CN104569404B - The method of direct competitive TRFIA method detection olaquindox - Google Patents

The method of direct competitive TRFIA method detection olaquindox Download PDF

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CN104569404B
CN104569404B CN201410787030.1A CN201410787030A CN104569404B CN 104569404 B CN104569404 B CN 104569404B CN 201410787030 A CN201410787030 A CN 201410787030A CN 104569404 B CN104569404 B CN 104569404B
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olaquindox
liquid
ola
micropore
cps
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CN104569404A (en
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金仁耀
桑永玉
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Zhejiang Gongshang University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Abstract

The method that the invention discloses a kind of direct competitive TRFIA method detection olaquindox, comprise the steps: to be separately added into olaquindox titer or the sample liquid handled well and europium labeled monoclonal antibody in each micropore of the pre-coated bar of antigen, mixing, hatch 1h for 37 DEG C, wash micropore 3～5 times with cleaning mixture；Each micropore adds 200 μ L and strengthens liquid, 37 DEG C of lucifuge oscillation incubation 10min；Its fluorescence intensity level cps is measured with time identifier；Draw standard curve；Cps according to each samplex/cps0Value calculates the olaquindox concentration of correspondence from standard curve, then is multiplied by corresponding extension rate, calculates olaquindox concentration actual in sample.Detection method is simple and convenient, highly sensitive, good stability, and its lowest detectable limit is up to 0.83ng mL‑1。

Description

The method of direct competitive TRFIA method detection olaquindox
Technical field
The present invention relates to the detection method of a kind of olaquindox, the time-resolved fluoroimmunoassay belonged in biotechnology divides Analysis technical field, is specially one olaquindox drug residue in feedstuff, water body and animal derived food direct The detection method of competition TRFIA.
Background technology
Olaquindox (Olaquindox, OLA) is a kind of antivirus somatotropic agent, is once widely used in aquaculture, Once it was referred to as " Aquatic product clenbuterol hydrochloride ".The toxic and side effects of olaquindox should not be underestimated, and there is obvious genetoxic And cumulative toxicity, therefore in succession formulate strict operating specification and residue limits standard both at home and abroad.Such as the U.S. and European Union prohibits the use of olaquindox, Japan's regulation olaquindox MRL in animal tissue and internal organs (MRL) it is 300 μ g kg-1, No. 168 bulletin " feedstuff medicine that the Ministry of Agriculture of China issued in calendar year 2001 Additive operating specification " in regulation feedstuff in addition must not be higher than 50mg kg-1, provide against simultaneously Fish, fowl and body weight use more than in the breeding process of 35kg pig.While it is true, antibacterial growth-promoting effect is good and Cheap olaquindox is added the most in violation of rules and regulations to be used.Therefore, strengthen the detection supervision of olaquindox, particularly add The research of strong olaquindox detection technique is the most necessary.
The method for detecting residue of olaquindox, mainly includes traditional Instrumental Analysis and the big class of immunoassay two.Wherein Instrumental method mainly includes spectrographic method, chromatography and LC-MS technology etc., and Instrumental Analysis accuracy is high, accurate Degree is strong, but its sample pretreatment process complexity length loaded down with trivial details, time-consuming, need professional and technical personnel to operate, instrument examination Agent etc. are expensive, it is impossible to greatly promoted in basic unit.Immuno analytical method by feat of it efficiently, quickly, The advantage such as high sensitivity and high specific is widely used in small-molecule drug residue detection.At present, enzyme connection is exempted from Epidemic disease adsorption experiment (ELISA) most widely used general, development the most ripe, about ELISA various detection report Road is the most, but at home and abroad there is no time resolved fluoro-immunoassay (TRFIA) detection about olaquindox Any report of aspect, therefore exploitation has the olaquindox time resolved fluoro-immunoassay of independent intellectual property right (TRFIA) detection method is significant.
