CN105136755A - Fluorescence polarization immunoassay method for detection of erythromycin - Google Patents
Fluorescence polarization immunoassay method for detection of erythromycin Download PDFInfo
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Abstract
The invention provides a fluorescence polarization immunoassay method for detection of erythromycin. The method includes: subjecting an erythromycin standard substance solution with a known concentration to mixed incubation respectively with a fluorescent marker solution and an erythromycin monoclonal antibody solution to carry out competing reaction, thus obtaining a fluorescence polarization value of the system; taking the measured fluorescence polarization value as the ordinate, and adopting the concentration of a series of sulfamethazine standard substances with known concentration as the abscissa to draw a standard curve; using a to-be-measured sample to replace the erythromycin standard substance in step 1), according to the operation in step 1), determining the fluorescence polarization value of the system after incubation, and according to the standard curve, calculating the erythromycin concentration of the to-be-measured sample. The invention provides the homogeneous and rapid erythromycin fluorescence polarization immunodetection method, which only needs sample adding, has no need of separation and washing operation, and can obtain the detection result within 10min.
Description
Technical field
The invention belongs to immuno analytical method and field of veterinary drug residue detection, being specifically related to a kind of fluorescence polarization immunoassay method for detecting erythromycin.
Background technology
Erythromycin (Erythromycin, ERY) is first Macrocyclolactone lactone kind medicine come out, and is mainly used in treating the livestock and poultry caused by gram-positive bacteria and mycoplasma, is also used as feed addictive to improve food conversion ratio, growth promoting effects.But this compounds is slow at livestock and poultry internal metabolism, easily remain in animal body, can have side effects to body, this causes certain threat to the health of people.Therefore, requirement has all been made to erythromycin residue limits in animal food in countries in the world.European Union forbids that from 2006 all microbiotic are used as somatotrophic feed addictive and use, EC1181/2002 specifies that the maximum residue limit(MRL) of erythromycin in RP-HPLC must not more than 200 μ g/kg, and No. 235, the Ministry of Agriculture of China is the clear stipulaties maximum residue limit(MRL) of erythromycin in the products such as muscle, milk and fat also.
What the method that in current monitoring animal derived food, erythromycin remains was appeared in the newspapers mainly contains instrument detection method.Mainly contain: efficient liquid phase-tandem mass spectrometry, high performance liquid chromatography, vapor-phase chromatography etc., although these instrumental method accuracy are high, reproducible, be applicable to the advantages such as quantitatively detection, but equipment price is expensive, complicated operation, need special technician, be difficult to the Large-scale Screening and the popularization that realize sample.
Sensitive with it, efficient, special, the easy advantage of immunologic detection method is widely used at medicament residue detection field.Wherein conventional Enzyme-linked Immunosorbent Assay method and immune flow measurement chromatographic technique, because needs multistep is separated and washing operation, and consuming time longer.
Summary of the invention
The present invention is directed in existing erythromycin immunoassay technology, the shortcoming such as complicated operation, length consuming time, set up a kind of fluorescence polarization immunoassay method for detecting erythromycin, whole testing process only needs a step competitive reaction, simple to operate, quick.
The object of this invention is to provide a kind of fluorescence polarization immunoassay method for detecting erythromycin.
Fluorescence polarization immunoassay method for detecting erythromycin provided by the present invention, comprises the steps:
1) solution of the erythromycin standard items of a series of concentration known is mixed with fluorescent marker solution and erythromycin monoclonal antibody solution respectively, hatch the reaction that is at war with, measure the fluorescence polarization value of gained system; With the fluorescence polarization value measured for ordinate, the concentration of the sulfamethazine standard items of described a series of concentration known is horizontal ordinate, drawing standard curve;
2) with testing sample replace step 1) in erythromycin standard items, according to step 1) in application of sample amount, described testing sample is mixed with described fluorescent marker solution and described erythromycin monoclonal antibody solution, hatch the reaction that is at war with, measure the fluorescence polarization value of gained system, according to step 1) in typical curve, calculate the concentration of erythromycin in described testing sample.
Said method step 1) in, described fluorescent marker is the conjugate of erythromycin haptens and fluorescein.
