods of the antibody and the cell line, and a kit for detecting olaquindox in a forage. According to the invention, the anti-olaquindox monoclonal antibody has a high specificity and sensitivity and a large linear range, and can be used for establishing the immunologic method for the rapid detection of the illegal addition of olaquindox in the forage, the application method of the anti-olaquindox monoclonal antibody comprises the indirect-competition ELISA kit and a colloidal gold test strip, and the method has low requirements on equipment and the personal operation, has a low detection cost and can satisfy the detection needs of a large batch of forage samples.
Description
Technical field
The present invention relates to small molecule immune chemical analysis and detection technique field, being specifically related to olaquindox monoclonal antibody and hybridoma cell strain, its preparation method and the test kit for detecting olaquindox in feed.
Background technology
Olaquindox (o-laquindox, OLA) also known as olaquindox, chemical name is 2-hydroxyethylamino formyl-3-methyl-quinoxalin-1,4-dioxide, there is good broad-spectrum antimicrobial effect, to the digestive tract diseases caused by the gram negative pathogenic bacteria such as intestinal bacteria, Salmonella, there is good curative effect, and can promote that livestock and poultry are to the digestibility and utilization of feed, improve the speed of growth, in animal food is produced, play certain effect.
A large amount of domestic and international research reports shows: the olaquindox residued in animal tissues is a kind of gene toxic agent and sexual gland mutagenic compound, animal not only can be made to occur poisoning or dead, and remains in livestock product and also have larger harm to human body.Therefore, " feed medicated premix operating specification " regulation of calendar year 2001 Ministry of Agriculture's issue, forbid that olaquindox is for poultry and aquaculture, and can only be used for the pig feed lower than 35 kg body weight, usage quantity is 50 g/t feeds.In recent years, some Feedstuff Enterprises and raiser are by economic profit incentive, phenomenon that is heavy dose of, life-time service olaquindox is still very serious in violation of rules and regulations, in order to ensure human security with healthy, carries out the work that in feed and fodder additives, Determination of olaquindox detects significant.
Detection method mainly high performance liquid chromatography, the Liquid Chromatography-Tandem Mass Spectrometry of olaquindox in feed, but this method not only needs expensive plant and instrument, professional operator, and complex operation step, time-consuming.Because Enzyme-linked Immunosorbent Assay method has high specificity, the advantage such as highly sensitive, easy, quick, in wild animal resources field, application is more and more.The domestic anti-olaquindox monoclonal antibody of indirect competitive ELISA test kit many employings import for detecting OLA at present, testing cost is very high; Also there is report to select anti-OLA polyclonal antibody and anti-OLA monoclonal antibody for olaquindox residue detection in domestic literature and patent, but be not very good, mainly due to problems such as the specificity of antibody are not strong, linearity range is little, specificity is inadequate.Such as Yang Shu is bright, Yu Hongxia selects about a kind of patent of invention (CN1888903) detecting the enzyme linked immunological kit of olaquindox is olaquindox polyclonal antibody; In the Song Chunmei master's Diplomarbeit-preparation of olaquindox monoclonal antibody in 2009 and the foundation of immunology quick detection method thereof, antibodies specific is strong, linearity range is little, and the method is mainly for the residue detection of olaquindox in animal food.
Summary of the invention
Object of the present invention is exactly for above-mentioned defect of the prior art, provides a kind of anti-olaquindox monoclonal antibody of high specific with high-titer and sensitivity.
To achieve these goals, technical scheme provided by the invention is: have the hybridoma cell strain that preserving number is CGMCC No.6260.
Above-mentioned hybridoma cell strain, its preserving number is: CGMCC No. 6260, and Classification And Nomenclature is mouse boosting cell-myelomatosis hybridoma; Depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center, and address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, and preservation date is on July 4th, 2012.
Second object of the present invention there is provided above-mentionedly has the preparation method that preserving number is the hybridoma cell strain of CGMCC No.6260, comprises the following steps:
1) prepare olaquindox derivative-carrier protein couplet thing, described carrier proteins is bovine serum albumin, oralbumin or keyhole limpet hemocyanin; The compound of described olaquindox derivative to be structural formula be formula I;
Formula I:
;
2) animal immune: the olaquindox derivative-carrier protein couplet thing prepared with step 1) is immunogen immune mouse, gets the serum of immunized mice, detects tiring of anti-olaquindox in serum, chooses the highest immune mouse of tiring;
3) cytogamy: get step 2) cultivate after the spleen cell of the highest immune mouse of tiring of anti-olaquindox and murine myeloma cell SP2/0 carry out cytogamy in the serum that obtains;
4) screening of hybridoma cell strain: detecting step 3) the tiring of cell conditioned medium liquid after the fusion that obtains, screening positive cell, after repeatedly clone positive rate be 100% cell be olaquindox specificity lymph hybridoma cell strain (1H9).