Time-resolved fluoroimmunoassay (TRFIA) is development in recent years a kind of high sensitivity inspection rapidly Survey means.The principle of TRFIA is the chelating agen utilizing and having bifunctional group structure, its one end and lanthanide series In conjunction with, the other end is then connected with the free amino group on antibody (or antigen), makes lanthanide series Eu3+Labelling resists Body (or antigen), it combines with the antigen (or antibody) in testing sample and generates antigen antibody complex.This Time immune complex fluorescence intensity the most weak, need to add and a kind of strengthen liquid, make Eu3+Dissociate down from complex Come, can be formed with another kind of chelating agen TTA under the synergism of TOPO, Triton X-100 etc. in strengthening liquid New complex, this complex can be launched the strongest fluorescence, make fluorescent effect strengthen up to a million times.Finally use Time resolution instrument measures its fluorescence intensity cps, i.e. can determine that the content of antigen in sample.
Summary of the invention
The invention provides the detection method of a kind of olaquindox direct competitive TRFIA, for qualitatively or quantitatively Detection feedstuff, water body are with the residual quantity of olaquindox in animal derived food, and time is short, average recovery rate in its detection Height, the coefficient of variation are less, have simplicity, fast and accurately feature.
The present invention is achieved through the following technical solutions:
The method of direct competitive TRFIA method detection olaquindox, it is characterised in that comprise the steps:
A. in each micropore of the pre-coated bar of antigen, it is separately added into the olaquindox titer of 50 μ L series concentration Or the sample liquid handled well and 50 μ L europium labeled monoclonal antibodies, mixing, hatch 1h for 37 DEG C, with washing Wash liquid washing micropore 3～5 times；
The most each micropore adds 200 μ L and strengthens liquid, 37 DEG C of lucifuge oscillation incubation 10min；
C. its fluorescence intensity level cps is measured with time identifier；
D. with [1-(cpsx/cps0)] * 100% value is for vertical coordinate, wherein cpsxRepresent variable concentrations olaquindox mark Fluorescent value corresponding to quasi-liquid, cps0Represent fluorescent value corresponding during zero standard concentration；With olaquindox concentration Logarithm value is that abscissa draws standard curve；Cps further according to each samplex/cps0Value calculates from standard curve Go out the olaquindox concentration of correspondence, then be multiplied by corresponding extension rate, calculate olaquindox concentration actual in sample.
The present invention uses Timed resolved fluoroimmunoassay (TRFIA) to detect olaquindox (OLA).TRFIA Technology mainly has two aspects: first, the preparation of specificity anti-olaquindox monoclonal antibody, through mouse immune, Cell merges, positive hybridoma cell strain is screened, clone, ascites is prepared and ascites purification, finally obtains mesh Mark monoclonal antibody (OLA-mAb)；Second, the anti-olaquindox monoclonal antibody of europium labelling (Eu3+-OLA-mAb) preparation.
Assay method is: take out the antigen coated lath being coated with OLA-OVA, by OLA standard solution or sample Product treatment fluid joins in respective micropore, adds Eu3+-OLA-mAb, oscillating reactions, free OLA Eu is jointly competed with the OLA-OVA on antigen coated lath3+-OLA-mAb, scrubbed liquid washs, does not connect The Eu connect3+-OLA-mAb is removed.After adding enhancing liquid oscillating reactions, under the exciting of ultraviolet light, transmitting is the strongest Fluorescence, measure its fluorescence intensity cps with time-resolved fluorescence instrument, fluorescence intensity becomes anti-with the concentration in sample Ratio, reference standard curve i.e. can determine that the content of olaquindox in sample.
The preparation method of anti-olaquindox monoclonal antibody is as follows: be first coupled to by olaquindox with succinic anhydrides (HS) On carrier protein BSA, synthetic immunogen (OLA-HS-BSA) immunity BALB/c mouse, take immunity little Mice spleen cell and SP2/0 myeloma cell fusion, filter out the sun of energy stably excreting anti-olaquindox monoclonal antibody Sexual cell strain amplification culture, injection cell induces ascites in entering Mice Body, and purification obtains the list of anti-olaquindox Clonal antibody.
