CN101988923A - Time resolution immunoassay detection kit of diethylstilbestrol residuals and detection method thereof - Google Patents
Time resolution immunoassay detection kit of diethylstilbestrol residuals and detection method thereof Download PDFInfo
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Abstract
The invention provides a time resolution immunoassay detection kit of diethylstilbestrol residuals and a detection method thereof. The kit of the invention is coated with an elisa plate of diethylstilbestrol antigen, a lanthanide-labeled goat-anti-rabbit or goat anti mouse antibody, a diethylstilbestrol antibody and the like. The invention also discloses a method using the kit for detecting the diethylstilbestrol residuals. In the kit for detecting the diethylstilbestrol provided by the invention, an indirect competitive time resolution immunoassay technology is adopted, so that immunoassay is high, stability is good, operating steps are simplified greatly, reaction time is shortened greatly, errors caused by complicated operation are reduced and the cost is lowered, thereby being suitable for screening for a large number of samples and having important realistic significance.
Description
Technical field
The invention belongs to technical field of immunoassay, relate to residual detection kit of a kind of Pollution by Chemicals and preparation method thereof, be specifically related to a kind of residual time resolution immunoassay kits of diethylstilbestrol and preparation method thereof that detects.
Background technology
Diethylstilbestrol (diethylatilbestrol, DES) belong to the synthetic hormone, diethylstilbestrol (vehicle economy S) is first oral activated synthetic estrogen, chemistry is called 1, two (4-2-hydroxyphenyl) 21, the 22 diethyl ethene of 2-2-, the popular name DES, being colourless crystallization or white crystalline powder, is the present domestic clinical medicine synthetic hormone of widespread use the most, and its structural formula is suc as formula shown in (I):
Diethylstilbestrol is obvious in aspect effects such as the anabolism that promotes animal protein, raising animal daily gain and minimizing fat.Therefore be used as a kind of animal growth promoting agent is used for animal and fowl fodders such as pig, ox, sheep, chicken always.As a kind of animal feed additive, diethylstilbestrol can be in animal derived food, as residual in liver, muscle, egg, the milk, animal is by polluting and enrichment surrounding environment by metabolism simultaneously, can cause juvenile's growth precocity and feminization by food chain, and the imbalance of people's hormone in vivo, diseases such as women with breast cancer, oophoroma.
Late 1970s, international cancer research institution (IARC) discovers that diethylstilbestrol is because the carcinogenicity of itself can produce serious threat to health.Find in the risk assessment to diethylstilbestrol that simultaneously diethylstilbestrol all has considerable influence to the incidence and the neurological problem of genital structure, fecundity and the cancer of human body, also have a strong impact on people's immune system and immunologic function simultaneously.The abuse of diethylstilbestrol in animal food caused extensive attention both domestic and external.At present, each country all forbids the application of diethylstilbestrol (DES) in animal produces both at home and abroad.In order to guarantee the healthy of people, effectively contain the abuse of diethylstilbestrol, the detection method of setting up diethylstilbestrol has very large meaning.
At present, detecting the residual method of DES mainly contains following several: high performance liquid chromatography (HPLC), gas chromatography-mass spectrography (GC-MS), liquid chromatography-mass spectrography (LC-MS), thin-layered chromatography, capillary zone electrophoresis method (CE) and immune analysis method (IA).Though instrumental method is sensitive accurately, needs expensive professional instrument, to analyze time-consumingly, cost is higher.Immune analysis method has simply, fast, sensitivity and the low characteristics of cost, can reach trace (μ gkg
-1) level, it is a kind of high flux prescreening method that present countries in the world are carried out and implemented, the time resolution immunologic detection method belongs to a kind of of immune analysis method, and it is actually and grows up on the basis of fluorescence analysis (FIA), is a kind of special fluorescence analysis.
The ultimate principle of time resolution immunoassay (TRFIA) be its to have adopted special lanthanide series metal be trivalent rare earth ions (Eu
3+, Tb
3+, Dy
3+, Sm
3+) and sequestrant (replacing fluorescent material, isotope or enzyme).Labelled protein, polypeptide, hormone, antibody, nucleic acid probe or biologically active cell, after question response system (as antigen-antibody reaction, nucleic acid probe reaction, the reaction of biotin Avidin, target cell and effector cell kill and wound reaction etc.) takes place, with the fluorescence intensity in the time resolved analysis instrument mensuration end product, power according to fluorescent value, the concentration of analyte in the judgement system reaches the purpose of quantitative test.
The time immunologic detection method has been widely used in clinical detection abroad, is applied in many frontiers in recent years, as immunohistochemistry, microarray, and can be used for multiple labeling immunoassays such as double-tagging, three marks and four marks.The fluorescence immunoassay detection method has been learned to labelled immune and has been brought a revolution as an emerging biology techniques with wide development potentiality.The present invention provides a kind of new way to the fast detecting that immunoassay technology further is applied to the food veterinary drug residue.
But still there is not the correlation technique report that adopts the time resolution immunoassay to detect diethylstilbestrol at present, more there is not a kind of kit that diethylstilbestrol time resolution immunoassay detects that is fit to carry out, so that realize high sensitivity, fast detecting in enormous quantities simple to operate.
Summary of the invention
An object of the present invention is to overcome the deficiencies in the prior art, a kind of residual time resolution immunoassay detection kit of diethylstilbestrol that detects is provided.
Another object of the present invention provides the residual method of described detection diethylstilbestrol, and high sensitivity, simple to operate can fast detecting in enormous quantities.
Purpose of the present invention is achieved by the following technical programs:
A kind of residual time resolution immunoassay detection kit of diethylstilbestrol that detects is provided, comprises by the ELISA Plate of diethylstilbestrol antigen, diethylstilbestrol antibody, lanthanide series mark goat-anti rabbit or sheep anti-mouse antibody (two is anti-); Described lanthanide series comprises Eu
3+, Tb
3+, Dy
3+Or Sm
3+Etc. rare earth element.