3rd object of the present invention there is provided the anti-olaquindox monoclonal antibody with high specific, is that the hybridoma cell strain being CGMCC No.6260 by preserving number produces.
4th object of the present invention there is provided the above-mentioned preparation method with the anti-olaquindox monoclonal antibody of high specific, comprises the following steps:
A) preparation of ascites: get adult BALB/C female mice abdominal injection whiteruss 0.5mL, within 10 days, pneumoretroperitoneum injects 2 × 10
6individual above-mentioned hybridoma cell strain, injects and to carry out aseptic technique after 10 ~ 14 days and gather ascites, by the ascites that collects with centrifugal 10 min of rotating speed 1000 rpm, collects middle layer clarified liq, namely obtains ascitic type monoclonal antibody.
B) antibody purification: step a) is obtained centrifugal after ascites supernatant saturated ammonium sulphate, obtain anti-olaquindox monoclonal antibody crude product; Carry out purifies and separates through Sephacryl S-200 chromatography column, collect the ascties protein of positive pipe, obtain anti-olaquindox monoclonal antibody.
5th object of the present invention there is provided the above-mentioned application of anti-olaquindox monoclonal antibody in quantitative and qualitative analysis detection olaquindox in feed with high specific.
6th object of the present invention there is provided the test kit that the above-mentioned anti-olaquindox monoclonal antibody with high specific detects olaquindox in feed, and described test kit comprises following composition:
1) enzyme plate of liquid bag quilt is buffered with the bag containing olaquindox derivative-oralbumin conjugate;
2) the anti-olaquindox monoclonal antibody solution of best effort concentration;
3) goat anti-mouse IgG-HRP of best effort concentration;
4) substrate A liquid: containing 0.1%H
2o
2pH be 5.0 sodium phosphate citric acid solution;
5) the TMB ethanol solution of substrate B liquid: 2mg/mL;
6) sulfuric acid of stop buffer: 2M;
7) olaquindox standard solution: be respectively 0,1,3,9,27,81,243ng/mL;
8) 2 times of concentrated sample working solvent: containing 0 ~ 50% methyl alcohol, pH is the PBS solution of 7.4;
9) 20 times of concentrated washing lotions: concentration to be 0.2M, pH be 7.4 PBST solution.
7th object of the present invention there is provided the using method that the above-mentioned anti-olaquindox monoclonal antibody with high specific detects the test kit of olaquindox in feed: take 1.0 g Feed Samples, grind, add 10mL deionized water, after shaking up, put into 80 DEG C of water-bath 40min; The centrifugal 10min of 4000r/min, sucking-off supernatant liquor, filter paper filtering; Dilute supernatant liquor with sample working solvent 10 times or 400 times (pig feeds), mix; Every hole adds olaquindox standard solution or sample to be tested each 50
, then add olaquindox monoclonal antibody 50
/ hole, 37 DEG C of reaction 30min; Open cover plate film, dry liquid in hole, PBST washing 4 ~ 5 times; Add ELIAS secondary antibody 100
/ hole, 37 DEG C of reactions 30min, PBST wash 4 ~ 5 times; Add substrate A liquid, substrate B liquid each 50
/ hole, 37 DEG C of reaction 10 ~ 15min; Add stop buffer 50
/ hole, measures OD by microplate reader
450nm.
Interpretation of result: with B/B
0% is ordinate zou, and with the semilog of olaquindox standard concentration for X-coordinate, drawing standard graphic representation, according to regression equation and Sample Dilution multiple, calculates the interpolation concentration of olaquindox in feed sample.Take B/B0% as ordinate zou, with the logarithmic value of olaquindox standard concentration for X-coordinate, drawing standard curve, calculates olaquindox concentration.
8th object of the present invention there is provided and a kind ofly use that above-mentioned olaquindox detection kit is a large amount of, add the judgment basis of the positive and negative sample of olaquindox in rapid screening feed in violation of rules and regulations: (1) is for the detection of olaquindox in pig feed, as inhibiting rate (B/BO) >88.17% of sample, sample is negative; As inhibiting rate 50.74<(B/BO) <88.17% time, containing olaquindox in sample, but meet limitation requirement; As inhibiting rate (B/BO) <50.74%, in sample, Determination of olaquindox exceeds standard; (2) for the detection of other olaquindox in feed, as the inhibiting rate when sample (B/BO) >91.88% of sample, sample is negative; As inhibiting rate (B/BO) <79.79%, containing olaquindox in sample, sample is positive.