Olaquindox standard solution obtains from the dilution of OLA sterling, and diluent is that the 0.01M pH containing 5% methanol is The phosphate buffer of 7.4, totally 12 bottles, OLA concentration is followed successively by: 0ng mL-1、0.3ng·mL-1、0.6 ng·mL-1、1.2ng·mL-1、2.4ng·mL-1、4.8ng·mL-1、9.6ng·mL-1、19.4ng·mL-1、38.8 ng·mL-1、77.5ng·mL-1、155.0ng·mL-1、310ng·mL-1。
Olaquindox monoclonal antibody (the Eu of europium labelling3+-OLA-mAb) preparation method as follows:
A. the good olaquindox monoclonal antibody (OLA-mAb) of 0.5mL purification and 0.5mL 0.01M pH are taken It it is the PBS solution 1:1 mixing of 7.4；
The most accurately weigh 3.0mg and be cyclized diethylene triamine pentaacetic acid acid anhydride, add 90 μ L DMSO and dissolve；
C. 90 μ L B are slowly dropped in A, adjust pH to 9.0, room temperature lucifuge with 0.125M NaOH Place 2h；
D. the reactant liquor that step C finally gives is transferred in bag filter, 0.01mol L-1PH7.4PBS is saturating Analysis is overnight；
The most accurately weigh 0.242g EuCl3·6H2O is made into 3.3 × 10 in 20mL water-2M EuCl3Solution；
F. take 100 μ L step E gained solution and add in step D gained dialysis solution, room temperature lucifuge reaction 3h Being placed in bag filter dialysis 24h～36h, subpackage is stored in-20 DEG C, i.e. obtains europium labelling olaquindox monoclonal Antibody (Eu3+-OLA-mAb)。
Strengthen liquid preparation method as follows: accurately weigh 120.0mg а-thenoyltrifluoroacetone (TTA) and 386.6mg trioctyl phosphine oxide (TOPO), adds 1.0mL dehydrated alcohol and is dissolved, then add wherein Enter 2.78g Potassium Hydrogen Phthalate and a small amount of deionized water, 40 DEG C to be dissolved after add 11.8mL glacial acetic acids and 5mL Triton X-100, is finally settled to 2000mL with water, adjusts pH to 3.0, use absorbent cotton sucking filtration, will Filtrate stands overnight, and 4 DEG C of refrigerators keep in Dark Place, standby.
Beneficial effects of the present invention: this detection method is simple and convenient, highly sensitive, good stability, it is Low detection limit is up to 0.83ng mL-1。
Detailed description of the invention
In order to make the purpose of the present invention, technical scheme, good effect clearer, pass through following example The present invention is further elaborated.Description below for specific embodiments is only used for explaining this Bright, do not limit the present invention.
Embodiment one:
Follow these steps to detect Feed Sample:
(1) coating antigen (OLA-OVA) and the preparation of immunogen (OLA-BSA):
First in three neck round bottom flask, accurately add 2.106g olaquindox and 1.6g succinic anhydrides, add 80mL pyridine, removes pyridine under reduced pressure after back flow reaction 4h at 115 DEG C, adds 60mL in remaining mixture Ice distilled water, 2mol L-1HCl adjusts pH to 2.0～3.0,4 DEG C of left overnight.Decompression sucking filtration also distills with ice Water is drained after washing 3 times, and faint yellow flour is OLA-HS；
The most secondly weigh 14.528mg OLA-HS to be dissolved in 0.8mL DMF, add 4.603mg NHS and 8.253mg DCC, under room temperature, after lucifuge stirring reaction 10h, 2000r/min is centrifuged 10min, centrifugal rear supernatant Liquid is A liquid.Weigh 20mg OVA (or BSA) and be dissolved in 5mL 0.01mol L-1Phosphate buffer (PBS, PH7.4), in, this is B liquid.At 4 DEG C, 0.6mL A liquid is added dropwise in the B liquid being slowly stirred, 4 DEG C Stirring reaction is overnight.Proceed to next day in bag filter, PBS 2d, centrifugal precipitation of abandoning, cross-linking products name For OLA-HS-OVA and OLA-HS-BSA.