To be carbodlimide method (EDC), active ester method (DCC, NHS), mixed anhydride method (isobutyl chlorocarbonate) carry out coupling with diethylstilbestrol haptens and carrier protein to described diethylstilbestrol antigen obtains, and described carrier protein comprises bovine serum albumin(BSA) (BSA), human serum albumins (HSA), keyhole limpet hemocyanin carrier proteins such as (KLH).
Diethylstilbestrol and sodium chloroacetate are carried out halogenating reaction, replace the hydroxyl on the diethylstilbestrol molecule, the synthetic diethylstilbestrol haptens that contains the carboxyl spacerarm of 2 carbon; Or the amino in maleic anhydride and the diethylstilbestrol carried out acylation reaction, the synthetic diethylstilbestrol haptens that contains the carboxyl spacerarm of 4 carbon.
The haptens that will contain the carboxyl spacerarm of 2 carbon is used for immunogenic preparation, and the haptens that will contain the carboxyl spacerarm of 4 carbon is used for the preparation of coating antigen, and immunogene and coating antigen carry out purifying by column chromatography, and purity is identified through the SDS-PAGE electrophoresis.Immunogene of the present invention adopts different haptens structures will more help improving the sensitivity of detection with coating antigen.
Described diethylstilbestrol antibody comprises monoclonal antibody, rabbit polyclonal antibody or genetic engineering antibody.
MONOCLONAL ANTIBODIES SPECIFIC FOR:
Animal immune: haptens and carrier protein couplet thing with the carboxyl spacerarm that contains 2 carbon are that immunogene is carried out the interval immunity to the Balb/c mouse, and immunoassay detects and obtain containing in the blood mouse spleen of diethylstilbestrol specific antibody indirectly.
Fusion of Cells and clone: get the Balb/c mouse boosting cell and the myeloma cell SP20 that produce specific antibody and merge, adopt indirect competition time resolution immunization method to measure cell conditioned medium liquid, screen positive hole.Utilize limiting dilution assay or microclone method that cloning is carried out in positive hole, obtain and set up the hybridoma cell strain that produces antibody.
Cell cryopreservation and recovery: get the hybridoma that is in exponential phase and make cell suspension, be sub-packed in frozen pipe, in the medium-term and long-term preservation of liquid nitrogen with cryopreserving liquid.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
The preparation of antibody and purifying: adopt in the body and induce method, Balb/c mouse (8 age in week) abdominal cavity is injected the sterilization paraffin oil, 7～14 days pneumoretroperitoneum injection hybridomas were gathered ascites after 7～10 days.Carry out the ascites purifying through sad-saturated sulfuric acid amine method or affinity chromatography, purity is identified through the SDS-PAGE electrophoresis, bottle packing ,-20 ℃ of preservations.
The preparation of diethylstilbestrol rabbit polyclonal antibody
Adopt new zealand white rabbit as immune animal, with diethylstilbestrol and carrier protein couplet thing is that immunogene is carried out immunity to new zealand white rabbit, repeatedly serum antibody titer is measured in the immunity back, and heart is taken a blood sample, and obtains the polyclonal antibody of purifying through sad-ammonium sulphate precipitation.
The preparation of diethylstilbestrol genetic engineering antibody
Genetic engineering antibody mainly refers to small molecular antibody, comprise: Fab (constituting), Fv (constituting), ScFv (single-chain antibody by VH and VL by complete light chain and Fd, be formed by connecting by a connection peptides between VH and the VL), single domain antibodies (only being made up of VH) etc. are through the improved antibody of technique for gene engineering.
The preparation method is: the RNA that extracts diethylstilbestrol monoclonal cell or the mouse boosting cell after the diethylstilbestrol immunogen immune, reverse transcription is cDNA, designerantibodies weight chain amplimer, utilize round pcr to amplify the weight chain gene of antibody, insert suitable expression plasmid, at expression in escherichia coli, utilize immune affine method to carry out purifying, purity is identified by the SDS-PAGE electrophoresis.
The preparation of described lanthanide series mark goat-anti rabbit or sheep anti-mouse antibody is with Eu
3+Mark goat-anti rabbit is an example, gets the 5mg/mL goat anti-rabbit igg 1ml that is dissolved in 0.05mol/L PBS pH7.0, and through the conversion buffered salt condition of PD-10 post, eluent is 50mmol/L pH 9.6NaCO
3-NaHCO
3Damping fluid.Collect protein peak, through the quantitative (1.46A of uv absorption analysis
280-0.74A
260), dilute the goat-anti rabbit to 2mg/mL with above-mentioned eluent.The goat anti-rabbit igg of getting after 500 μ L dilute adds the Eu that contains 0.2mg
3+-N
2-[p-isocyanic acid-benzyl]-oxalic acid alkene triamine tetraacethyl (Eu
3+In-DTTA) the brown vial, place 28 ℃ of constant temperature ovens to react 48h, (1 * 30cm) chromatography is measured the photofluorometer numerical value of every pipe to reactant liquor with the SepharoseCL-6B of 50mmol/L Tris-HCl pH 7.8 damping fluid balances, detect first counting peak after diluting, the dilution back is standby.
Mark rate=mark number/IgG molecular number
For convenience of execute-in-place and batch detection, described kit can also comprise the diethylstilbestrol standard solution, strengthen liquid, concentrated cleaning solution, diluted sample concentrate.
Preferably, can also comprise that box body, bag are cushioned liquid, lavation buffer solution, confining liquid and cover plate film.
Described diethylstilbestrol standard solution is that concentration is the DES standard items stock solution of 1000 μ g/L, and being made into concentration gradient before the use again is diethylstilbestrol (DES) the standard items working fluid of 8 μ g/L, 4 μ g/L, 2 μ g/L, 1 μ g/L, 0.5 μ g/L, 0 μ g/L.
Described enhancing liquid comprises beta-diketon body, trioctylphosphine oxide (TOPO), TritonX-100, glacial acetic acid and Potassium Hydrogen Phthalate (the pH value is 2.0～3.2); With the Potassium Hydrogen Phthalate adjust pH to 3.2 of 0.1mol/L, (1mL Triton X-100 adds tri-distilled water and is settled to 1L for β-NTA), 50 μ mol trioctyl phosphine oxides to add 15 μ mol beta-diketon bodies with the 6mL glacial acetic acid.
