CN104597243A - Antibody chip kit and method for detecting sulfonamide residues in foods - Google Patents
Antibody chip kit and method for detecting sulfonamide residues in foods Download PDFInfo
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Abstract
The invention discloses an antibody chip kit for detecting sulfonamide residues in foods. The kit comprises a chip, an antibody, a Cy3 labeled second antibody and an extraction reagent, wherein the antibody is a monoclonal antibody secreted from a hybridoma cell strain 4E5 with the preservation number: CCTCC NO: C201148; a sulfonamide coating antigen is fixed in the chip. The invention further discloses an antibody chip method for detecting sulfonamide residues in foods. The method can detect 16 sulfonamide drugs at the same time, so that a rapid, sensitive and high throughput method is provided for detecting sulfonamide residues in foods is provided. The detection method established in the invention is high in accuracy, good in precision and high in detection efficiency. Compared with the prior art, a lot of time can be shortened and a lot of cost can be lowered, and the kit and method have a good market application value.
Description
Technical field
The invention belongs to biochip technology field, particularly relating to a kind of antibody chip kit for detecting sulfa drug residue in food and method.
Background technology
Sulfa drugs is the broad spectrum antibiotic of a class Prof. Du Yucang, on veterinary clinic, is widely used in the diseases such as treatment meningitis, gonorrhoea, urinary tract infection.In husbandry sector, as feed addictive, can growth of animals or poultry be promoted, improve product quality.Some, in order to pursue interests abuse sulfa drugs, cause these medicines to be accumulated in animal food.There is the bad reactions such as allergic reaction, kidney damage, leukopenia in people after eating these foods polluted.The abuse of a large amount of sulfa drugs also can cause bacterial resistance occurred, destroys ecologic environment.Stable in order to underwriter's based food safety and ecologic environment, China specifies that the maximum residue limit(MRL) of sulfa drugs in all edible foods is 100 μ g/kg.
The residual method of the detection sulfa drugs that current China takes in animal tissue has instrument analytical method, microbial process, method of immunity.There is expensive equipment, complicated operation, detection time long shortcoming in instrumental method, is not easy to promote.Microbial method Chang Zuowei screening technique, can detect multi-medicament simultaneously, but accuracy and specificity have much room for improvement.Immunoassays depend on antigen-antibody reaction, have highly sensitive, high specificity, simple operation and other advantages, are detection methods most widely used at present.From experimental principle, ELISA method, colloidal gold strip, antibody chip technology, immune microsphere technology all belong to method of immunity, ELISA method only can detect one-component, accuracy and the specificity of colloidal gold strip are bad, and immune microsphere technology exists the shortcoming that preparation process is loaded down with trivial details, detection time is long.
CN102608318A discloses monoclonal antibody for detecting sulfa drugs and enzyme-linked immunoassay method and kit, monoclonal antibody prepared by this invention can identify 16 kinds of sulfa drugss, the enzyme-linked immunoassay method set up and kit are using sulphadiazine and ovalbumin conjugate as coating antigen, but the detection sensitivity of these 16 kinds of sulfa drugss of the method is not high, such as, to the IC of sulfadimidine
50be 5.8 μ g/L, to the IC of sulphadiazine
50be 0.92 μ g/L, to the IC of 5-methoxysulfadiazine
50be 0.76 μ g/L.The lowest detectable limit (LOD) of kit is 17.47 μ g/Kg.
Antibody chip technology is the detection means developed rapidly in recent years, has both had having superiority of other method of immunity, combines again the high-throughout advantage of chip technology, is suitable for very much the residue detection of multiple agricultural and veterinary chemicals in great amount of samples.
Summary of the invention
First object of the present invention is to provide a kind of antibody chip kit for detecting sulfa drug residue in food.
This kit comprises two anti-, extraction reagent of chip, antibody, Cy3 mark, the monoclonal antibody secreted by hybridoma cell strain 4E5 that described antibody is is CCTCCNO:C201148 by preserving number, described chip secures sulfamido coating antigen, and described sulfamido coating antigen is 2-sulfonamido-4-methylpyrimidine-5-carboxylic acid and ovalbumin conjugate.
Described preserving number is open in the patent of " for detecting the monoclonal antibody of sulfa drugs and enzyme-linked immunoassay method and kit " that the hybridoma cell strain 4E5 of CCTCC NO:C201148 has applied on February 27th, 2012 the applicant, publication number is 102608318A, and publication date is on July 25th, 2012.