Beneficial effect of the present invention is: anti-olaquindox monoclonal antibody provided by the invention has comparatively high specific and sensitivity, and linearity range is large, can be used in setting up the immunological method that rapid detection olaquindox in feed illegally adds, its application method is indirect competitive ELISA enzyme linked immunological kit and colloidal gold strip, the requirement of the method to plant and instrument and human users is lower, testing cost is low, can meet the needs detected Feed Sample in enormous quantities.
Accompanying drawing explanation
Fig. 1 is the ultraviolet detection figure of immunity holoantigen OLA-HS-BSA, can be used for the evaluation of immune holoantigen synthesis.
Fig. 2 is that bag is by the ultraviolet detection figure with holoantigen OLA-HS-OVA.Can be used for wrapping the evaluation of being synthesized by holoantigen.
Fig. 3 is monoclonal antibody 1H9 hypotype qualification figure, can be used for the qualitative evaluation that hybridoma cell strain builds strain.
Fig. 4 is the polyacrylate hydrogel electrophorogram of monoclonal antibody 1H9, wherein 1 is mark, and 2,3,4 is the purified antibodies of 10 microlitres, and 5,6,7 is the purified antibodies of 5 microlitres, 8,9 is the non-purifying ascites of 20 microlitres, can be used for the quality evaluating antibody purification effect and manufacture order clonal antibody.
Fig. 5 is the evaluation map of tiring of monoclonal antibody 1H9 ascites and purifying protein, can be used for the quality evaluating monoclonal antibody.
Fig. 6 is that olaquindox suppresses graphic representation, can be used for the quality evalution of olaquindox ELISA detection kit.
Embodiment
embodiment 1:
One, the preparation of anti-olaquindox monoclonal antibody:
1, the preparation of olaquindox derivative-carrier protein couplet thing
(1) building-up process of olaquindox derivative is as follows:
，
Detailed process is:
A. in molar ratio for 1:2 takes olaquindox and succinyl oxide, round-bottomed flask is placed in, 90 DEG C of heating reflux reaction 8h;
B. underpressure distillation removing organic solvent, adds 100 ~ 200mL distilled water hold over night;
C. be extracted with ethyl acetate 3 next day, extract organic phase with sodium bicarbonate aqueous solution, in triplicate, collect aqueous phase, then be 1 ~ 2 with concentrated hydrochloric acid adjust pH, separate out tawny syrup;
D. abandon acid layer, after distillation washing 5 times, obtain Tan solid through vacuum-drying, i.e. the crude product of haptens olaquindox hemisuccinic acid ester (OLA-HS);
E. with column chromatography silica gel (140 order) dry column-packing, column packed is highly 30cm, the crude product (being dissolved in 2ml moving phase) of applied sample amount 20mg olaquindox hemisuccinic acid ester (OLA-HS), TLC monitors, collect elutriant, the tawny powder obtained after volatilizing, is haptens olaquindox hemisuccinic acid ester (OLA-HS) after purifying;
(2) preparation of olaquindox derivative-carrier protein couplet thing (immunizing antigen):
A. taking 10 ~ 20mg olaquindox hemisuccinic acid ester (OLA-HS) is dissolved in 1mL dimethyl formamide (DMF), add 24mg NHS(N-N-Hydroxysuccinimide again) and 31mg N, N-dicyclohexylcarbodiimide (DCC), 25 DEG C of lucifuges react 24 h;
B. 6 ~ 12mg BSA(bovine serum albumin is taken next day) be dissolved in the PBS solution of 2mL 0.01 mol/L, obtain carrier protein solution;
C. reaction soln in step a is dropwise added in the carrier protein solution of step b, 37 DEG C of shaken overnight;
D. the reaction solution that step c obtains is put into ultra-filtration centrifuge tube (30000Dal), with pH 7.4, the PBS damping fluid of 0.1 mol/L, 4 DEG C, 3000r/min, 10min fully wash away unreacted small-molecule substance completely, repeat 3 ~ 5 times;
E. with pH 7.4 PBS reaction buffer dilution antigen extremely first volume ,-20 DEG C of preservations.
Carrier proteins can be bovine serum albumin (BSA), oralbumin (OVA), keyhole limpet hemocyanin (KLH) or associated protein, be the ultraviolet detection figure of holoantigen OLA-HS-BSA, OLA-HS-OVA as shown in Figure 1, 2, for the qualification of holoantigen.