Described concentrated washing lotion comprises that (pH value 7.4 0.1mol/L), is 15～25 times of conventional working concentration for the phosphate buffer of 0.5～1.5% (volume by volume concentration) polysorbas20.
The Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) of described concentration and dilution liquid pH value 7.4～8.0,0.1～0.25mol/L is 5～15 times of conventional working concentration.
ELISA Plate is 96 hole ELISA Plate, adopts polystyrene micropore plate, and it is the anti-diethylstilbestrol antigens of 75 μ g/L that this coated in microporous plate has concentration, and closed porosity surface adsorption site not; With the sealing of confining liquid room temperature, described confining liquid comprises skimmed milk power solution (the 5g skimmed milk power: 100mL PBS) that is dissolved in PBS.With the calf serum 3mL (deactivation) of diluted to final concentration 3% (volume by volume concentration).
The present invention provides simultaneously and has detected the residual method of diethylstilbestrol, may further comprise the steps:
(1) sample pre-treatments;
A. urine specimen is handled
Limpid urine sample can directly carry out check and analysis.If urine sample is muddy shape, centrifugal 5min of 2000r/min or filtration detect with supernatant.
B. feed
Feed is pulverized, taken by weighing 1g and place the 50mL test tube, add 10mL ethyl acetate, fully mixing vibration 10min.The centrifugal 10min of 5000r/min.Get organic phase 1mL organic phase in another test tube, vacuum rotary steam is done, and adds 2mL n-hexane dissolution sample; PBS-methyl alcohol (3: 2) the solution 2mL that adds pH7.0, vortex oscillation 5min, the centrifugal 10min of 4000r/min.Remove the upper strata normal hexane, it is to be measured to take off layer solution 50 μ L.
C. organize (detection) with muscle, liver or kidney etc.
With historrhexis, with Ultrasound Instrument or analogous instrument tissue is homogenized, take by weighing the tissue after 3g homogenizes, add ethyl acetate 9mL, anhydrous calcium oxide 0.3g, vortex oscillation 10min, the centrifugal 10min of 4000r/min.Remove supernatant 6mL evaporate to dryness, add normal hexane 2mL dissolved residue, add PBS-methyl alcohol (3: 2) the solution 1mL of pH7.0, vortex oscillation 5min, the centrifugal 10min of 4000r/min.Remove the upper strata normal hexane, it is to be measured to take off layer solution 50 μ L.
(2) utilize kit of the present invention that standard solution and sample are detected.
(3) according to typical curve and sample solution fluorescent value calculation sample concentration.
Main agents of the present invention provides with the form of working fluid, saves the running time, and described detection specifically may further comprise the steps:
(1) kit is taken out from cold storage environment, place 20～24 ℃ of environment balances to be no less than 30min, ELIAS strip is fixed, do two parallel laboratory tests, number in order;
(2) add 50 μ L standard solution in the standard items hole, sample well adds 50 μ L testing samples, and every then hole adds 50 μ L diethylstilbestrol antibody working fluids, mixing; Oscillating reactions 45min;
(3) wash plate behind the question response six times, and on thieving paper, pat dry, to guarantee to remove fully the liquid in the hole; Every hole adds 100 μ L europiums and marks two anti-working fluids, oscillating reactions 45min; Described Eu3+ mark goat-anti rabbit, mark rate is 11.2, concentration is 0.15mg/mL;
(4) wash plate behind the question response six times, and on thieving paper, pat dry, to guarantee to remove fully the liquid in the hole; Every hole adds 200 μ L and strengthens liquid, mixing, and the lucifuge room temperature is patted vibration 5min;
(5) differentiate immunoassay (TRFIA) detector with the time and measure each hole fluorescent value;
(6) do typical curve according to standard solution fluorescent value and concentration semilog, according to typical curve and sample solution fluorescent value calculation sample concentration.
Testing result is handled and is analyzed:
With the mean value calculation percentage fluorescent value of obtained standard model light absorption value, be ordinate with the percentage fluorescent value, the semilog of diethylstilbestrol concentration of standard solution is a horizontal ordinate drawing standard curve, obtains straight-line equation.The use the same method percentage fluorescent value of calculation sample solution is obtained the diethylstilbestrol concentration of counter sample according to equation.The calculating formula of described percentage fluorescent value is:
Percentage fluorescent value (%)=(B/B
0) * 100
Wherein, B is the mean fluorecence value of standard solution or sample, B
0It is the mean fluorecence value of 0 μ g/L standard solution.
The analysis of testing result can also utilize computer professional software to calculate and analyze, and is 0.1～8.1 μ g/L to the diethylstilbestrol linear detection range, and detectability can reach 0.01 μ g/L, and whole testing process needs 2h just can finish.
Beneficial effect of the present invention:
The present invention adopts the lanthanide series thing of marking, and DTTA is a sequestrant, has set up the quantitative analysis method to diethylstilbestrol, and this method operating process is short, simple, fast, and precision and accuracy are all better, and sample pre-treatments is simple.The present invention preferably adopts europium (Eu
3+) thing that serves as a mark, effective, cost is low.
The mode that the present invention adopts sealing to preserve guarantees that the fluorescent value of the blank plate of described ELISA Plate is lower than 1000, guarantees not have the pollution of rare earth element.
Concentrated cleaning solution is that (pH7.4 0.1mol/L), is 15～25 times of normal working concentration for the phosphate buffer that contains 0.5～1.5% polysorbas20; Described diluted sample concentrate is the phosphate buffer of pH7.4～8.0,0.1～0.25mol/L, be 5～15 times of normal working concentration, realize effectively reducing volume under the prerequisite of effect of the present invention, conveniently be equipped with in kit, it is more reasonable that kit is equipped with.
The present invention uses the acid liquid that strengthens, and makes europium ion (Eu
3+) lanthanide ion that waits mark to use disintegrates down free Eu from chelate
3+Down collaborative at trioctyl phosphine oxide, form a kind of new chelate that can accept exciting light expeditiously with the beta-diketon body again.Under excitation, the europium ion capacitation, the process electronic transition is also returned ground state, just can launch extremely strong fluorescence signal, reads its fluorescent value on time resolution immunity (TRFIA) analyser, and its sensitivity can reach 16 * 10
-18Mol Eu
3+
Description of drawings
Fig. 1 typical curve
Embodiment
Further describe the present invention below in conjunction with the drawings and specific embodiments.Employed test method is conventional method if no special instructions among the following embodiment; Employed material, reagent etc. if no special instructions, are the reagent and the material that can obtain from commercial channels.