Described erythromycin haptens be selected from following any one: erythromycylamine (ERM) and the improved erythromycin haptens (ERY-CMO) of ethyloic azanol.
Described fluorescein be selected from following any one: fluorescein isothiocynate (FITC), dichlorotrazinylaminofluorescein (DTAF), AF647 carboxylic acid succinimide ester (AF647) and 4 '-aminomethyl fluorescein (4 '-AMF).
Particularly, described fluorescent marker has the structure shown in table 1.
The structure of each fluorescent marker of table 1
Preferably, described fluorescent marker is ERM-FITC.
Described erythromycin monoclonal antibody is selected from the monoclonal antibody 5B2 secreted by laboratory hybridoma cell strain 5B2,6C1 and 6D9, any one (ZhanhuiWangetal., FoodChemistry, 2015,171, the 98-107) in 6C1 and 6D9.
Preferably, described erythromycin monoclonal antibody is 5B2
.
The solute of the solution of the erythromycin standard items of described a series of concentration known is erythromycin standard items, and solvent is borate buffer solution.
The volumetric molar concentration of described borate buffer solution is 45-55mmol/L, and pH value is 8.0-8.5.
Described a series of concentration known is 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.625ng/mL, 7.8125ng/mL, 3.906ng/mL and 1.953ng/mL10 gradient concentration.
The concentration of described fluorescent marker solution is working concentration, and concentration corresponding when being background value (buffer solution) 10 times that the working concentration of described fluorescent marker is the fluorescent value of label is 15nM.
The concentration of described erythromycin monoclonal antibody solution is working concentration, in described antibody binding curve 50% antibody in conjunction with time antibody dilution multiple as the working concentration of antibody, be 8ug/mL.
Step 1) and step 2) in, the solution of described erythromycin standard items, described fluorescent marker solution and described erythromycin monoclonal antibody solution mix with equal-volume, and described volume specifically can be 70 μ L-100 μ L, specifically can be 70 μ L.
Step 1) and step 2) in, the temperature of described competitive reaction is 20-25 DEG C, and be preferably 25 DEG C, the time is 1-5min, is preferably 2min.
Step 1) and step 2) in, the condition determination of described fluorescence polarization value is: when described fluorescent marker is ERM-FITC, ERM-DTAF or ERY-CMO-4 '-AMF time, excitation wavelength is 485nm, and emission wavelength is 530nm, cutoff value is 515nm; When described fluorescent marker is ERM-AF647, excitation wavelength is 644nm, and emission wavelength is 685nm, cutoff value is 665nm.
The drafting of described typical curve is carried out according to the quadruplex parameters in OriginPro8.0 software.
Step 2) in, described testing sample can be animal food, specifically can be milk.
The application of said method in animal food in erythromycin content detection also belongs to protection scope of the present invention.
Described animal food specifically can be milk.
The invention has the beneficial effects as follows:
Existing erythromycin detection technique complex operation is consuming time, can not meet the requirement that high flux detects fast, and other immune analysis methods is mostly heterogeneous reaction, needs multistep to hatch and washing step, length consuming time, complicated operation.Not enough for these, the invention provides a kind of homogeneous phase, erythromycin fluorescence polarization immunoassay method fast.Method only needs application of sample, without the need to being separated and washing operation, just can obtain testing result in ten minutes.The present invention, by the screening to three kinds of monoclonal antibodies and four kinds of erythromycin fluorescent markers, establishes efficient, sensitive erythromycin fluorescence polarization immunoassay method.Method is 7.39ng/mL in the sensitivity of borate buffer solution standard curve, and sensing range is 1.85-29.47ng/mL, and because milk substrate dilutes 14 times, the detection in milk is limited to 14.08 μ g/L; Quick, the easy high flux of the method, and the residue detection limitation requirement that can meet erythromycin, be applicable to the detection demand of erythromycin in milk very much.
The fluorescence polarization immunoassay technology (FluorescencePolarizationImmunoassay, FPIA) that the present invention applies is a kind of homogeneous immunoassay method not needing to be separated and to wash.Due to simple to operate, quick, be more suitable for the advantages such as the high flux screening of robotization, become clinical gradually, common technology that the field small molecular such as food and environment is analyzed.And the FPIA Fast Detection Technique that in milk, erythromycin is residual have not been reported.