Preferably, described chip is prepared as follows: using brilliant core macromolecule substrate G as substrate, with fence, substrate is divided into 12 conversion zones, each conversion zone establishes vertical totally 9 microarraies of three rows three, by sulfamido coating antigen solution and two kinds of contrast solutions---the oralbumin solution difference point sample of oralbumin solution and Cy3 mark one is arranged on microarray wherein, fixing 0.5h after point sample completes, closes 0.5h with 2% (percentage by weight) bovine serum albumin solution.
Preferably, the concentration of described sulfamido coating antigen solution is 10 μ g/mL, and the solvent of described sulfamido coating antigen solution is the pH value containing 5% (percentage by weight) glycerine is the carbonate buffer solution of 9.16.
Preferably, the point sample order on described microarray is from top to bottom, from left to right.
Preferably, described Cy3 mark two anti-be sheep anti mouse or the rabbit anti-mouse antibody of Cy3 mark.
Preferably, described extraction reagent is prepared as follows: accurately take KH
2pO
40.41g, K
2hPO
45.59g, ultrapure water dissolves, and is settled to 1000mL.
Second object of the present invention is to provide described antibody chip kit in the application detecting sulfa drug residue in food.
3rd object of the present invention is to provide a kind of antibody chip method for detecting sulfa drug residue in food, and the method comprises the following steps:
1) sample pre-treatments: add after sample homogenization and extract reagent extraction;
2) sample liquid and the antibody diluted are secured sulfamido coating antigen solution and contrast solution by joining after the volume ratio mixing of 1: 1---on the chip of the oralbumin solution of oralbumin solution and Cy3 mark, in 37 DEG C of reaction 0.5h, wash 3 times, dry;
3) marked by Cy3 two anti-are added to chip, in 37 DEG C of reaction 0.5h, washs 3 times, drying;
4) scan chip with InnoScan 700A scanner, the oralbumin solution marked with oralbumin solution and Cy3 contrasts, and carries out interpretation of result with Mapix analysis software,
Described antibody is by the monoclonal antibody of preserving number secreted by the hybridoma cell strain 4E5 of CCTCC NO:C201148;
Described sulfamido coating antigen is 2-sulfonamido-4-methylpyrimidine-5-carboxylic acid and ovalbumin conjugate;
Described Cy3 mark two anti-be sheep anti mouse or the rabbit anti-mouse antibody of Cy3 mark.
Preferably, described extraction reagent is prepared as follows: accurately take KH
2pO
40.41g, K
2hPO
45.59g, ultrapure water dissolves, and is settled to 1000mL.
Preferably, described antibody dilutes with the phosphate buffer of pH7.4, and the dilutability of described antibody is 1:800; The concentration of described sulfamido coating antigen solution is 10 μ g/mL, and the solvent of described sulfamido coating antigen solution is the carbonate buffer solution of the pH9.16 containing 5% glycerine.
The invention has the beneficial effects as follows:
1) antibody chip kit of the present invention can detect 16 kinds of sulfa drugss simultaneously---sulfadimidine, daimeton, sulphadiazine, 5-methoxysulfadiazine, cistosulfa, sulfaclozine, sulfamethoxypyridazine, sulphathiazole, fanasil, sulfamethyldiazine, sulfaisodimidine, sulfaquinoxaline, sulfadimethoxine, Sulfamethoxazole, ayerlucil and NU-445, thus provide quick, sensitive, a high-throughout method for the sulfa drug residue in food detects.
2) the antibody chip kit set up of the present invention and detection method accuracy high, precision is good, detection efficiency is high.To the IC of sulfadimidine
50be 2.91 μ g/L, to the IC of sulphadiazine
50be 0.43 μ g/L, to the IC of 5-methoxysulfadiazine
50be 0.68 μ g/L, to the IC of sulfaclozine
50be 2.73 μ g/L, to the lowest detectable limit (LOD) of pork and milk lower than 9 μ g/kg, enzyme-linked immunoassay method disclosed in CN102608318A and kit be all better than to the detection sensitivity of 16 kinds of sulfa drugss.
Accompanying drawing explanation
Accompanying drawing 1 is Technology Roadmap of the present invention.
Accompanying drawing 2 is results that substrate is optimized, and in figure, 1 is the comparison of substrates of different relative fluorescence signal intensity, and 2 is comparisons of substrates of different background value.