Get 0.55g (2mmol) DES and be dissolved in 20-50ml acetone, be stirred to dissolving fully, add 1.88g load agent K
2CO
3-Al
2O
3, stirring 1～5h, 0.15ml (1mmol) 2-methyl bromoacetate dropwise adds after being dissolved in 10ml acetone, reaction 8h, reacting liquid filtering, filtrate obtains white solid at the rotation evaporate to dryness, and above-mentioned white is carried out column chromatography (CCl
3H: CH
3OH=10: 1), column chromatography gained monoesters product is dissolved in the 25ml absolute ethyl alcohol, add 15ml water, after adding excessive slightly NaOH solid dissolving, with above-mentioned solution rotating evaporation, remove the ethanol in the solution, obtain aqueous solution and place frozen water to cool off, in this solution, add the 1N hydrochloric acid solution, transfer pH 3～6, white precipitate appears, (3 * 20mL), (2 * 20mL), anhydrous magnesium sulfate dewaters saturated common salt water washing organic phase with ethyl acetate extraction, evaporate to dryness promptly obtains product haptens hapten 1, productive rate 26%.
The haptenic synthetic route of diethylstilbestrol is as follows:
Haptenic nuclear-magnetism qualification result: MS (APCI negative) m/z:325[M-H]
-.MS
2(APCI) m/z:298.9[M-CH
2CH
3], 267[M-CH2COOH].
1HNMR(MeOD-d
6，600MHz)
7.10-7.00(d，2H)，6.93-6.79(d，2H)，4.65(s，1H)，2.13(d，2H)，1.98(d，2H)，0.73(m，3H)
The preparation of embodiment 2 antigens
The preparation of diethylstilbestrol antigen:
A. 50 μ mol/L diethylstilbestrol haptens are dissolved in the dimethyl formamide (DMF) of 1mL, in this solution, add equimolar dicyclohexylcarbodiimide (DCC) and N-hydroxy-succinamide (NHS) then, be allowed to condition to react under the room temperature and spend the night;
B. centrifugal, get supernatant 800 μ L, slowly join among the BSA or OVA carrier protein carbonate buffer solution of 4mL 15mg/mL, under magnetic agitation, react 4h then;
C. after question response was finished, the bag filter of packing into was used distill water dialysis 2 times earlier, uses 0.8% (quality is than concentration) normal saline dialysis then, gets product;
D. adopt UV scanning to measure in conjunction with than (Chen Xinjian etc., 1998), antigen is concentrated preserve or freeze-drying is preserved and obtained diethylstilbestrol immunogene and coating antigen at last, packing is stored in-20 ℃ the refrigerator.
The preparation of embodiment 3 antibody
MONOCLONAL ANTIBODIES SPECIFIC FOR:
Animal immune: haptens and carrier protein couplet thing with the carboxyl spacerarm that contains 2 carbon are that immunogene is carried out the interval immunity to the Balb/c mouse, and immunoassay detects and obtain containing in the blood mouse spleen of diethylstilbestrol specific antibody indirectly.
Fusion of Cells and clone: get the Balb/c mouse boosting cell and the myeloma cell SP20 that produce specific antibody and merge, adopt indirect competition time resolution immunization method to measure cell conditioned medium liquid, screen positive hole.Utilize limiting dilution assay or microclone method that cloning is carried out in positive hole, obtain and set up the hybridoma cell strain that produces antibody.
Cell cryopreservation and recovery: get the hybridoma that is in exponential phase and make cell suspension, be sub-packed in frozen pipe, in the medium-term and long-term preservation of liquid nitrogen with cryopreserving liquid.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
The preparation of antibody and purifying: adopt in the body and induce method, Balb/c mouse (8 age in week) abdominal cavity is injected the sterilization paraffin oil, 7～14 days pneumoretroperitoneum injection hybridomas were gathered ascites after 7～10 days.Carry out the ascites purifying through sad-saturated sulfuric acid amine method or affinity chromatography, purity is identified through the SDS-PAGE electrophoresis, bottle packing ,-20 ℃ of preservations.
(2) preparation of diethylstilbestrol rabbit polyclonal antibody
Adopt new zealand white rabbit as immune animal, with diethylstilbestrol and carrier protein couplet thing is that immunogene is carried out immunity to new zealand white rabbit, first immunisation is with 100 μ L antigens and the fully emulsified back immunity of 100 μ L Freund's complete adjuvants, second and third, four immunity are with 100 μ L antigens and the fully emulsified immunity of 100 μ L Freunds, serum antibody titer and inhibition are measured in the back, heart is taken a blood sample, and obtains the polyclonal antibody of purifying through sulfuric acid amine fractional precipitation.
(3) preparation of diethylstilbestrol genetic engineering antibody
Genetic engineering antibody mainly refers to small molecular antibody, comprise: Fab (constituting), Fv (constituting), ScFv (single-chain antibody by VH and VL by complete light chain and Fd, be formed by connecting by a connection peptides between VH and the VL), single domain antibodies (only being made up of VH) etc. are through the improved antibody of technique for gene engineering.
The preparation method is: the RNA that extracts diethylstilbestrol monoclonal cell or the mouse boosting cell after the diethylstilbestrol immunogen immune, reverse transcription is cDNA, designerantibodies weight chain amplimer, utilize round pcr to amplify the weight chain gene of antibody, insert suitable expression plasmid (TG1 bacterial strain, commercial), at expression in escherichia coli, utilize immune affine method to carry out purifying, purity is identified by the SDS-PAGE electrophoresis.
