CN103344758B - Sulfadimidine chemiluminescence enzyme immunoassay detection method and kit - Google Patents
Sulfadimidine chemiluminescence enzyme immunoassay detection method and kit Download PDFInfo
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Abstract
The invention discloses a kind of sulfadimidine chemiluminescence enzyme immunoassay detection method and kit, indirect exzyme immunoassay and chemiluminescence combine by this method, to be applied in animal derived food during Sulfamethazine Residues detects, there is special, quick, sensitive, feature accurately, the IC of kit
50be 4.006 μ g/L, the recovery is 94.42 ~ 102.73%, is 100% with the identical rate of existing detection method, and linear detection range is wide simultaneously, lowest detectable limit is low, is suitable for trace analysis and the batch detection of sulfadimidine, has a extensive future.
Description
Technical field
The invention belongs to chemiluminescence enzyme immunoassay technical field, particularly relate to a kind of sulfadimidine chemiluminescence enzyme immunoassay detection method and kit.
Background technology
Sulfadimidine (Sulfadimidine, SM
2) also known as sulfamethazine, arnosulfan pyrimidine, it is one of sulfa drugs most widely used in livestock breeding industry, the conventional medicine doing feed addictive and bacteriosis, long-term interpolation or abuse can cause the medicament residue in animal food, threaten to human health.Research finds, human body is taken in can be accumulated in vivo by any approach, affects the immune system of body, destroys the tissues such as muscle, kidney, thyroid gland, can bring out thyroid cancer simultaneously; And this medicine also can be shifted to fetus or neonate from parent by placenta and milk, and make the Thyroid Hormones Levels in its blood plasma and brain tissue reduce, this has profound influence to the brain growth of human and animal.On the other hand, the drug resistance of the residual easy Induction of bacterial of sulfadimidine, makes medicine lose the value of disease therapy.Therefore, the regulations such as Codex Committee on Food of the United Nations and European Union, in food and feed, sulfa drugs is single all must not more than 100 μ g/kg; The maximum residue limit(MRL) of Japan's regulation is 10 μ g/kg; In Dec, 2002, the Ministry of Agriculture of China announced No. 235 file regulation, and in the muscle of all food animals, fat, liver and kidney, sulfamido maximum residue limit(MRL) is 100 μ g/kg, and is classified as residue of veterinary drug monitoring emphasis.
At present, the method detecting sulfadimidine has microbial method, physico-chemical method (as high performance liquid chromatography, thin-layer chromatography etc.) and immunoassay (radio immunoassay, enzyme linked immunosorbent assay, chemiluminescence immunoassay etc.).Wherein, microbial method is easy, quick, cheap, and be applicable to basic unit's field screening, but be subject to other biotic influence, sensitivity is low, loss is high; Physico-chemical method precision is high, accuracy is high, but expensive equipment, strongly professional, detection time is long, can be used for the confirmation of doubtful sample; Immunoassay is easy and simple to handle, quick, special, highly sensitive, has been applied to the detection that multi-medicament is residual in recent years, but the Chemiluminescence quantitative immunoassay of related detection Sulfamethazine Residues there is not been reported.
Summary of the invention
The technical problem to be solved in the present invention be to provide a kind of economy, easy, quick, special, sensitive, accurately sulfadimidine chemiluminescence enzyme immunoassay detection method and kit and this kit Sulfamethazine Residues in animal derived food detect in application.
Another technical matters that the present invention will solve is to provide a kind of sulfadimidine monoclonal antibody of high specificity.
For solving the problems of the technologies described above, the present invention by the following technical solutions: sulfadimidine chemiluminescence enzyme immunoassay detection method, comprises the following steps:
<1> process testing sample;
<2> is by the conjugate (SM of haptens sulfadimidine and carrier protein ovalbumin
2-OVA) as antigen coated in luminous solid phase carrier, after closing, add series standard sample or the testing sample through pre-treatment respectively, then add sulfadimidine monoclonal antibody and react;
<3>, after step <1>, adds ELIAS secondary antibody and reacts, and then add chemical luminescence for liquid, measures the luminous value of series standard sample and testing sample;
The luminous value that <4> records with step <3> calculates inhibiting rate, drawing standard curve, and the content calculating sulfadimidine in testing sample according to the regression equation of typical curve and the inhibiting rate of testing sample.