(2)Eu3+The preparation of-OLA-mAb:
A.5mL the olaquindox monoclonal antibody (OLA-mAb) that purification is good with 0.5mL 0.01M pH is The PBS solution 1:1 mixing of 7.4；
B. weigh 3.0mg and be cyclized diethylene triamine pentaacetic acid acid anhydride, add 90 μ L DMSO and dissolve；
C. 90 μ L B are slowly dropped in A, adjust pH to 9.0, room temperature lucifuge with 0.125M NaOH Place 2h；
D. reactant liquor final for C is transferred in bag filter, 0.01mol L-1PH7.4PBS dialysed overnight；
The most accurately weigh 0.242g EuCl3·6H2O is made into 3.3 × 10 in 20mL water-2M EuCl3Solution；
F. taking 100 μ L E and add in D, room temperature lucifuge reaction 3h is placed in bag filter dialysis 24h～36h, Subpackage is stored in-20 DEG C, i.e. obtains europium labelling olaquindox monoclonal antibody (Eu3+-OLA-mAb)。
(3) preparation of antigen coated lath:
With 0.05M sodium carbonate buffer (CBS) that pH is 9.6, OLA-OVA being diluted to concentration is 1.0 μg·mL-1, every micropore 100 μ L joins in microwell plate, and 37 DEG C are coated 2h；Pour out microwell plate is coated slow After rushing liquid, 300 μ L cleaning mixture are joined in each micropore, after 20S, again pours out liquid in hole, repeat behaviour Make 3～5 times, completely remove the liquid in micropore for the last time；300 μ L are added containing 2% defat in microwell plate The 0.01M PBS of milk powder, closes 0.5h for 37 DEG C；Discard confining liquid, wash 3～5 times, pat dry, obtain anti- Primordial covering lath, is sealed at 4 DEG C and saves backup.
(4) preparation of various reagent:
A. olaquindox titer: concentration is followed successively by 0ng mL-1、0.3ng·mL-1、0.6ng·mL-1、1.2 ng·mL-1、2.4ng·mL-1、4.8ng·mL-1、9.6ng·mL-1、19.4ng·mL-1、38.8ng·mL-1、 77.5ng·mL-1、155.0ng·mL-1、310ng·mL-1, diluting from OLA sterling and obtain, diluent is The phosphate buffer of the 0.01M pH7.4 containing 5% methanol.
B. buffer it is coated: i.e. 0.05M pH is the carbonate buffer solution of 9.6, weighs Na2CO31.49g, NaHCO32.93g, adjusts PH to 9.6, and ultra-pure water is settled to 1000mL.
C. confining liquid: the i.e. phosphate buffer that 0.01M pH is 7.4 (PBS) containing 2% defatted milk powder.
D. cleaning mixture: the i.e. phosphate buffer that 0.01M pH is 7.4 (PBS) containing 0.05% tween 20.
E. diluent: the i.e. phosphate buffer that 0.01M pH is 7.4 (PBS) containing 5% methanol.
F. liquid is strengthened: accurately weigh 120.0mg а-thenoyltrifluoroacetone (TTA) and 386.6mg tri- Octyl group phosphine oxide (TOPO), adds 1.0mL dehydrated alcohol and is dissolved, then it is adjacent to be added thereto to 2.78g Potassium hydrogen phthalate and a small amount of deionized water, 40 DEG C of rear addition 11.8mL glacial acetic acids to be dissolved and 5mL Triton X-100, is finally settled to 2000mL with water.Adjust pH to 3.0, use absorbent cotton sucking filtration, filtrate was stood At night, 4 DEG C of refrigerators keep in Dark Place, standby.