V wherein
H(Back), V
H(For) two primers antibody heavy chain variable region gene V that is used to increase
HV
L(Back), V
L(For) two primers antibody chain variable region gene V that is used to increase
LTwo primers of Linker1, Linker2 are used for overlap extension PCR method splicing V as the joint primer
H, V
LGenetic fragment becomes the scFv genetic fragment, and this joint primer includes coding connection peptides (Gly
4Ser)
3Genetic fragment, 5 ' end and V
H3 ' the complementation of fragment end, 3 ' end and V
LFragment 5 ' end is complementary.RS (Back), two primers of RS (For) are used for secondary PCR amplification total length scFv genetic fragment, introduce BGl I and Not I restriction enzyme site simultaneously.Two primers of R1, R2 are primer on the carrier, are used for the PCR evaluation that carrier inserts fragment.
V
H(Back) 5
-CAG?GTS?MAR?CTG?CAG?SAG?TCW?GG-3
V
H(For) 5
-TGA?GGA?GAC?GGT?GAC?CGT?GGT?GCC-3
V
L(Back) 5
-GAC?ATC?GAG?CTC?ACT?CAG?TCT?CCA-3
V
L(For) 5
-CCG?TTT?TAT?TTC?CAG?CCT?GGT?CCC-3
Linker1 5
-GGC?ACC?ACG?GTC?ACC?GTC?TCC?TCA?GGT?GGA
GGC?GGT?TCA?GGC?GGA?GGT?GGC?TCT?GG-3
Linker2 5
-TGG?AGA?CTG?AGT?GAG?CTC?GAT?GTC?CGATCC?GCC
ACC?GCC?AGA?GCC?ACC?TCC?GCC-3
RS(Back) 5
-GTC?CTC?GCA?ACT?GCG?
GCC?CAG?CCG?GCC?ATG
(containing Bgl I site) GCC CAG GTC AAA CTG CAG GAG TCA GG-3
RS(For) 5
-GAG?TCA?TTC?T
GC?GGC?CGC?CCG?TTT?TAT?TTC
(containing Not I site) CAG CCT GGT CCC-3
R1 5
-CCA?TGA?TTA?CGC?CAA?GCT?TTG?GAG?CC-3
R2 5
-CGA?TCT?AAA?GTT?TTG?TCG?TCT?TTC?C-3
V
LAmplification condition: reaction solution vortex mixing, of short duration centrifugal after, carry out pcr amplification reaction, course of reaction and condition are: 94 ℃ * 5min sex change, add 1 μ L high-fidelity Pfu enzyme (2.5U), and carry out following circulation: 94 ℃ * 30s, 54 ℃ * 1min, 72 ℃ * 1min, totally 25 circulations, last 72 ℃ are extended 10min.After reaction finishes, V
LReaction product is got 5 μ L and is carried out the detection of 1% agarose gel electrophoresis.
V
HAmplification condition: reaction solution vortex mixing, of short duration centrifugal after, carry out pcr amplification reaction, course of reaction and condition are: 94 ℃ * 5min sex change, add 1 μ L high-fidelity Pfu enzyme (2.5U), and carry out following circulation: 94 ℃ * 30s, 60 ℃ * 1min, 72 ℃ * 1min, totally 30 circulations, last 72 ℃ are extended 10min.After reaction finishes, V
HReaction product is got 5 μ L and is carried out the detection of 1% agarose gel electrophoresis.
(1) splicing of scFv genetic fragment and pcr amplification:
The overlap extension reaction
At first carry out the pre-splicing of linker by following reaction system:
Linker1 primer (10 μ mol/L) 0.5 μ L
Linker2 primer (10 μ mol/L) 0.5 μ L
10×PCR?Buffer 5μL
dNTP(2.5mmol/L) 4μL
Deionized water 34.5 μ L
Cumulative volume 44.5 μ L
Reaction solution vortex mixing, of short duration centrifugal.The pre-sex change of 94 ℃ * 5min on the PCR instrument adds 0.5 μ L high-fidelity Pfu enzyme, carries out following circulation: 94 ℃ * 45s, and 50 ℃ * 1min, 72 ℃ * 1min, totally 3 circulations.
Add V in the system in the above
H, V
LCarry out the splicing of scFv:
V
HPurified product (50ng) 3 μ L
V
LPurified product (50ng) 2.5 μ L
Linker splices solution 44.5 μ L in advance
Cumulative volume 50 μ L
Successively with V
H, V
LAfter the adding, with rifle mixing gently.0.5 μ L high-fidelity Pfu enzyme is added in the pre-sex change of 94 ℃ * 5min on the PCR instrument, carries out following circulation: 94 ℃ * 45s, and 50 ℃ * 1min, 72 ℃ * 1min, totally 30 circulations, 72 ℃ are extended 10min.
(2) pcr amplification of scFv gene
Adopt following reaction system:
Splicing product 4 μ L
5×PCR?Buffer 10μL
dNTP(2.5mmol/L) 4μL
RS (Back) primer (10 μ mol/L) 1 μ L
RS (For) primer (10 μ mol/L) 1 μ L
Sterile pure water 30 μ L
Cumulative volume 50 μ L
Mix, of short duration centrifugal after, on the PCR instrument, the pre-sex change of 94 ℃ * 2min adds 0.5 μ L high-fidelity Pfu enzyme, carries out following circulation: 94 ℃ * 45s, 60 ℃ * 1min, 72 ℃ * 1min, totally 30 circulations, last 72 ℃ are extended 10min.Get 5 μ L reaction product and carry out electrophoresis detection.
Embodiment 4Eu
3+The preparation of mark sheep anti mouse and purifying
Get the 5mg/mL goat anti-rabbit igg 1ml that is dissolved in 0.05mol/L PBS pH7.4, through the conversion buffered salt condition of PD-10 post, eluent is 50mmol/L pH 9.6NaCO
3-NaHCO
3Damping fluid.Collect protein peak, through the quantitative (1.46A of uv absorption analysis
280-0.74A
260), dilute the goat-anti rabbit to 2mg/mL with above-mentioned eluent.The goat anti-rabbit igg of getting after 500 μ L dilute adds the Eu that contains 0.2mg
3+-N
2-[p-isocyanic acid-benzyl]-oxalic acid alkene triamine tetraacethyl (Eu
3+In-DTTA) the brown vial, place 28 ℃ of constant temperature ovens to react 48h, (1 * 30cm) chromatography is measured the photofluorometer numerical value of every pipe to reactant liquor with the SepharoseCL-6B of 50mmol/L Tris-HCl pH 7.8 damping fluid balances, detect first counting peak after diluting, the dilution back is standby.