Step <1> carries out with reference to No. 1025, Ministry of Agriculture bulletin-24-2008 standard; Step <2> is undertaken by following operation: with coating buffer by 1:100000 dilute envelope antigen, every hole adds 100 μ L, 4 DEG C hatch 10h after, incline coating buffer, with washing trigger washing chemistry luminous plaque 3 times, pats dry; Then, every hole adds 350 μ L confining liquids, hatches 1h for 37 DEG C, and incline deblocking liquid, washing 3 times, patting dry with washing trigger; After 37 DEG C of oven dry, use masking foil vacuum seal, 4 DEG C of preservations; Chemiluminescent plate is taken out from 4 DEG C, balances to room temperature, according to every hole 50 μ L, series standard sample solution and testing sample are added Chemiluminescent plate, 3 holes are respectively established to repeat, then every hole adds 50 μ L sulfadimidine monoclonal antibodies, and mixing, hatches 30min for 37 DEG C; Incline liquid, washing 3 times, patting dry with washing trigger; Step <3> is undertaken by following operation: every hole adds the HRP-goat anti-mouse IgG that 50 μ L1:2500 dilute, and hatches 45min for 37 DEG C; Incline liquid, washing 5 times, patting dry with washing trigger; Add 100 μ L chemical luminescence for liquid in every hole, mixing, measures each hole luminous value as early as possible.
Confining liquid is the skimmed milk power of 5%; The extension rate of sulfadimidine monoclonal antibody is 1:1500; The concentration of series standard sample solution is 0.1 μ g/L, 1 μ g/L, 10 μ g/L, 100 μ g/L, 1000 μ g/L; Coating buffer is the carbonate buffer solution of pH9.6.
Sulfadimidine chemiluminescence enzyme immunoassay detection kit, comprises and is coated with envelope antigen (SM
2-OVA) Chemiluminescent plate, sulfadimidine series standard sample solution, sulfadimidine monoclonal antibody working fluid, concentrated cleaning solution, HRP-goat anti-mouse IgG (ELIAS secondary antibody) working fluid, chemical luminescence for liquid; Envelope antigen is the conjugate of sulfadimidine and carrier protein ovalbumin; Sulfadimidine series standard sample solution is 6 concentration gradients, is 0 μ g/L, 0.1 μ g/L, 1 μ g/L, 10 μ g/L, 100 μ g/L, 1000 μ g/L respectively; Concentrated cleaning solution is by NaCl8.00g, KH
2pO
40.20g, Na
2hPO
412H
2o2.90g, KCl0.20g, Tween-20 0.50mL, adding water is settled to 100mL and makes; Chemical luminescence for liquid is from super quick ECL chemical luminescence reagent kit P0018--BeyoECLPlus.
Application during mentioned reagent box Sulfamethazine Residues in animal derived food detects.
Sulfadimidine monoclonal antibody, by following operation preparation: with haptens sulfadimidine and the clear albuminous conjugate (SM of carrier proteins Bovine
2-BSA) as immunogene, immunity female BAl BIc/c mouse in 6 week age, conventionally carry out the screening of Fusion of Cells and positive strain, continuous clone is involved in a criminal case 3 times to the positive cell filtered out, through qualification, frozen and after building strain, carry out the preparation of ascites, then by purifying ascites, obtain the monoclonal antibody of energy specific recognition sulfadimidine.
Above-mentioned sulfadimidine monoclonal antibody, by following operation preparation:
<1> animal immune
By 6 of health week age female BAl BIc/c mouse by after fundamental immunity and 5 booster immunizations, impacts is carried out to the strongest the highest, competitive mouse of tiring immune;
<2> Fusion of Cells and colony screening
Impact immunity after 3 days, put to death after extracing eyeball of mouse bloodletting, collect blood, asepticly get spleen, cell-fusion techniques is utilized mouse immune splenocyte and Sp2/0 cell to be merged, cell suspension after fusion adds to and is covered with in 96 orifice plates of feeder cells in advance, adopts HAT Selective agar medium screening fused cell;
When Growth of Cells is to culture hole area 1/10, aseptic accurate absorption 100 μ L supernatant, adds to envelope antigen SM
2in the ELISA Plate of-OVA; Survey cells and supernatant with indirect elisa method to tire, limiting dilution assay is adopted to clone 3 times continuously to positive cell, until when positive rate is 100%, cell line is carried out that expansion is cultivated, qualification, frozen and build strain, carry out Continuous Cultivation to cell line after qualification to go down to posterity more than 2 months, detect its stability and antibody-secreting ability every 5 generations;
<3> cell cryopreservation and recovery
With cryopreserving liquid, the hybridoma being in exponential phase is made cell suspension, be sub-packed in cryopreservation tube, be placed in liquid nitrogen and preserve; Take out cryopreservation tube during recovery, put into 37 DEG C of water-bath middling speeds immediately and melt, centrifugal segregation cryopreserving liquid, move in cell bottle and cultivate;
The preparation and purification of <4> ascites
Adopt in body and induce method, sterilizing paraffin oil is injected Balb/c mouse peritoneal in 8 week age, injects hybridoma after 7-14 days, after 7-10 days, collect ascites; The ascites of collecting, after saturated ammonium sulfate just pure and mild affinity chromatography, obtains sulfadimidine monoclonal antibody.