2, animal immune:
Add equivalent Freund's complete adjuvant with above-mentioned olaquindox hemisuccinic acid ester-bovine serum albumin and make water in oil emulsifying agent, first immunisation only adds equivalent Freund's complete adjuvant by 0.2 mL/, abdominal cavity, neck dorsal sc multi-point injection; After 2 weeks, be according to dosage that 0.1 mL/ only adds equivalent Freund's incomplete adjuvant, abdominal cavity, neck dorsal sc multi-point injection; Repeat immunity twice, every minor tick 2 weeks; Final immunization is the immunizing antigen of abdominal injection normal saline dilution; Measure serum titer with indirect elisa method, choose the highest mouse of serum titer for cytogamy.
3, hybridoma is prepared:
(1) preparation of feeder cell:
Merge first 1 day, get healthy BALB/c mouse, hole bloodletting under socket of the eye, cervical dislocation is put to death, immerse in 75% alcohol 5 min that sterilize, aseptic abdominal cut skin, expose peritonaeum, the HAT(histone acetyltransferase of 5 mL precoolings is extracted with syringe) squeeze into mouse peritoneal, to extrude after belly extracted liquid again gently, centrifugal 5 min of 1000 r/min, abandon supernatant, cell to 2 × 10 are adjusted with HAT substratum (sigma company, H0262)
5individual/mL, adds in 6 piece of 96 porocyte culture plate, and every hole 100 μ L, is placed in 37 DEG C, 5% CO
2cultivate in incubator;
(2) merge:
A. select the mouse myeloma SP2/0 cell strain being in logarithmic phase, collect mouse myeloma SP2/0 cell, centrifugal 10 min of 1000 r/min, abandon supernatant, use 0.4% trypan exclusion stain afterwards with 5 mLDMEM basic mediums are resuspended, counting, can carry out cytogamy when being greater than 90%;
B. get the highest mouse of serum titer, spurt immunity is after 4 days, and under socket of the eye, hole is taken a blood sample, be separated positive serum ,-20 DEG C frozen for subsequent use, and cervical dislocation is put to death, immerse in 75% alcohol 5 min that sterilize, aseptic taking-up spleen, divests fat and reticular tissue, be placed on 200 mesh sieves and shred, gently after grinding, rinse with 15 Ml DMEM basic mediums, proceed in 50 mL centrifuge tubes, centrifugal 10 min of 1000 r/min, abandon supernatant, resuspended with 5 mL DMEM basic mediums, 0.4% trypan blue solution dyeing, counting;
C. mouse myeloma SP2/0 cell and splenocyte are mixed in 50 mL centrifuge tubes in the ratio of 1:8, centrifugal 10 min of 1000 r/min, abandon supernatant, touch at the bottom of pipe and break up cell mass, be placed in 37 DEG C of water-baths, with the 50% PEG(polyoxyethylene glycol of 1 mL suction pipe by pre-temperature, v/v) solution slowly adds in fusion pipe, limit edged rotates fusion pipe gently, add in 2 min, leave standstill 1 min in a water bath, slowly drip basic medium 15 mL of 37 DEG C of pre-temperature immediately, adding step is: 1mL/min, after leaving standstill 1 min, 2mL/min, 3 mL/ min, 9 mL/ min, centrifugal 8 min of 1000 r/min, abandon supernatant, add 60 mL HAT substratum suspension cells, join in the 96 porocyte culture plates containing feeder cell after mixing, every hole 100 μ L, be placed in 37 DEG C, 5% CO
2cultivate in incubator,
(3) positive-selecting of hybridoma:
After cytogamy, in time growing hybridoma rapidly and grown to about 1/2 of covering 96 orifice plate floorage, adopt indirect elisa method screening positive hybridoma cell, concrete steps are as follows:
A. draw treat gaging hole supernatant 100 μ L correspondence add bag by good check-out console, 37 DEG C hatch 0.5 h after wash;
B. add the goat anti-mouse IgG-HRP of 1:10000, every hole 100 μ L, 37 DEG C hatch 0.5 h after wash;
C. TMB(3 is added, 3', 5,5'-tetramethyl benzidine) substrate nitrite ion, every hole 100 μ L, 37 DEG C of lucifuges develop the color 15 min;
D. add 2 mol/L sulfuric acid stop buffers, every hole 50 μ L, measure OD450nm value;
E. to merge the positive serum of mouse for positive control; With the cell conditioned medium in amixia knurl growth hole for negative control, take cell culture medium as blank.