Mark rate=mark number/IgG molecular number
The preparation of embodiment 5 time resolution immunoassay kits components
(1) preparation of thickening and washing damping fluid: (pH7.4 0.1mol/L), is 15～25 times of normal working concentration to contain the phosphate buffer of 0.5～1.5% polysorbas20.
(2) Tris-HCl of the preparation of diluted sample concentrate: pH7.4～8.0,0.1～0.25mol/L is 5～15 times of normal working concentration.
(3) preparation of confining liquid: skimmed milk power 1.0～5.0g is dissolved in 100mL distilled water.
(4) preparation of enhancing liquid: the 6mL glacial acetic acid adds 15 μ mol with the Potassium Hydrogen Phthalate adjust pH to 3.2 of 0.1mol/L
-two ketoboidies (
-NTA), and 50 μ mol trioctyl phosphine oxides (TOPO), 1mL Triton X-100 adds tri-distilled water and is settled to 1L.
(5) the bag quilt of ELISA Plate microwell plate: envelope antigen pH9.6,0.05mol/L carbonate buffer solution (contain 1～2g sodium carbonate and 2～4g sodium bicarbonate, distilled water 1L) is diluted to 0.1～5ug/mL, every hole in ELISA Plate adds 100uL, 37 ℃ of bags by 1h after 4 ℃ down bag spent the night, coating buffer inclines, with PBST washing 3 times, pat dry, in every hole, add 200uL 1.0～5.0% skimmed milk powers then, wash 3 times with PBST after putting into 37 ℃ of incubator 1h, 4 ℃ of preservations in the aluminium foil bag are enclosed in dry back.
(6) preparation of diethylstilbestrol standard solution: accurately take by weighing diethylstilbestrol standard specimen 8.1mg, be dissolved in the 0.1L damping fluid (the PBS-methyl alcohol (3: 2) of pH7.0), prepare 8 μ g/L, 4 μ g/L, 2 μ g/L, 1 μ g/L, 0.5 μ g/L, 0 μ g/L diethylstilbestrol solution respectively with the damping fluid dilution then, damping fluid is prepared 0 μ g/L in the same old way in addition, 4 ℃ of preservations.
(8) reagent packing: all ingredients is prepared on request, measures the aseptic subpackaged diethylstilbestrol antibody working fluid 7mL/ bottle in qualified back, and concentration is 12.5mg/mL; Diethylstilbestrol standard model 1mL/ bottle, two anti-working fluid 10mL/ bottles, concentration is 2.0mg/mL; Strengthen liquid 20mL/ bottle, concentrate washing lotion 50mL/ bottle, concentrating sample dilution 50mL/ bottle.Label after the packing, indicate the lot number and the term of validity, 4 ℃ of preservations.
(9) assembling of kit: will detachably wrap respectively by 1 of the microwell plate of good envelope antigen, diethylstilbestrol antibody working fluid, two anti-working fluids, diethylstilbestrol standard solution, strengthen liquid, concentrate washing lotion, each 1 bottle of concentrating sample dilution, 6 bottles of diethylstilbestrol standard solution, operation instructions are put assigned address in the kit for 1 part.Kit encapsulates after the assay was approved, 4 ℃ of preservations.
Set up the time resolution immunoassay kits that detects diethylstilbestrol, comprise that other working fluids can be equipped with at control laboratory by the ELISA Plate of diethylstilbestrol antigen, diethylstilbestrol antibody, lanthanide series mark goat-anti rabbit or sheep anti-mouse antibody.
If for diethylstilbestrol in the test sample fast in enormous quantities, the time resolution immunoassay kits of the detection Enrofloxacin of establishment comprises following component:
(1) wraps by the ELISA Plate of diethylstilbestrol antigen 96 holes;
(2) diethylstilbestrol antibody, the 7mL/ bottle; Concentration is 12.5mg/mL;
(3) europium is marked two anti-working fluids, 10mL/ bottle; Concentration is 2.0mg/mL;
(4) the diethylstilbestrol standard solution is 6 bottles, and concentration is respectively 8 μ g/L, 4 μ g/L, 2 μ g/L, 1 μ g/L, 0.5 μ g/L, each 1mL/ bottle of 0 μ g/L;
(5) strengthen liquid, 20mL/ bottle;
(6) concentrated cleaning solution, the 50mL/ bottle;
(7) concentrating sample dilution, the 50mL/ bottle;
(8) operation instructions, 1 part;
(9) cover plate film, 2;
(10) valve bag (containing drying agent), 1.
The preparation method of the fluorescence immunoassay kit that detection diethylstilbestrol of the present invention is residual may further comprise the steps:
(1) bag is by the preparation method of ELISA Plate of diethylstilbestrol antigen: it is 75 μ g/L that DES-OVA is cushioned the liquid dilution with bag, in the ELISA Plate micropore, add antigen 1 00 μ L, putting into 4 ℃ of refrigerator bags is spent the night, wash plate behind the equilibrium at room temperature twice, with 37 ℃ of sealings of skimmed milk power confining liquid (250 μ L) 1h, wash plate three times, with patting dry on the dustless thieving paper, preserve with the vacuum seal of aluminium film dry back.Fixedly the diethylstilbestrol antigen vectors is a kind of in the following material, for example polystyrene, cellulose nitrate, tygon, polypropylene, polyacrylamide, cross-linked glucose, glass, silicon rubber, Ago-Gel etc.
(2) preparation method of DES antibody diluent: with DES antibody with sad-ammonium sulfate method purifying after, it is standby to be diluted to the 1mg/mL packing with PBS.
(3) Eu
3+Mark goat-anti rabbit: with 200 times of the pH 7.85Tris-HCl dilutions that contains 0.2%BSA;
(4) preparation of diethylstilbestrol standard solution: with concentration be the DES of 1000 μ g/L as stock solution, being made into concentration gradient before the use again is the DES standard items working fluid of 8 μ g/L, 4 μ g/L, 2 μ g/L, 1 μ g/L, 0.5 μ g/L, 0 μ g/L.