Accompanying drawing explanation
Fig. 1 is the IC of four kinds of fluorescent markers and three kinds of antibody Criterion curves
50value compares.
Fig. 2 is the typical curve of erythromycin fluorescence polarization immunoassay in borate buffer solution.
Embodiment
Below by specific embodiment, the present invention will be described, but the present invention is not limited thereto.
The experimental technique used in following embodiment if no special instructions, is conventional method; Reagent used in following embodiment, biomaterial etc., if no special instructions, all can obtain from commercial channels.
Any one (ZhanhuiWangetal., FoodChemistry, 2015,171,98-107) in the monoclonal antibody of the erythromycin monoclonal antibody used in following embodiment secreted by hybridoma cell strain 5B2,6C1 and 6D9.
Example 1, preparation based on the fluorescent marker of haptens erythromycylamine
For ERM-FITC, synthetic method is as follows:
Take 0.2mg erythromycylamine standard items and 1mgFITC powder, join vibration in 200 μ LDMF and extremely dissolve; Add 50 μ L triethylamines, room temperature lucifuge reacts 7 days; Get 50 μ L reactant liquors thin-layered chromatography (TLC) to be separated, with pure FITC for contrast, developping agent is methylene chloride/methanol (v:v, 1:1); Scraping silica gel plate scrapes R
fthe yellow band of=0.5, methanol-eluted fractions, detects for subsequent use.Through Mass Spectrometric Identification, ERM-FITC product peak m/z is 1123.4.
The fluorescent marker of other erythromycylamines as: the reactions steps of DTAF, tetramethylrhodamine sulfonic acid chloride, AF647 and the labeling method of FITC similar, fluorescent marker leaves 4 DEG C in.
Example 2, based on the preparation of the haptenic fluorescent marker of erythromycin after transformation
Step 1: the haptenic transformation of erythromycin
Will containing 44mg ethyloic azanol NaHCO
3the 2mL distilled water between pH to 5-6 is regulated dropwise to join in the 1mL absolute ethyl alcohol containing 100mg erythromycin raw material; Put stirring reaction 5h in 50 DEG C of waters bath with thermostatic control; After reaction terminates, naturally cool to room temperature, add water cancellation, regulates solution ph to be acid; With dichloromethane extraction twice, combined dichloromethane layer, adds anhydrous Na
2sO
4jolting, hold over night; Get dichloromethane layer, 40 DEG C of vacuum rotary steams, to dry, dissolve with 5mLDMF ,-20 DEG C of preservations.
Step 2: the synthesis of erythromycin fluorescent marker (ERY-CMO-4 '-AMF) after transformation
The DMF solution taking 200 μ LERY-CMO mixes with the DMF solution of the 200 μ L containing 0.4mgNHS and 0.8mgEDC, jolting 8h under room temperature; Get the above-mentioned solution of 200 μ L and add 2mg4 '-Aminofluorescein vibration to powder dissolution.Add 50 μ L triethylamines, room temperature lucifuge is spent the night; Get 50 μ L reactant liquors thin-layered chromatography (TLC) to be separated, with pure 4 '-AMF for contrast, developping agent is: methylene chloride/methanol (v:v, 1:3).R on scraping silica gel plate
fthe yellow band of=0.7, methanol-eluted fractions, detects for subsequent use.Through Mass Spectrometric Identification, ERY-CMO-4 '-AMF product peak m/z is 1151.6.