(4) hybridoma clone, build strain:
Positive cell hole is to double screening, adopts limiting dilution assay to clone, and evaluate according to the OD450nm value detected and corresponding number of cell clones.The hole that preferred OD450nm value is high, number of cell clones is few, by being cloned into continuous 3 time cloning positive rates all reaches 100% for several times, obtains hybridoma cell strain 2 strain.Respectively by 2 strain of hybridoma enlarged culturing, through repeatedly going down to posterity, after freezing and thawing, ELISA detects cell conditioned medium.Selection is tired a strain preservation of height, high specificity, good stability.
Wherein the supernatant of hybridoma cell strains is tired as 1:6.92 × 10
4; Detect through mouse monoclonal antibody homotype Rapid ELISA detection kit, determine that 1H9 monoclonal antibody belongs to for IgG2a/ κ (see figure 3); Called after anti-olaquindox specificity lymph hybridoma cell strain 1H9.This cell strain has delivered the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, and preservation date is on July 4th, 2012, preserving number CGMCC No. 6260.
4, the preparation and purification of monoclonal antibody:
(1) preparation of ascitic type monoclonal antibody:
Get adult BALB/C female mice by 0.5ml/ abdominal injection whiteruss, 10 d pneumoretroperitoneums inject hybridoma, and 1 ~ 5 × 10
6individual/only, when visible mouse web portion obviously expands and is slow in action after injection 7 ~ 10 d, carry out aseptic technique after 10 ~ 14 d and gather ascites, centrifugal 10 min of 1000 rpm, collect middle layer clarified liq, are ascitic type monoclonal antibody, and mensuration is tired ,-20 DEG C of preservations.The results are shown in Figure 5, purified antibodies is tired as 1:3.2 × 10
7.
(2) pre-treatment of monoclonal antibody:
Get ascitic type monoclonal antibody 5 mL, add the saturated (NH of 3 times of mL normal saline and 4 times of volumes respectively
4)
2sO
4solution, mixing; After 4 DEG C of precipitation 2h, there is a large amount of white flock precipitate in solution, centrifugal 30 min of 4000rpm; Abandoning supernatant, resolution of precipitate, in the physiological saline of 5 mL, adds the saturated (NH accounting for cumulative volume 45%
4)
2sO
4solution, after 4 DEG C of precipitation 2 h, centrifugal 30 min of 4000 rpm; Abandoning supernatant, precipitation is dissolved in the physiological saline of 5 mL;
(3) purifying of monoclonal antibody:
Choose Sephacry S-200 and make post material (GE Medical Group, 17-0584-10), dress post, the ascitic type monoclonal antibody of getting saturated ammonium sulphate after balance carries out loading, and moving phase is 0.2M PBS, flow velocity 0.8mL/min, until when the UV light absorption value of display starts to rise, mensuration antibody protein is tired, and collects the ascties protein of positive pipe ,-20 DEG C of preservations;
(4) evaluation of monoclonal antibody:
Adopt the anti-olaquindox monoclonal antibody of SDS-PAGE electroresis appraisal, the results are shown in Figure 4, heavy chain, the light chain bands of purified antibodies are clear, and assorted band is less;
After adopting BCA kit measurement mouse ascites, ammonium sulfate process, the protein concentration of purified antibodies be respectively antibody protein concentration 50.14,28.59,3.24mg/mL, after recording purifying, olaquindox monoclonal antibody tires as 64000(P/N>2.0).
Use the mouse monoclonal antibody homotype Rapid ELISA detection kit of Pierce company, detect Hybridoma Cell Culture supernatant liquor, result shows that this anti-olaquindox monoclonal antibody subclass is IgG2a/ κ.
embodiment 2:
The foundation of racing ELISA detecting method:
1. indirect competitive ELISA method
(1) bag quilt: after detectable antigens OLA-OVA bag being buffered liquid dilution, every hole 100 μ L, add in 96 hole enzyme plates, 4 DEG C are spent the night, and PBST washs 5 times;
(2) close: every hole adds 200 μ L confining liquids, and hatch 1 h for 37 DEG C, PBST washs 5 times;
(3) add measuring samples: add measuring samples, every hole 50 μ L, arrange olaquindox standard control and the blank of different concns simultaneously, then add the olaquindox monoclonal antibody of working concentration, every hole 50 μ L, mixing, hatch 0.5 h for 37 DEG C, PBST washs 5 times;
(4) add goat anti-mouse IgG-HRP: the goat anti-mouse IgG-HRP adding working concentration, every hole 100 μ L, hatch 0.5 h for 37 DEG C, PBST washs 5 times;
(5) add nitrite ion: add freshly prepared TMB nitrite ion, every hole 100 μ L, 37 DEG C of lucifuges develop the color 15 min;
(6) add stop buffer: add 2 mol/L H2SO4 stop buffers, every hole 50 μ L, termination reaction, reads OD450nm value by microplate reader;
(7) result judges: with blank control wells zeroing, do typical curve with different concns olaquindox standard substance denary logarithm value and the OD450nm value recorded.