Accompanying drawing 3 is optimum results of 4 kinds of point sample orders and point sample order, and in figure, B, C, D, E are 4 kinds of point sample orders, and A is point sample effect schematic diagram, and C is optimum point sample precedence diagram.
Accompanying drawing 4 is optimum results of spotting solution and confining liquid, in figure, 1 is the optimum results of different buffer solution as sampling liquid, 2 is CBS optimum results as sampling liquid of different pH, and 3 is comparisons of the fixed effect adding different surfaces activity, and 4 is optimum results of confining liquid.
Accompanying drawing 5 is optimum results of antibody diluent and temperature of reaction, and in figure, 1 is the optimum results of primary antibodie dilution, and 2 is optimum results of two anti-dilutions, and 3 is optimum results of reaction temperature.
Accompanying drawing 6 is the optimum results of each time in Dispersal risk chip processes, and in figure, 1 is the optimum results of set time, and 2 is optimum results of off-period, and 3 is the optimum results in primary antibodie reaction time, and 4 is the optimum results in two anti-reaction time.
Accompanying drawing 7 is the solid phase carrier that adopts of the present invention and subregion schematic diagram, in figure 1: slide; 2: brilliant core macromolecule substrate G trim; 3:12 subregion fence.
Accompanying drawing 8 is the typical curves set up.
Accompanying drawing 9 is stability test results of antibody chip, and in figure, 1 is 37 DEG C of accelerated stability results, and 2 is 4 DEG C of long-time stability results.
Embodiment
Below by embodiment, the invention will be further described, but do not limit the present invention.
The synthesis of embodiment 1 sulfamido coating antigen
Take 62mg haptens 2-sulfonamido-4-methylpyrimidine-5-carboxylic acid, 34mg NHS, 45mg DCC, add 1.5mLDMF and dissolve, under magnetic agitation, in 4 DEG C of reaction 18h, cross and filter precipitation, filtrate is for connecting albumen.
Get the PBS damping fluid that 100mg ovalbumin is dissolved in 10mL, under magnetic stirring, above-mentioned active ester 1mL is dropwise joined in protein solution, 4 DEG C of reaction 21h.
Loaded by reactant liquor in bag filter, dialyse 3 days in PBS damping fluid, every 6h changes a dislysate.Last freeze-drying is preserved.
The selection of embodiment 2 chip parameter
1) comparison of substrates of different: respectively at poly-D-lysine slide, positive charge slide, 0.5 μ g/mL Cy3-OVA put by brilliant core macromolecule substrate G, aldehyde radical substrate, Agarose modification slide, by InnoScan 700A scanner scanning and storage data, the results are shown in accompanying drawing 2.Obviously can see that the relative fluorescence signal intensity of brilliant core macromolecule substrate G is the highest from Fig. 2-1, show that the adsorbability of brilliant core macromolecule substrate G to sample is best; The background value of its background value lower than aldehyde radical can be found out from Fig. 2-2.Consider adsorbability and the business-like brilliant core macromolecule substrate G of background value two aspect this experimental selection of factor as substrate.
2) selection of different point sample order: be from right to left according to following four kinds of mode: B respectively, from bottom to top; C is from left to right, from the top down; D is from left to right, from the top down, has first put first row conversion zone, then has put second row conversion zone; E is from right to left, from bottom to top, has first put second row conversion zone, then has put first row conversion zone, and the schematic diagram of each point sample loading mode is shown in that accompanying drawing 3, A is final point sample effect.Result shows: relative to other modes, the relative fluorescence signal value of mode C is in steady state (SS) substantially, the coefficient of variation minimum (7.96%), illustrates that the some homogeneity which is pointed out is better, so select mode C as the point sample mode of optimum chip.