The present invention has inquired into the chemiluminescence enzyme immune mechanism detecting sulfadimidine first, indirect exzyme immunoassay and chemiluminescence combine by inventor, successfully establish sulfadimidine chemiluminescence enzyme immunoassay detection method and kit, this method to be applied in animal derived food during Sulfamethazine Residues detects, there is special, quick, sensitive, feature accurately, the IC of kit
50be 4.006 μ g/L, the recovery is 94.42 ~ 102.73%, is 100% with the identical rate of existing detection method, is suitable for trace analysis and the batch detection of sulfadimidine.Linear detection range of the present invention (0.1 ~ 1000 μ g/L) is wider than ELISA, when detecting the sample of sulfadimidine severe overweight (content more than ELISA sensing range lower than 1000 μ g/L), without the need to diluting further sample and duplicate detection, decreasing workload, saving testing cost and time; Meanwhile, lowest detectable limit of the present invention (0.17 μ g/L) reduces 10 times than ELISA, more meets trace analysis and batch detection, has good application prospect.
Accompanying drawing explanation
Fig. 1 is the typical curve of embodiment 2 sulfadimidine chemiluminescence enzyme immunoassay detection method.
In figure: X-axis is the logarithm value of sulfadimidine standard model concentration, Y-axis is that the luminous value of each concentration sulfadimidine standard model is divided by " 0 " concentration hole luminous value (RLU/RUL
0%).
Embodiment
The preparation of embodiment 1 sulfadimidine monoclonal antibody
1.1 animal immune
With haptens sulfadimidine and the clear albuminous conjugate (SM of carrier proteins Bovine
2-BSA) as immunogene, by 10 healthy 6 week age female BAl BIc/c mouse be divided into 2 groups at random, wherein 1 group is control group; After fundamental immunity and 5 booster immunizations, blood sampling measures serum titer, and to tiring, the strongest the highest, competitive mouse carries out impact immunity;
1.2 Fusion of Cells and colony screening
Impact immunity after 3 days, put to death after extracing eyeball of mouse bloodletting, collect blood, asepticly get spleen, cell-fusion techniques is utilized No. 5 mouse immune splenocytes and Sp2/0 cell to be carried out merging (fusion rate is 71.4%), cell suspension after fusion adds to and is covered with in 96 orifice plates of feeder cells in advance, adopts HAT Selective agar medium screening fused cell;
When Growth of Cells is to culture hole area 1/10, aseptic accurate absorption 100 μ L supernatant, adds to envelope antigen SM
2in the ELISA Plate of-OVA; Survey cells and supernatant with indirect elisa method to tire, limiting dilution assay is adopted to clone 3 times continuously to positive cell, until when positive rate is 100%, cell line is carried out that expansion is cultivated, qualification, frozen and build strain, carry out Continuous Cultivation to cell line after qualification to go down to posterity more than 2 months, detect its stability and antibody-secreting ability every 5 generations;
1.3 cell cryopreservations and recovery
With cryopreserving liquid, the hybridoma being in exponential phase is made cell suspension, be sub-packed in cryopreservation tube, be placed in liquid nitrogen and preserve; Take out cryopreservation tube during recovery, put into 37 DEG C of water-bath middling speeds immediately and melt, centrifugal segregation cryopreserving liquid, move in cell bottle and cultivate;
The preparation and purification of 1.4 ascites
Adopt in body and induce method, sterilizing paraffin oil is injected Balb/c mouse peritoneal in 8 week age, injects hybridoma after 7-14 days, after 7-10 days, collect ascites; The ascites of collecting, after saturated ammonium sulfate just pure and mild affinity chromatography, through SDS-PAGE electroresis appraisal, confirms that the sulfadimidine monoclonal antibody 1E7 obtained reaches electrophoresis pure.