(5) preparation of enhancing liquid: the 6mL glacial acetic acid adds 15 μ mol with the Potassium Hydrogen Phthalate adjust pH to 3.2 of 0.1mol/L
-two ketoboidies (
-NTA), and 50 μ mol trioctyl phosphine oxides (TOPO), 1mL Triton X-100 adds tri-distilled water and is settled to 1L.
(6) bag is cushioned the preparation of liquid: get Na
2CO
30.375g, NaHCO
30.732g, NaCl 2.250g, NaN
30.100g adding distil water gets the 0.1mol/L carbonate buffer solution to 250mL.
(7) preparation of lavation buffer solution (PBST): get KH
2PO40.4g, Na
2HPO
412H
2O5.8g, NaCl 16.0g, KCl 0.4g, Tween-201.0mL, adding distil water is to 2000mL.
(8) confining liquid: 1) skimmed milk power 5g is dissolved in 100mL PBS; 2) calf serum 3mL (deactivation), with diluted to final concentration 3%.
(9) dilution (50mmol/LTris-HCl, pH 7.85): get Tris-base 6.4g, NaCl 9.0g, NaN
30.4g adjusting its pH value with dense HCl (12M) is 7.85, adding distil water is settled to 1L.
Embodiment 7 sample pre-treatments
A. urine specimen is handled
Limpid urine sample can directly carry out check and analysis.If urine sample is muddy shape, centrifugal 5min of 2000r/min or filtration, supernatant is used for detecting.
B. feed
Feed is pulverized, taken by weighing 1g and place the 50mL test tube, add 10mL ethyl acetate, abundant mixing vibration 10min, the centrifugal 10min of 5000r/min gets organic phase 1mL organic phase in another test tube, and vacuum rotary steam is done, and adds 2mL n-hexane dissolution sample; PBS-methyl alcohol (3: 2) the solution 2mL that adds pH7.0, vortex oscillation 5min, the centrifugal 10min of 4000r/min.Remove the upper strata normal hexane, it is to be measured to take off layer solution 50 μ L.
C. organize in (muscle, liver, kidney etc.)
With historrhexis, with Ultrasound Instrument or analogous instrument tissue is homogenized, take by weighing the tissue after 3g homogenizes, add ethyl acetate 9mL, anhydrous calcium oxide 0.3g, vortex oscillation 10min, the centrifugal 10min of 4000r/min.Remove supernatant 6mL evaporate to dryness, add normal hexane 2mL dissolved residue, add PBS-methyl alcohol (3: 2) the solution 1mL of pH7.0, vortex oscillation 5min, the centrifugal 10min of 4000r/min.Remove the upper strata normal hexane, it is to be measured to take off layer solution 50 μ L.
The detection method of embodiment 8 kits
(1) kit is taken out from cold storage environment, place 20～24 ℃ of environment balances to be no less than 30min, ELIAS strip is fixed, do two parallel laboratory tests, number in order.
(2) add 50 μ L standard solution in the standard items hole, sample well adds 50 μ L testing samples, and every then hole adds 50 μ L diethylstilbestrol antibody working fluids, mixing; Oscillating reactions 45min.
(3) wash plate behind the question response six times, and on thieving paper, pat dry, to guarantee to remove fully the liquid in the hole.Every hole adds 100 μ L europiums and marks two anti-working fluids, oscillating reactions 45min.
(4) wash plate behind the question response six times, and on thieving paper, pat dry, to guarantee to remove fully the liquid in the hole.Every hole adds 200 μ L and strengthens liquid, mixing, and the lucifuge room temperature is patted vibration 5min.
(5) differentiate immunoassay (TRFIA) detector with the time and measure each hole fluorescent value.
(6) do typical curve according to standard solution fluorescent value and concentration semilog, according to typical curve and sample solution fluorescent value calculation sample concentration.
Testing result is handled and is analyzed:
With the mean value calculation percentage fluorescent value of obtained standard model light absorption value, be ordinate with the percentage fluorescent value, the semilog of diethylstilbestrol concentration of standard solution is a horizontal ordinate drawing standard curve, obtains straight-line equation, straight-line equation is y=54.367-41.34x, R
2=0.99356.The use the same method percentage fluorescent value of calculation sample solution is obtained the diethylstilbestrol concentration of counter sample according to equation.The calculating formula of described percentage fluorescent value is:
Percentage fluorescent value (%)=(B/B
0) * 100
Wherein, B is the mean fluorecence value of standard solution or sample, B
0It is the mean fluorecence value of 0 μ g/L standard solution.
The analysis of testing result can also utilize computer professional software to calculate and analyze, and is 0.1～8.1 μ g/L to the diethylstilbestrol linear detection range, and detectability can reach 0.01 μ g/L, and whole testing process needs 2h just can finish.
Embodiment 9 kit precision and accuracy tests
1, standard solution replica test
From 3 batches of ELISA Plate according to the preparation of the method the embodiment 4 (6), each extracts 20 micropores out, measures the fluorescent value (CPS value) of 1.0 μ g/L standard solution, repeats 20 times, calculates coefficient of variation CV%, the results are shown in Table 1.
Table 1 standard solution replica test
The result shows the variation within batch coefficient scope of kit standard items detection between 2.8～5.2%, and interassay coefficient of variation is 8.9%.
2, sample repeatability and accuracy test
Accuracy is meant the matching degree of measured value and true value, and in immune analysis determination, accuracy often represents with the recovery that precision is often represented with the coefficient of variation.In blank pig urine, chicken, it is 1 μ g/L (μ g/kg), 5 μ g/L (μ g/kg) that diethylstilbestrol is added into final concentration, and in blank feed, it is 50 μ g/kg, 100 μ g/kg that diethylstilbestrol is added into final concentration, each 10 of each concentration are parallel, measure 3 batches.Calculating mean value, the interpolation recovery and batch interior and interassay coefficient of variation.The results are shown in Table 2.
Table 2 sample repeatability and accuracy test result
The result shows the interpolation recovery of urine sample, pork liver, feed sample between 87.0～110%, and the variation within batch coefficient is between 6.3～9.1%, and interassay coefficient of variation is between 14.2～18.3%.