3) selection of point sample dilution: select pH7.4 phosphate buffer (PBS) respectively, pH9.6 carbonate buffer solution (CBS), pH7.4Tris-HCl buffer salt solution (TBS), containing 10% (percentage by weight, the pH7.4PBS (PBS+10%G) of glycerine down together), containing the pH9.6CBS (CBS+10%G) of 10% glycerine, pH7.4TBS (TBS+10%G) containing 10% glycerine prepares 10 μ g/mL sulfamido coating antigen solution respectively as sampling liquid and carries out point sample, prepare chip, the results are shown in accompanying drawing 4-1, can find out and use CBS+10% glycerine, CBS as the relative fluorescence signal intensity of sampling liquid apparently higher than other sampling liquid, so tentatively select CBS to be the spotting solution of brilliant core macromolecule substrate G, be optimized the pH of CBS, result, as shown in accompanying drawing 4-2, can show the increase along with CBS pH value, and fluorescence signal intensity presents reduction trend, illustrates with the CBS of pH9.16 relatively good as sampling liquid, investigate the impact of different surfaces activating agent on fixed effect, the results are shown in accompanying drawing 4-3, can find out with containing 0.5%Trehalose, do not add surfactant and substantially similar containing the relative fluorescence signal intensity of 1.5%Mannitol, all than other height, so intend selecting pH9.16CBS as sampling liquid, consider that low concentration glycerine (5-10%) can reduce the volatilization of sample spot, stable point shape, so final choice contains the pH 9.16CBS of 5% glycerine as sampling liquid simultaneously.
4) selection of set time: will be set to 0.5 respectively the set time, 1,2,4,6h, the suitable set time is sifted out according to relative fluorescence signal intensity, the results are shown in accompanying drawing 6-1, the prolongation along with the set time can be found out, relative fluorescence signal intensity sharply declines, so select 0.5h as the set time.
5) selection of confining liquid: select 2% (percentage by weight, down together) ovalbumin (OVA), 2% bovine serum albumin(BSA) (BSA), 25% hyclone (FCS) screen respectively as confining liquid, the results are shown in accompanying drawing 4-4, can find out 2%BSA when being confining liquid relative fluorescence signal intensity higher than other two, so select 2%BSA to be confining liquid.
6) selection of off-period: will be set to 0.5 respectively off-period, 1,1.5,2h, filter out according to relative fluorescence signal intensity the off-period that one reaches the signal intensity of needs within a short period of time.The results are shown in accompanying drawing 6-2, can find out the prolongation along with off-period, its relative fluorescence signal intensity declines gradually, so select 0.5h as off-period.
7) selection of antibody diluent: with pH7.4PBS, pH7.4TBS, pH9.6CBS tri-kinds of conventional buffer solution are respectively as primary antibodie and two anti-dilutions, the results are shown in accompanying drawing 5-1 and 5-2, known pH 7.4PBS is the highest as relative fluorescence signal intensity during antibody diluent, illustrate that antigen-antibody reaction is better under the environment of pH7.4PBS, so select pH7.4PBS as antibody diluent.
8) selection of temperature of reaction: carry out antigen-antibody reaction respectively at 4,25,37 DEG C, the results are shown in accompanying drawing 5-3, obviously can find out and just can carry out more thorough 37 DEG C of antigen-antibody reactions, so choose 37 DEG C as temperature of reaction.
9) selection in reaction time: be 0.5 respectively by antigen-antibody reaction set of time, 1,1.5,2,3h, sift out the suitable antibody response time according to relative fluorescence signal intensity.Accompanying drawing 6-3 surveys optimum results in the primary antibodie reaction time, and when the known primary antibodie reaction time is 1.5h, its signal intensity is higher, illustrates that antigen-antibody reaction reaches optimum condition when 1.5h.Consider that chip is as a kind of method for quick, select 0.5h as the primary antibodie reaction time, and relative fluorescence signal intensity will be optimized by point sample concentration and antibody dilution.Accompanying drawing 6-4 surveyed optimum results in two anti-reaction time, can find out that relative fluorescence signal intensity roughly strengthens along with the prolongation in two anti-reaction time, but strengthen be not clearly, so select 0.5h as two anti-reaction time.
The preparation of embodiment 3 antibody chip
1) optimization of coating antigen and antibody concentration
The selection of coating antigen concentration: the concentration of sulfamido coating antigen solution is from left to right set to 2.5,5,10,20, a 40 μ g/mL5 concentration, the sulfanilamide (SN) antibody adding 1:8000,1:4000,1:2000,1:1000,1:500 from right to left is successively optimized, found that when sulfanilamide (SN) coating buffer is 10 μ g/mL, signal intensity during antibody dilution 1:500 is about 1.7 ten thousand, and differ larger with its signal intensity all around, so select 10 μ g/mL to be the concentration of sulfamido coating antigen solution, tentatively determine that antibody dilution is 1:500.