2. the determination of envelope antigen and olaquindox monoclonal antibody best effort concentration:
Adopt square formation volumetry, by the detected result of indirect elisa method in table 1, as can be seen from the table, when the extension rate of OLA mAb is 4000, when OLA-HS-OVA dilutes 20000 times, OD450nm is 1.022, monoclonal antibody addition in binding reagents box, consider that antibody concentration can reduce by half in OLA-Kit operation, therefore determines that OLA mAb and OLA-HS-OVA working concentration are respectively 1:2000 and 1:20000.
Table 1
。
3. the determination of goat anti-mouse IgG-HRP best effort concentration:
Being prepared check-out console with the bag of the OLA-OVA determined by concentration, is positive control with the olaquindox monoclonal antibody of working concentration, and the ascites prepared with murine myeloma cell SP2/0 (1:1000) is negative control; With enzyme diluent, goat anti-mouse IgG-HRP is diluted 5000,10000,15000,20000 times respectively, measured by indirect elisa method, finally according to the OD of each group of positive hole and negative hole
450nmvalue and ratio (P/N value) thereof, goat anti-mouse IgG-HRP extent of dilution corresponding to maximum value is the best effort concentration of goat anti-mouse IgG-HRP, and as shown in table 2, the result in table 2 shows, the P/N when 1:10000 is the highest for enzyme working concentration.
Table 2
。
Embodiment 3:
Indirect ELISA method detects antibody sensitivity, linearity range, specificity:
1, indirect ELISA method detects antibody sensitivity, linearity range:
With PBS liquid preparation olaquindox standard solution, concentration is respectively 0,1,3,9,27,81,243 ng/mL, adopts indirect competitive ELISA method to detect.As shown in table 3, with olaquindox concentration denary logarithm value for X-coordinate, with inhibiting rate B/B0, for ordinate zou, (B is OD450nm value corresponding to OLA different standards concentration, OD450nm value corresponding when being 0 that B0 is OLA concentration), drawing standard suppresses curve (see figure 6), carry out correlation regression analysis, calculate monoclonal antibody to the IC50 of olaquindox.The results are shown in Table 3, determine that the IC50 of antibody is 15.30+6.99, linearity range is 0 ~ 243 ng/mL.
Table 3
。
2, indirect ELISA method detects antibodies specific:
Using olaquindox ex hoc genus anne thing Quinocetone, mequindox, 3-Jia based quinoxaline-2-carboxylic acid, (MQCA), Oxoquinoxaline-2-carboxylic acid (QCA) and terramycin, paraxin, florfenicol, lincomycin, lactic norfloxacini, Clenbuterol hydrochloride, as competitor, measure monoclonal antibody to the IC of each competitor with indirect competitive ELISA method
50.According to formula, calculate cross reacting rate.Cross reacting rate (CR%)=IC
50(olaquindox)/IC
50(competitor) × 100%.As shown in Table 4, the specificity of anti-olaquindox monoclonal antibody is high, particularly marker no cross reaction residual with it.
Table 4
。
embodiment 4:
The composition of olaquindox detection kit and using method:
1. the composition of olaquindox test kit
(1) preparation of enzyme plate: by bag by antigen OLA-HS-OVA as shown in Figure 2, dilute 10000 times with the sodium carbonate-bicarbonate damping fluid of pH 9.6, every hole 100
/ hole, 4 DEG C of bags are spent the night, and secondary daily 5% skimmed milk solution is by 200
/ hole, 37 DEG C of closed 2h, discard liquid, dry, envelope;
(2) determination of sample working solvent: choose the methyl alcohol of 9 kinds of different concns and water, methyl alcohol and PBS solution as sample working solvent, investigate typical curve and the rate of recovery; Determine that the PBS solution containing 0 ~ 50% methyl alcohol is as sample working solvent;
(3) protein concentration is 1.62
the monoclonal antibody working fluid of/mL, solvent is pH7.4 PBS;
The goat anti-mouse IgG of the horseradish peroxidase-labeled of (4) 10000 times of dilutions;
(5) substrate A liquid is for containing 0.1%H
2o
2pH 5.0 sodium phosphate citric acid solution;
(6) substrate B liquid is the TMB ethanol solution of 2mg/mL; Olaquindox standard solution 7 bottles, 0,1,3,9,27,81,243ng/mL;
(7) stop buffer is the H of 2M
2sO
4;
(8) 2 times of concentrated sample working solvent are for containing 0 ~ 50% methyl alcohol, and pH is the PBS solution of 7.4;
(9) 20 times of concentrated washing lotions are contain the PBST solution that 0.2M, pH are 7.4.