The test of embodiment 10 storage lives
(1) kit is positioned over 2～8 ℃, get 0,2,4,6,8,9,10,11 and 12 months kit respectively, to fluorescent value, 50% inhibition concentration of diethylstilbestrol standard model (0.1 μ g/L), add the recovery, each parameter of variation within batch coefficient is measured.
(2) kit was placed 12 days under the condition of 37 ℃ of preservations, measure fluorescent value, 50% inhibition concentration, the interpolation recovery, each parameter of variation within batch coefficient of diethylstilbestrol standard model (0.1 μ g/L) every day.
(3) kit was preserved 12 days at-20 ℃ of refrigerators, measure fluorescent value, 50% inhibition concentration, the interpolation recovery, each parameter of variation within batch coefficient of diethylstilbestrol standard model (0.5 μ g/L) every day.
Can find out that from the result preserve test through three kinds of conditions, the fluorescent value of diethylstilbestrol standard model (0.5 μ g/L) descends less than 5%, and CPS is not less than 10000; 50% inhibiting rate is between 0.5～1.0 μ g/L; Add the recovery between 80～110%; The variation within batch coefficient is less than 10%, and between 6.3～9.1%, interassay coefficient of variation is between 14.2～18.3%.Every index all conforms to quality requirements, and therefore, kit can be preserved 12 months at 2～8 ℃.
Claims (10)
1. one kind is detected the residual time resolution immunoassay detection kit of diethylstilbestrol, it is characterized in that comprising by the ELISA Plate of diethylstilbestrol antigen, diethylstilbestrol antibody, lanthanide series mark goat-anti rabbit or sheep anti-mouse antibody; Described lanthanide series is Eu
3+, Tb
3+, Dy
3+Or Sm
3+
2. kit according to claim 1 is characterized in that described diethylstilbestrol antigen is that diethylstilbestrol and carrier protein couplet are obtained; Described diethylstilbestrol antibody comprises monoclonal antibody, rabbit polyclonal antibody or genetic engineering antibody.
3. kit according to claim 1 is characterized in that also comprising the diethylstilbestrol standard solution, strengthens liquid, concentrated cleaning solution and diluted sample concentrate.
4. kit according to claim 3 is characterized in that described diethylstilbestrol standard solution is is that the concentration gradient that the diethylstilbestrol standard items of 1000 μ g/L are made into is the diethylstilbestrol standard items working fluid of 8 μ g/L, 4 μ g/L, 2 μ g/L, 1 μ g/L, 0.5 μ g/L, 0 μ g/L with concentration.
5. kit according to claim 3 is characterized in that described enhancing liquid comprises that beta-diketon body, trioctylphosphine oxide (TOP0), Triton X-100, glacial acetic acid and pH value are 2.0～3.2 Potassium Hydrogen Phthalate.
6. kit according to claim 3 is characterized in that described concentrated washing lotion comprises the phosphate buffer of 0.5～1.5% polysorbas20, and described phosphate buffer pH value is 7.4, and concentration is 0.1mol/L; Described concentration and dilution liquid is the Tris-HCl of pH value 7.4～8.0,0.1～0.25mol/L.
7. time resolution immunoassay kits according to claim 1 is characterized in that described ELISA Plate is 96 hole ELISA Plate, and being coated with concentration is the anti-diethylstilbestrol antigens of 75 μ g/L, and closed porosity surface adsorption site not; Described confining liquid comprises the skimmed milk power solution that is dissolved in PBS.
8. the detection method of a diethylstilbestrol is characterized in that may further comprise the steps:
(1) sample pre-treatments;
(2) detect with each described kit of claim 1～7;
(3) result treatment and analysis.
9. detection method according to claim 8 is characterized in that described sample pre-treatments is:
(1) urine specimen is handled:
Limpid urine sample can directly carry out check and analysis; If urine sample is muddy shape, centrifugal 5min of 2000r/min or filtration detect with supernatant;
Or (2) feed sample process:
Feed is pulverized, taken by weighing 1g and place the 50mL test tube, add 10mL ethyl acetate, abundant mixing vibration 10min, the centrifugal 10min of 5000r/min gets organic phase 1mL organic phase in another test tube, and vacuum rotary steam is done, and adds 2mL n-hexane dissolution sample; PBS-methyl alcohol (3: 2) the solution 2mL that adds pH7.0, vortex oscillation 5min, the centrifugal 10min of 4000r/min removes the upper strata normal hexane, and it is to be measured to take off layer solution 50 μ L;
Or (3) tissue samples is handled:
Described tissue comprises muscle, liver or kidney; With historrhexis and homogenizing, take by weighing the tissue after 3g homogenizes, add ethyl acetate 9mL, anhydrous calcium oxide 0.3g, vortex oscillation 10min, the centrifugal 10min of 4000r/min removes supernatant 6mL evaporate to dryness, add normal hexane 2mL dissolved residue, PBS-methyl alcohol (3: 2) the solution 1mL that adds pH7.0, vortex oscillation 5min, the centrifugal 10min of 4000r/min, remove the upper strata normal hexane, it is to be measured to take off layer solution 50 μ L.
10. detection method according to claim 5 is characterized in that may further comprise the steps:
(1) kit is taken out from cold storage environment, place 20～24 ℃ of environment balances to be no less than 30min, ELIAS strip is fixed, do parallel laboratory test, number in order;
(2) add 50 μ L standard solution in the standard items hole, sample well adds 50 μ L testing samples, and every then hole adds 50 μ L diethylstilbestrol antibody working fluids, mixing; Oscillating reactions;
(3) wash plate after the described oscillating reactions of step (2), pat dry, every hole adds 100 μ L europiums and marks two anti-working fluids, oscillating reactions;
(4) wash plate after the described oscillating reactions of step (3), pat dry, every hole adds 200 μ L and strengthens liquid, mixing, vibration;
(5) differentiate the immunoassay detector with the time and measure each hole fluorescent value;
(6) do typical curve according to standard solution fluorescent value and concentration semilog, according to typical curve and sample solution fluorescent value calculation sample concentration.
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