Optimum antibody is dilution to be determined: prepare sulfanilamide (SN) antibody chip with selected concentration, add 1:500,1:1000 sulfanilamide (SN) antibody successively to react, when found that sulfanilamide (SN) antibody dilution is 1:500, inhibition is not obvious, analyzing reason should be that antibody excess causes, and when contrast antibody dilution is 1/1000, inhibition is more obvious, drafting obtains typical curve, calculates IC
50be 2.91 μ g/L, standard curve range is 1-16 μ g/L, and high concentration suppresses the signal intensity optimized lower, and analyzing reason, should to be that antibody is too low cause, so sulfanilamide (SN) antibody dilution is adjusted to 1:800.
2) sulfamido antibody chip processed
Carrier fence is divided into 12 (2 × 6) individual conversion zone as shown in Figure 7.With AD3200 point sample instrument, 1%OVA solution, 10 μ g/mL sulfamido coating antigen solution, Cy3-OVA solution are carried out point sample in each region respectively, 20nL/ point, each region forms the dot matrix of 13 × 3.Chip after point sample is placed in airtight wet box, is placed in 37 DEG C of constant temperature ovens and fixes 0.5h.Chip is put into containing PBST (measuring to flood slide to be as the criterion) sluicing pipe, with shaking table jolting/manually shake and wash 2min, in triplicate, then put into containing ddH
2with shaking table jolting/manually shake and wash 2min in O (being as the criterion to flood slide) sluicing pipe, in triplicate, finally by slide with 2000r/min rotating speed 4 DEG C of centrifugal 1min.Then add 20 μ L/ hole 2%BSA confining liquids, put into airtight wet box, be placed in 37 DEG C of constant temperature ovens and close 0.5h, after washing, 37 DEG C dry 5min, vacuumize encapsulation be stored in 4 DEG C for subsequent use.
The Performance Assessment of embodiment 4 antibody chip
1) foundation of typical curve
Preparation 1:800 sulfamido antibody, with PBS, sulfadimidine (SM2) standard items are mixed with 6 concentration such as 0,1,2,4,8,16 μ g/L, respectively 10 μ L standard solutions and 10 μ L antibody are added on chip, react 0.5 hour, make the medicine of artificial antigen fully and in sample fixed jointly compete specific antibody, washing, centrifuge dripping, adds the Cy3 mark goat anti-mouse igg that 20 μ L 1:200 dilute, reacts 0.5 hour, washing, centrifuge dripping.Use InnoScan700A scanner to scan the chip of drying, analyze with Mapix analysis software.With the logarithm value of competitor concentration be horizontal ordinate, corresponding inhibiting rate F/F
0for ordinate, drawing standard curve, is shown in accompanying drawing 8, IC
50value is 2.91 μ g/L, and the range of linearity is 1-16 μ g/L.Same procedure, replication 5 batches, obtains IC
50mean value, and calculate in sheet and the coefficient of variation between sheet, obtain the coefficient of variation between sheet internal sheet and be all less than 15%, illustrates that repeatability is well.
2) sensitivity
Measure 20 part of 0 μ g/L competitor standard solution, F value is substituted into respectively typical curve regression equation calculation corresponding concentration value, ask 20 parts of mean values measuring concentration value
with standard deviation (SD).According to formula
calculate Z value, as the Judging index of antibody chip detection method sensitivity.The sensitivity that result obtains sulfanilamide (SN) antibody chip detection method is 0.49 μ g/L, well below maximum residue limit.
3) specificity
Respectively its analog standard items doubling dilution of sulfadimidine is become a series of concentration drawing standard curve, calculate IC
50value, with standard items IC
50value contrast obtains cross reacting rate.The results are shown in Table 1, this antibody chip can identify sulfadimidine, daimeton, sulphadiazine, 5-methoxysulfadiazine, cistosulfa, sulfaclozine, sulfamethoxypyridazine, sulphathiazole, fanasil, sulfamethyldiazine, sulfaisodimidine, sulfaquinoxaline, sulfadimethoxine well, and also has certain identification to Sulfamethoxazole, ayerlucil and NU-445.