(5) Feed Sample pretreatment:
The feedstuff sample pulverizer bought on market is pulverized, crosses 60 mesh testing sieves, accurately weigh 1g feedstuff sample Product, add the 3mL phosphate buffer that 0.01M pH is 7.4 containing 5% methanol, and vortex oscillation extracts 1 Min, 4000r/min are centrifuged 10min, take supernatant 10000r/min recentrifuge 10min, take on 50 μ L Clear for TRFIA analysis.
(6) the TRFIA detection of olaquindox
Take out the antigen coated lath of OLA-OVA, at the OLA series concentration standard solution of 50 μ L or sample Reason liquid joins in respective micropore, and each standard specimen and sample solution must use new suction nozzle, add and use diluent The Eu of 1:1000 dilution3+-OLA-mAb 50 μ L, hatches 1h for 37 DEG C, and cleaning mixture washs 3～5 times, adds 200 μ L measures after strengthening liquid lucifuge oscillating reactions 10min.The OLA content in sample is calculated according to standard curve, In the sample treatment liquid of the present embodiment, OLA concentration is 12ng mL-1, it is shown in Table 1.
Table 1
Embodiment two:
Follow these steps to detect pond water sample:
(1) detection preliminary preparation is with (1)～(4) of embodiment 1
(2) pond water sample pretreatment:
First pond water is filtered impurity such as removing algae, the most accurately measure the pond water after 1mL filters, add Entering the 2mL phosphate buffer that 0.01M pH is 7.4 containing 5% methanol, whirlpool mixes, is directly used in TRFIA detects.
(3) the TRFIA detection of olaquindox
Take out the antigen coated lath of OLA-OVA, at the OLA series concentration standard solution of 50 μ L or sample Reason liquid joins in respective micropore, and each standard specimen and sample solution must use new suction nozzle, add and use diluent The Eu of 1:1000 dilution3+-OLA-mAb 50 μ L, hatches 1h for 37 DEG C, and cleaning mixture washs 3～5 times, adds 200 μ L measures after strengthening liquid lucifuge oscillating reactions 10min.The OLA content in sample is calculated according to standard curve, In the sample treatment liquid of the present embodiment, OLA concentration is 2ng mL-1, it is shown in Table 2.
Table 2
Several specific embodiments being only the present invention listed above.The present invention is not limited to above example, institute Directly to derive or to associate the detection method of deformation gained from the disclosure of invention, all it is considered as the present invention's Protection domain.

Claims (5)

1. the method for direct competitive TRFIA method detection olaquindox, it is characterised in that comprise the steps:
1). in each micropore of the pre-coated bar of antigen, it is separately added into the olaquindox standard of 50 μ L series concentration Liquid or the sample liquid handled well and olaquindox monoclonal antibody Eu of 50 μ L europium labellings3+-OLA-mAb, Mixing, hatches 1h for 37 DEG C, washs micropore 3～5 times with cleaning mixture；The olaquindox monoclonal anti of described europium labelling Body Eu3+The preparation method of-OLA-mAb is as follows:
(A) good olaquindox monoclonal antibody OLA-mAb of 0.5mL purification and 0.5mL 0.01M pH are taken It is the PBS solution 1:1 mixing of 7.4, obtains A liquid；
(B) accurately weigh 3.0mg and be cyclized diethylene triamine pentaacetic acid acid anhydride, add 90 μ L DMSO and dissolve, Obtain B liquid；
(C) 90 μ L B liquid are slowly dropped in A liquid, adjust pH to 9.0 with 0.125M NaOH, Room temperature lucifuge places 2h；
(D) reactant liquor that step (C) finally gives is transferred in bag filter, 0.01mol L-1pH7.4 PBS is overnight；
(E) 0.242g EuCl is accurately weighed3·6H2O is made into 3.3 × 10 in 20mL water-2M EuCl3Molten Liquid；
(F) taking 100 μ L step (E) gained solution to add in step (D) gained dialysis solution, room temperature is kept away Photoreaction 3h is placed in bag filter dialysis 24h～36h, and subpackage is stored in-20 DEG C, i.e. obtains europium labelling Olaquindox monoclonal antibody Eu3+-OLA-mAb；
2). each micropore adds 200 μ L and strengthens liquid, 37 DEG C of lucifuge oscillation incubation 10min；
3). measure its fluorescence intensity level cps with time identifier；
4). with [1-(cpsx/cps0)] * 100% value is for vertical coordinate, wherein cpsxRepresent variable concentrations olaquindox Fluorescent value corresponding to titer, cps0Represent fluorescent value corresponding during zero standard concentration；With olaquindox concentration Logarithm value be abscissa draw standard curve；Cps/cps further according to each sample0Value is counted from standard curve Calculate the olaquindox concentration of correspondence, then be multiplied by corresponding extension rate, calculate olaquindox actual in sample dense Degree.