The screening of example 3, best fluorescent marker and Antibody Combination
Step one: respectively with various fluorescent marker and three kinds of erythromycin monoclonal antibody (5B2 of synthesis, 6C1 and 6D9) combine, the concentration (15nM) of fluorescent marker corresponding when first the working concentration of each fluorescent marker being all set as that fluorescence intensity is 10 times of buffer solution background fluorescence intensity, each antibody borate buffer solution is according to 1/100, 1/200, 1/400, 1/800, 1/1600, 1/3200, 1/6400, 1/12800, 1/25600 and 1/51200 dilution, draw antibody binding curve, obtain maximum changing value δ mP (the δ mP=mP of signal intensity
max-mP
min), (ERM-FITC, ERM-DTAF and ERY-CMO-4 '-AMF excitation wavelength 485nm, emission wavelength 530nm, cutoff value 515nm, ERM-AF647 excitation wavelength 644nm, emission wavelength 685nm, cutoff value 665nm) wherein, the signal intensity value of ERM-FITC is maximum.Experimental result is as shown in table 2:
The signal intensity of table 2 four kinds of fluorescent markers and three kinds of antibody
Step 2: the concentration (15nM) of fluorescent marker corresponding when first the working concentration of each fluorescent marker being all set as that fluorescence intensity is 10 times of BB solution background fluorescence intensity, under 50% antibody dilution (8ug/mL) condition, set up typical curve that different label and different antibodies combine and calculate IC
50, according to the IC of each typical curve
50value selects best fluorescent marker.Experimental result as shown in Figure 1.
As seen from the figure: the final best fluorescent marker selected and Antibody Combination are the monoclonal antibody 5B2 secreted by ERM-FITC and hybridoma cell strain 5B2.
The foundation of example 4, FPIA method
Step one: competition FPIA; Borate buffer solution is utilized to configure the erythromycin standard items of 1000,500,250,125,62.5,31.25,15.625,7.8125,3.906 and 1.953ng/mL10 gradient concentration respectively, 96 black low in conjunction with microwell plate in add 70 μ L erythromycin standard items, the fluorescent marker of 70 μ L working concentrations (15nM) and the erythromycin monoclonal antibody solution of 70 μ L working concentrations (8ug/mL) respectively, room temperature lucifuge measures fluorescence polarization value after hatching 10min; ERM-FITC, ERM-DTAF and ERY-CMO-4 '-AMF excitation wavelength 485nm, emission wavelength 530nm, cutoff value 515nm; ERM-AF647 excitation wavelength 644nm, emission wavelength 685nm, cutoff value 665nm.
Step 2: drawing standard curve: after competitive reaction terminates, with each hole fluorescence polarization value measured for ordinate, the concentration of erythromycin standard items is horizontal ordinate, utilizes the four parameter model fit standard curve of Origin8.0.
Erythromycin fluorescence polarization typical curve in borate buffer solution is shown in accompanying drawing 2.
The typical curve set up sensitivity be 7.39ng/mL, sensing range is 1.85-29.47ng/mL.Because milk substrate has carried out 14 times of dilutions, so the erythromycin in milk detects be limited to 14.08 μ g/L; China and European Union etc. all in regulation milk erythromycin series maximum residue limit(MRL) be 40 μ g/kg; Therefore method of the present invention can be good at the needs meeting detection sensitivity.
Example 5, sample detection application examples
Step one: 10% acetonitrile solution adding 2ml milk and 2ml in 10ml centrifuge tube, whirling motion 5min, leaves standstill 30min, the centrifugal 10min of 10000g under 4 DEG C of freezing conditions, detects after getting the seven times of dilutions of middle clarification part 1mL borate buffer solution for FPIA.
Step 2: TIANZHU XINGNAO Capsul measures; Blank milk sample is added with the erythromycin standard items of 10000ng/mL, make its final concentration be 50ng/mL, 100ng/mL, 150ng/mL, each concentration three is parallel, detects, according to following formulae discovery TIANZHU XINGNAO Capsul according to above-mentioned sample treatment process.
TIANZHU XINGNAO Capsul (%)=(measured value/add value) × 100%
Utilize and calculate the accuracy that the detection recovery obtained evaluates the erythromycin fluorescence polarization immunoassay method that the present invention sets up; Experimental result is shown in Table 3:
Add in table 3 milk and reclaim result (n=3)
From table 3, the average TIANZHU XINGNAO Capsul of erythromycin in milk is between 96.08-107.77%, and the coefficient of variation is less than 17.59%; Show that erythromycin fluorescence polarization immunoassay method that the present invention sets up can meet the residue detection requirement of erythromycin in milk; And the present invention rapidly and efficiently, is quick on the draw, solve the shortcoming of traditional immunization method complicated operation, length consuming time, quick, the high flux that can perform well in erythromycin detect.