The cross reacting rate result of table 1 sulfanilamide (SN) antibody chip
| Medicine | IC 50(μg/L) | Cross reacting rate (%) |
| Sulfadimidine | 2.91 | 100.0 |
| Daimeton | 0.87 | 334.5 |
| Sulphadiazine | 0.43 | 676.7 |
| 5-methoxysulfadiazine | 0.68 | 427.9 |
| Cistosulfa | 6.29 | 46.3 |
| Sulfaclozine | 2.73 | 106.6 |
| Sulfamethoxypyridazine | 5.43 | 53.6 |
| Sulphathiazole | 2.16 | 134.7 |
| Fanasil | 0.3 | 970.0 |
| Sulfamethyldiazine | 1.6 | 181.9 |
| Sulfaisodimidine | 1.2 | 242.5 |
| Sulfaquinoxaline | 2.9 | 100.3 |
| Sulfadimethoxine | 4.6 | 63.3 |
| Sulfamethoxazole | 24 | 12.1 |
| Ayerlucil | 32 | 9.1 |
| NU-445 | 39 | 7.5 |
4) stability study
By the antibody chip prepared, carry out 37 DEG C of accelerated tests (Arrhenius law) and 4 DEG C of long-term stable experiments.Within 0th, 2,4,6,8 days/month, taking out antibody chip respectively at acceleration adopts indirect competitive to detect, and drawing standard curve, calculates IC
50value.Will speed up " 0 " hole relative fluorescence signal strength values and the IC of test
50the test findings of value and 0 day/month contrasts, (more than 50%) or IC if " 0 " hole relative fluorescence signal strength values obviously declines
50value obviously raises (more than 1 times), then judge the biologically active drop by half of antibody chip, antibody chip can not be used again, " 0 " hole relative fluorescence signal strength values and IC
50the shelf-life (agriculture doctor send out [2005] No. 17,2005) that be during this period of time antibody chip of value in variable range, the results are shown in accompanying drawing 9.Within 8th day, still F is remained on 37 DEG C of accelerated tests by the relative fluorescence signal strength values in accompanying drawing 9-1 known " 0 " hole
0more than 60%, IC
50value is floated in the scope of ± 20%, shows that sulfamido antibody chip at least can preserve 6 months at 4 DEG C.4 DEG C of long-term stable experiment results (accompanying drawing 9-2) also demonstrate this point.
5) sample-pretreating method
The preparation of sample reagent: accurately take KH
2pO
40.41g, K
2hPO
45.59g, ultrapure water dissolves, and is settled to 1000mL.
Milk sample disposal route: measure milk sample 2mL in 4mL centrifuge tube, after vortex 2min, gets part milk and extracts reagent dilutions 20 times, 12000r/min, 4 DEG C of centrifugal 10min, gets final product loading after removing upper-layer fat particle.
Pork sample treatment: take homogenate muscle samples 2.00 ± 0.02g in 50mL centrifuge tube, adds and extracts reagent 10mL, and concussion 10min makes it mixing, 6000r/min, 4 DEG C of centrifugal 5min, get supernatant extraction reagent dilute 4 times again after sample detection.
6) sulfamido antibody chip detecting step
Indirect competitive sulfamido antibody chip is adopted to detect sample.10 μ L extracts and 10 μ L antibody diluents are added on chip, react 0.5 hour, the medicine of artificial antigen fully and in sample fixed is made jointly to compete specific antibody, washing, centrifuge dripping, adds the Cy3 mark goat anti-mouse igg that 20 μ L 1:200 dilute, reacts 0.5 hour, washing, centrifuge dripping.Used by the chip of drying InnoScan 700A scanner to scan, analyze with Mapix analysis software.
7) lowest detectable limit and quantitative limit
Instrument of learning from else's experience is detected as each 20 parts negative of tissue to be measured (pork and milk) sample, after above-mentioned sample-pretreating method process, adopts indirect competitive to detect, measures F value, calculate blank sample F
0the mean value of value
.Will
substitute on typical curve and find corresponding concentration (C), and calculate standard deviation (SD).Calculate Z value according to formula Z=C+3 × SD, this is the lowest detectable limit (LOD) of method for organizing.Calculate Q value according to formula Q=C+10 × SD, this is the minimum quantitative limit (LOQ) of method for organizing.The results are shown in Table 2, the lowest detectable limit (LOD) in pork and milk lower than 9 μ g/kg, quantitative limit (LOQ) lower than 15 μ g/kg, lower than MRL (100 μ g/kg).