All reagent is stored in 2 ~ 8 DEG C.
2. Sample pretreatment:
Ground by Feed Sample, claim the Feed Sample that 1.0 ± 0.05g grinds, add 10ml deionized water, vibration shakes up, and puts into 70 DEG C of water-bath 30min; The centrifugal 15min of 4000r/min, sucking-off supernatant liquor; With sample working solvent 10 times or 4000 times of dilution supernatant liquors, mix, get 50
analyze.
3. the detection method of test kit:
(1) olaquindox standard solution and the corresponding micropore of feed sample to be measured are numbered according to the order of sequence, it is parallel that each standard substance and sample do 2 holes, and the position at record standard hole and sample aperture place;
(2) in the micropore of correspondence, olaquindox standard solution and sample to be tested 50 is added successively
, then add monoclonal antibody working fluid 50
/ hole, mixing of vibrating gently, with the rearmounted 37 DEG C of reaction 30min of cover plate film shrouding;
(3) carefully open cover plate film, liquid in hole is dried, with washings 250
/ hole, fully washs 4 ~ 5 times, and each washing room, every the several seconds, pats dry with thieving paper;
(4) ELIAS secondary antibody 100 is added
/ hole, mixing of vibrating gently, with the rearmounted 37 DEG C of reaction 30min of cover plate film shrouding, washes version, method same with (3);
(5) be taken up in order of priority and add substrate A liquid, substrate B liquid each 50
/ hole, mixing of vibrating gently, with the rearmounted 37 DEG C of reaction 10 ~ 15min of cover plate film shrouding;
(6) stop buffer 50 is added
/ hole, mixing of vibrating gently, measures OD by microplate reader
450nm.
4. the interpretation of result of test kit:
(1) sxemiquantitative decision method: because the content of sample of color or light absorption value and olaquindox is negative correlation, so color or the OD that can use sample aperture
450nmwith color or the OD of olaquindox standard orifice
450nmcompare, thus judge the concentration range of olaquindox in sample.
(2) quantitative analysis:
A. according to formula, percentage light absorption ratio is calculated:
Percentage absorbance (%)=B/B
0× 100%;
The mean absorbance values of B-standardized solution or sample solution;
B
0the mean absorbance values of-0 ng/mL standardized solution.
B. drawing standard curve, calculating concentration of specimens: with the percentage light absorption ratio of olaquindox standard substance for ordinate zou, with the semilog of olaquindox standard concentration for X-coordinate, drawing standard graphic representation, according to regression equation and Sample Dilution multiple, calculates the interpolation concentration of olaquindox in feed sample.
embodiment 5:
Detect the test strip of olaquindox in feed:
1. the preparation of olaquindox colloidal gold strip:
After anti-olaquindox monoclonal antibody colloid gold label, preparation colloidal gold pad, lines goat anti-mouse IgG-HRP and OLA-HS-OVA respectively and catches line and control line, test strip loaded together with siccative in aluminium foil bag, sealed type storage;
2. detect the preparation of sample:
Feed Sample is ground, adds appropriate amount of deionized water, shaken well, put into 70 DEG C of water-bath 30min; The centrifugal 15min of 4000r/min, sucking-off supernatant liquor is used for detecting;
3. using method
Test strip is inserted in analyte sample fluid, after 2 ~ 4 minutes, observe detected result, occur a red trace line, judge that sample is as positive (+); There are two red trace lines, judge that sample is as negative (-); If there is no red trace line, then show that test strip lost efficacy.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment to invention has been detailed description, for a person skilled in the art, it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (6)
1. there is mouse boosting cell-myelomatosis hybridoma that preserving number is CGMCC No.6260.
2. having the anti-olaquindox monoclonal antibody of high specific, it is characterized in that, is that the hybridoma cell strain being CGMCC No.6260 by preserving number produces.