2. the method for claim 1, it is characterised in that the preparation method of described antigen pre-coating plates As follows:
(1). it is coated: be that the sodium carbonate buffer of 9.6 is by coating antigen with being coated buffer i.e. 0.05M pH OLA-HS-OVA is diluted to 1.0 μ g mL-1, every micropore 100 μ L joins in polystyrene micropore plate, 37 DEG C It is coated 2h；
(2). washing: pour out after being coated buffer in microwell plate, 300 μ L cleaning mixture are joined each micro- Again pour out liquid in hole, repetitive operation 3～5 times after Kong Zhong, 20s, completely remove for the last time in micropore Liquid；Described cleaning mixture is the phosphate buffer that 0.01M pH is 7.4 containing 0.05% tween 20；
(3). close: in microwell plate, add 300 μ L containing the 0.01M PBS of 2% defatted milk powder, 37 DEG C Close 0.5h；
(4). wash micropore 3～5 times by step (2)；
(5). clappers: after clapping except all liq, obtain antigen pre-coating plates.
3. the method for claim 1, it is characterised in that the preparation side of described olaquindox monoclonal antibody Method is as follows: be first coupled on carrier protein BSA by olaquindox with succinic anhydrides (HS), synthetic immunogen OLA-HS-BSA, equivalent volumes incomplete Freund's adjuvant immunity BALB/c mouse, take immune mouse spleen cell With SP2/0 myeloma cell fusion, filter out the positive cell strain of energy stably excreting olaquindox monoclonal antibody also Amplification culture, injection cell induces ascites in entering Mice Body, and purification obtains the monoclonal antibody of olaquindox.
4. the method for claim 1, it is characterised in that described olaquindox titer is from OLA sterling Dilution obtains, and diluent is the phosphate buffer that 0.01M pH is 7.4 containing 5% methanol, totally 12 bottles, OLA concentration is followed successively by: 0ng mL-1、0.3ng·mL-1、0.6ng·mL-1、1.2ng·mL-1、2.4ng·mL-1、 4.8ng·mL-1、9.6ng·mL-1、19.4ng·mL-1、38.8ng·mL-1、77.5ng·mL-1、155.0ng·mL-1、 310ng·mL-1。
5. the method for claim 1, it is characterised in that the preparation method of described enhancing liquid is as follows: accurate Really weigh 120.0mg а-thenoyltrifluoroacetone and 386.6mg trioctyl phosphine oxide, add 1.0mL without Water-ethanol is dissolved, then is added thereto to 2.78g Potassium Hydrogen Phthalate and a small amount of deionized water, 40 DEG C Add 11.8mL glacial acetic acid and 5mL Triton X-100 after to be dissolved, be finally settled to 2000mL with water, Adjusting pH to 3.0, use absorbent cotton sucking filtration, filtrate stood overnight, 4 DEG C of refrigerators keep in Dark Place, standby.
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