The lowest detectable limit of sulfa drugs in table 2 tissue sample
8) recovery
Instrument of learning from else's experience is detected as negative tissue to be measured (pork and milk) sample, adds the concentration that maximum residue limit requires, each sample concentration establish 3 parallel.After sample preparation, adopt indirect competitive to measure drug concentration, repeat 3 times, according to formula:
calculate the recovery, and calculate batch interior interassay coefficient of variation.Result (table 3, table 4) the display recovery, between 80%-110%, with interassay coefficient of variation < 12% in batch, shows that antibody chip detection accuracy is good.
Table 3 sulfa drugs TIANZHU XINGNAO Capsul in milk
TIANZHU XINGNAO Capsul in table 4 sulfa drugs pork
Claims (9)
1. one kind for detecting the antibody chip kit of sulfa drug residue in food, comprise two anti-, extraction reagent of chip, antibody, Cy3 mark, it is characterized in that: described antibody is by the monoclonal antibody of preserving number secreted by the hybridoma cell strain 4E5 of CCTCC NO:C201148, described chip secures sulfamido coating antigen, and described sulfamido coating antigen is 2-sulfonamido-4-methylpyrimidine-5-carboxylic acid and ovalbumin conjugate.
2. as claimed in claim 1 for detecting the antibody chip kit of sulfa drug residue in food, it is characterized in that described chip is prepared as follows: using brilliant core macromolecule substrate G as substrate, with fence, substrate is divided into 12 conversion zones, each conversion zone establishes vertical totally 9 microarraies of three rows three, by sulfamido coating antigen solution and two kinds of contrast solutions---the oralbumin solution difference point sample of oralbumin solution and Cy3 mark one is arranged on microarray wherein, fixing 0.5h after point sample completes, closes 0.5h with 2% bovine serum albumin solution.
3. as claimed in claim 2 for detecting the antibody chip kit of sulfa drug residue in food, it is characterized in that: the concentration of described sulfamido coating antigen solution is 10 μ g/mL, the solvent of described sulfamido coating antigen solution is pH 9.16 carbonate buffer solution containing 5% glycerine.
4. as claimed in claim 1 for detecting the antibody chip kit of sulfa drug residue in food, it is characterized in that two of described Cy3 mark anti-be sheep anti mouse or the rabbit anti-mouse antibody of Cy3 mark.
5. as claimed in claim 1 for detecting the antibody chip kit of sulfa drug residue in food, it is characterized in that described extraction reagent is prepared as follows: accurately take KH
2pO
40.41g, K
2hPO
45.59g, ultrapure water dissolves, and is settled to 1000mL.
6. the antibody chip kit of claim 1-5 described in any one is in the application detecting sulfa drug residue in food.
7., for detecting an antibody chip method for sulfa drug residue in food, it is characterized in that comprising the following steps:
1) sample pre-treatments: add after sample homogenization and extract reagent extraction;
2) sample liquid and the antibody diluted are secured sulfamido coating antigen solution and contrast solution by joining after the volume ratio mixing of 1: 1---on the chip of the oralbumin solution of oralbumin solution and Cy3 mark, in 37 DEG C of reaction 0.5h, wash 3 times, dry;
3) marked by Cy3 two anti-are added to chip, in 37 DEG C of reaction 0.5h, washs 3 times, drying;
4) scan chip with InnoScan 700A scanner, the oralbumin solution marked with oralbumin solution and Cy3 contrasts, and carries out interpretation of result with Mapix analysis software,
Described antibody is by the monoclonal antibody of preserving number secreted by the hybridoma cell strain 4E5 of CCTCC NO:C201148;
Described sulfamido coating antigen is 2-sulfonamido-4-methylpyrimidine-5-carboxylic acid and ovalbumin conjugate;
Described Cy3 mark two anti-be sheep anti mouse or the rabbit anti-mouse antibody of Cy3 mark.
8. as claimed in claim 7 for detecting the antibody chip method of sulfa drug residue in food, it is characterized in that described extraction reagent is prepared as follows: accurately take KH
2pO
40.41g, K
2hPO
45.59g, ultrapure water dissolves, and is settled to 1000mL.
9. as claimed in claim 7 for detecting the antibody chip method of sulfa drug residue in food, it is characterized in that: described antibody dilutes with the phosphate buffer of pH7.4, and the dilutability of described antibody is 1:800; The concentration of described sulfamido coating antigen solution is 10 μ g/mL, and the solvent of described sulfamido coating antigen solution is the carbonate buffer solution of the pH9.16 containing 5% glycerine.
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