The qualification of 1.5 monoclonal antibodies
1.5.1 the mensuration of protein content
The protein content measuring the monoclonal antibody of purifying with ultraviolet spectrophotometer is 4.536mg/mL.
1.5.2 the mensuration of affinity
By non-competing ELISA, respectively with OD
450value is ordinate, with SM
2the concentration of-McAb is horizontal ordinate, drawing standard curve, and the affinity costant with the formula under the different antigen coated concentration of 1 calculating, the affinity costant (Ka) drawing sulfadimidine monoclonal antibody of averaging is 0.12 × 10
7l/mol.
1.5.3 the qualification of subclass
Measuring 1E7 subclass according to the mouse monoclonal Ig class/subgroup identification ELISA kit at the logical experiment material center of Luoyang one hundred Austria is IgG2b.
The foundation of the chemiluminescence enzyme immunoassay detection method of embodiment 2 Sulfamethazine Residues
The preparation of 2.1 related reagents
Carbonate buffer solution (pH9.6, i.e. coating buffer): accurately take Na
2cO
31.59g, NaHCO
32.93g, after ultrapure water dissolves, regulates pH to 9.6, is settled to 1000mL.
Cleansing solution (pH7.4): accurately take NaCl8.00g, KH
2pO
40.20g, Na
2hPO
412H
2o2.90g, KCl0.20g, after ultrapure water dissolves, regulate pH to 7.4, add Tween-20 0.50mL, be settled to 1000mL.
Phosphate buffer (PBS) (pH7.4): accurately take NaCl8.00g, KH
2pO
40.20g, Na
2hPO
412H
2o2.90g, KCl0.20g, after ultrapure water dissolves, regulate pH to 7.4, be settled to 1000mL.
Confining liquid: accurately take skimmed milk power 1.0g, adds 20mL phosphate buffer, stirs to dissolving completely.
The super quick ECL chemical luminescence reagent kit of BeyoECLPlus(, P0018,100mL): purchased from green skies biotechnology research institute.
The determination of 2.2 antigen coated conditions
Use the conjugate (SM of haptens sulfadimidine and carrier protein ovalbumin
2-OVA) as envelope antigen, this antigen is wrapped quilt respectively by after 1:25000,1:50000,1:75000,1:100000 and 1:125000 dilution under 37 DEG C of 10h, 25 DEG C of 10h, 4 DEG C of 10h and 37 DEG C 3h, 25 DEG C of 3h and 4 DEG C of 3h conditions, selects reaction normal curve IC
50(concentration of medicine during 50% inhibiting rate, the sensitivity of namely reacting) minimum condition is as the antigen coated condition of the best.Press 1:100000 by table 1 result determination envelope antigen to dilute, hatch 10h for best at 4 DEG C.
The optimum results of the antigen coated condition of table 1
The determination of 2.3 sealing conditions
With the antigen coated condition bag of the best by Chemiluminescent plate, using the PBS of 20mM, 5% skimmed milk power, 1% BSA, 5% hyclone as closed material, select reaction normal curve IC
50minimum, as reaction sealed liquid; Then, the skimmed milk power using 5% is as confining liquid, and setting off-period is 37 DEG C of 45min, 37 DEG C of 1h, 37 DEG C of 1.5h, 37 DEG C of 2h, calculates the IC under different condition
50, select reaction normal curve IC
50minimum, as reaction sealed condition, determine that skimmed milk power 37 DEG C of 1h of 5% are best by table 2 result.
The optimum results of table 2 sealing condition
The determination of 2.4 antibody dilution multiples
Under the best bag quilt, sealing condition, by sulfadimidine monoclonal antibody respectively according to 1:1000,1:1500,1:2000 and 1:2500 dilution, with reaction normal curve IC
50as Judging index, determine reaction optimum antibody extension rate.Determine that 1:1500 is antibody optimum diluting multiple by table 3 result.
The determination of 2.5 competitive reaction times
Under aforementioned fixed top condition, select 15min, 30min and 60min as the competitive reaction time respectively, with reaction normal curve IC
50as Judging index, select the competitive reaction time.Determine that 30min is the best competitive reaction time by table 3 result.