Claims (10)
1., for detecting a fluorescence polarization immunoassay method for erythromycin, comprise the steps:
1) solution of the erythromycin standard items of a series of concentration known is mixed with fluorescent marker solution and erythromycin monoclonal antibody solution respectively, hatch the reaction that is at war with, measure the fluorescence polarization value of gained system; With the fluorescence polarization value measured for ordinate, the concentration of the sulfamethazine standard items of described a series of concentration known is horizontal ordinate, drawing standard curve;
2) with testing sample replace step 1) in erythromycin standard items, according to step 1) in method, described testing sample is mixed with described fluorescent marker solution and described erythromycin monoclonal antibody solution, hatch the reaction that is at war with, measure the fluorescence polarization value of gained system, according to step 1) in typical curve, calculate the concentration of erythromycin in described testing sample.
2. method according to claim 1, is characterized in that: step 1) in, described fluorescent marker is the conjugate of erythromycin haptens and fluorescein;
Described erythromycin haptens be selected from following any one: erythromycylamine and the improved erythromycin haptens of ethyloic azanol;
Described fluorescein be selected from following any one: fluorescein isothiocynate, dichlorotrazinylaminofluorescein, AF647 carboxylic acid succinimide ester and 4 '-aminomethyl fluorescein.
3. method according to claim 1 and 2, is characterized in that: described fluorescent marker be following any one: erythromycylamine-fluoresceinisothiocyanate thing, erythromycylamine-dichlorotrazinylaminofluorescein conjugate, erythromycylamine-AF647 carboxylic acid succinimide ester and the improved erythromycin-4 of ethyloic azanol '-aminomethyl fluorescein conjugates.
4. the method according to any one of claim 1-3, is characterized in that: any one in the monoclonal antibody of described erythromycin monoclonal antibody secreted by hybridoma cell strain 5B2,6C1 and 6D9: 5B2,6C1 and 6D9.
5. the method according to any one of claim 1-4, is characterized in that: step 1) in, the solute of the solution of the erythromycin standard items of described a series of concentration known is erythromycin standard items, and solvent is borate buffer solution;
The volumetric molar concentration of described borate buffer solution is 45-55mmol/L, and pH value is 8.0-8.5;
Described a series of concentration known is 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.625ng/mL, 7.8125ng/mL, 3.906ng/mL and 1.953ng/mL10 gradient concentration.
6. the method according to any one of claim 1-5, is characterized in that: step 1) in, the working concentration of described fluorescent marker is for being 15nM;
The concentration of described erythromycin monoclonal antibody solution is working concentration is 8 μ g/mL;
Step 1) and step 2) in, the solution of described erythromycin standard items, described fluorescent marker solution and described erythromycin monoclonal antibody solution mix with equal-volume, and described volume is 70 μ L-100 μ L.
7. the method according to any one of claim 1-6, is characterized in that: step 1) and step 2) in, the temperature of described competitive reaction is 20-25 DEG C, and the time is 1-5min;
Step 1) and step 2) in, the condition determination of described fluorescence polarization value is: when described fluorescent marker is ERM-FITC, ERM-DTAF or ERY-CMO-4 '-AMF time, excitation wavelength is 485nm, and emission wavelength is 530nm, cutoff value is 515nm; When described fluorescent marker is ERM-AF647, excitation wavelength is 644nm, and emission wavelength is 685nm, cutoff value is 665nm.
8. the method according to any one of claim 1-7, is characterized in that: step 1) in, the drafting of described typical curve is carried out according to the quadruplex parameters in OriginPro8.0 software.
9. the method according to any one of claim 1-8, is characterized in that: step 2) in, described testing sample is animal food, specifically can be milk.
10. the application of the method according to any one of claim 1-9 in animal food in erythromycin content detection, described animal food specifically can be milk.
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| CN108551763A (en) * | 2015-12-18 | 2018-09-18 | 瓦里泰细胞有限公司 | A kind of method of determining sample target abundance |
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| CN108362882A (en) * | 2018-03-05 | 2018-08-03 | 杨博易 | A kind of fluorescence polarization immunoassay method for detecting ochratoxin |
| CN114166807A (en) * | 2021-11-22 | 2022-03-11 | 中国农业大学 | Fluorescence polarization immunoassay method for detecting polymyxin |
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