3. the preparation method with the anti-olaquindox monoclonal antibody of high specific according to claim 2, is characterized in that, comprise the following steps:
A) preparation of ascites: get adult BALB/C female mice by 0.5ml/ abdominal injection whiteruss, within 10 days, pneumoretroperitoneum injects hybridoma cell strain described in claim 1, and injection rate is 1 ~ 5 × 10
6individual/only, inject and carry out aseptic technique after 10 ~ 14 days and gather ascites, by the ascites that collects with centrifugal 10 min of rotating speed 1000 rpm, collection middle layer clarified liq, namely obtains ascitic type monoclonal antibody;
B) antibody purification: step a) is obtained centrifugal after ascites supernatant saturated ammonium sulphate, obtain anti-olaquindox monoclonal antibody crude product; Carry out purifies and separates through Sephacryl S-200 chromatography column, collect the ascties protein of positive pipe, obtain anti-olaquindox monoclonal antibody.
4. the anti-olaquindox monoclonal antibody with high specific according to claim 2 detects the application in olaquindox in feed at quantitative and qualitative analysis.
5. the anti-olaquindox monoclonal antibody with high specific that the hybridoma that utilization is CGMCC NO.6260 by preserving number according to claim 1 is secreted detects a test kit for olaquindox in feed, and it is characterized in that, described test kit comprises following composition:
1) enzyme plate of liquid bag quilt is buffered with the bag containing olaquindox derivative-oralbumin conjugate;
2) protein concentration is the anti-olaquindox monoclonal antibody solution of 1.62 μ g/mL, and solvent is pH7.4 PBS;
3) goat anti-mouse IgG of the horseradish peroxidase-labeled of 10000 times of dilutions;
4) substrate A liquid: be 0.1%H containing volumn concentration
2o
2pH be 5.0 sodium phosphate citric acid solution;
5) substrate B liquid: 2mg/mL 3,3 ', 5,5 '-tetramethyl benzidine ethanol solution;
6) sulfuric acid of stop buffer: 2M;
7) olaquindox standard solution: be respectively 0,1,3,9,27,81,243ng/mL;
8) 2 times of concentrated sample working solvent: containing volumn concentration 0 ~ 50% methyl alcohol, pH is the PBS solution of 7.4;
9) 20 times of concentrated washing lotions: concentration to be 0.2M, pH be 7.4 PBST solution.
6. test kit according to claim 5 is detecting the application in olaquindox, and it is characterized in that, the using method of test kit is:
1) pre-treatment of sample: take 1.0 g Feed Samples, grinds, adds 10mL deionized water, after shaking up, put into 80 DEG C of water-bath 40min; The centrifugal 10min of 4000r/min, sucking-off supernatant liquor, filter paper filtering; With sample working solvent 10 times or 400 times of dilution supernatant liquors, mix, get 50 μ L and analyze;
2) working method: by olaquindox standard solution and the corresponding micropore numbering of feed sample to be measured, record; Every hole adds olaquindox standard solution or each 50 μ L of sample to be tested, then adds olaquindox monoclonal antibody 50 μ L/ hole, 37 DEG C of reaction 30min; Open cover plate film, dry liquid in hole, PBST washing 4 ~ 5 times; Add ELIAS secondary antibody 100 μ L/ hole, 37 DEG C of reactions 30min, PBST wash 4 ~ 5 times; Add substrate A liquid, each 50 μ L/ holes of substrate B liquid, 37 DEG C of reaction 10 ~ 15min; Add stop buffer 50 μ L/ hole, measure OD by microplate reader
450nm.
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| CN104558185B (en) * | 2014-12-26 | 2018-08-03 | 华中农业大学 | Monoclonal antibody and enzyme-linked immunoassay method for detecting mequindox and olaquindox metabolite and kit |
| CN106754732A (en) * | 2015-11-20 | 2017-05-31 | 中国检验检疫科学研究院 | Olaquindox hybridoma cell strain and monoclonal antibody and complex immunity adsorbent and immune affinity column and kit and its application |
| CN110927382A (en) * | 2019-10-08 | 2020-03-27 | 杭州佰昕科技有限公司 | Time-resolved fluorescence immunoassay kit for detecting olaquindox and application thereof |
| CN110927375A (en) * | 2019-10-08 | 2020-03-27 | 杭州佰昕科技有限公司 | Fluorescent microsphere immunochromatography test strip for detecting olaquindox residue and application thereof |
| CN111060690B (en) * | 2019-10-08 | 2023-07-07 | 浙江工商大学 | Time-resolved fluoroimmunoassay kit for detecting olaquindox and application thereof |
| CN110927383A (en) * | 2019-10-08 | 2020-03-27 | 杭州佰昕科技有限公司 | Fluorescent microsphere immunochromatography test strip for detecting olaquindox residue 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 |
| 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 |
| CN111551748A (en) * | 2020-05-29 | 2020-08-18 | 临沂大学 | Enzyme-linked immunosorbent assay kit for trichosanthin and use method thereof |
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