Table 3 antibody dilution multiple, the optimum results of competitive reaction time
The determination of 2.6HRP-goat anti-mouse IgG extension rate
Under above optimal conditions, respectively HRP-goat anti-mouse IgG is diluted according to 1:1000,1:2000,1:4000 and 1:8000, with reaction normal curve IC
50as Judging index, determine the best two anti-extension rates of reaction.Result shows, and when diluting by 1:1000, values of chemiluminescence is too high, and error is larger; When diluting by 1:6000, values of chemiluminescence is too little, and result not easily judges; And during 1:2000 and 1:3000 dilution, luminous numerical values recited is light-emitting appearance optimum detection scope.Consider, determine that 1:2500 is the best dilute concentration of HRP-goat anti-mouse IgG.
The foundation of 2.7 typical curves, sensitivity and lowest detectable limit
In optimal conditions, sulfadimidine is made into 0.1 ~ 1000 μ g/L series concentration, each concentration 3 hole is repeated, and detects.With the logarithm value of standard model solution concentration for horizontal ordinate, with the luminous value of each concentration sulfadimidine standard items divided by " 0 " concentration hole luminous value (RLU/RUL0%) for ordinate, drawing standard curve.As shown in Figure 1, the regression equation of typical curve is y=-22.13x+63.338, R
2=0.9904, curve presents good linear relationship between 0.1 ~ 1000 μ g/L, and lowest detectable limit is 0.17 μ g/L, IC
50be 4.006 μ g/L.
2.8 specificitys of the present invention
With analytical review method of the present invention, some common antibiotics (sulphoamidine, sulphadiazine, sulfanilamide (SN) five first pyrimidine, penicillin, Ofloxacin, chloromycetin, diethylstilbestrol) are detected, measure IC
50, according to formula 2, calculate the cross reacting rate of each medicine.From table 4, the cross reacting rate of the method that the present invention sets up and common antibiotics is all less than 0.01%, shows that this law specificity is better.
Cross reacting rate=IC
50(sulfadimidine)/IC
50(other drug) × 100%(formula 2)
Table 4 specific assay result of the present invention
2.9 accuracy of the present invention
Recovery experiment is carried out with basic, normal, high three concentration (1 μ g/L, 10 μ g/L, 100 μ g/L), each sample sets 3 holes and repeats, and measures its values of chemiluminescence, the mean value obtained is substituted into the regression equation of typical curve, obtain respective standard sample concentration value, calculate the recovery.Determine that the recovery of the present invention is between 94.42 ~ 102.73% by table 5 result, show that this law has reliable accuracy.
The measurement result of table 5 accuracy of the present invention
The development of embodiment 3 Sulfamethazine Residues chemiluminescence enzyme immunoassay detection kit
According to the result of study of embodiment 1 and 2, assembling Sulfamethazine Residues chemiluminescence enzyme immunoassay detection kit.
3.1 kit compositions
A is coated with envelope antigen (SM
2-OVA) Chemiluminescent plate;
B sulfadimidine series standard sample solution: 0 μ g/L, 0.1 μ g/L, 1 μ g/L, 10 μ g/L, 100 μ g/L, 1000 μ g/L;
C sulfadimidine monoclonal antibody 1E7 working fluid;
DHRP-goat anti-mouse IgG working fluid;
E concentrated cleaning solution: NaCl8.00g, KH
2pO
40.20g, Na
2hPO
412H
2o2.90g, KCl0.20g, Tween-20 0.50mL, add water and be settled to 100mL;
F chemical luminescence for liquid A liquid, B liquid (super quick ECL chemical luminescence reagent kit P0018--BeyoECLPlus);
The bag quilt of 3.2 Chemiluminescent plates
With coating buffer press 1:100000 dilute envelope antigen, every hole adds 100 μ L, 4 DEG C hatch 10h after, incline coating buffer, with washing trigger washing chemistry luminous plaque 3 times, pats dry; Then every hole adds 350 μ L confining liquids, and hatch 1h for 37 DEG C, incline deblocking liquid, washing 3 times, patting dry with washing trigger; After 37 DEG C of oven dry, use masking foil vacuum seal, 4 DEG C of preservations.
The application of embodiment 4 Sulfamethazine Residues chemiluminescence enzyme immunoassay detection kit
The preparation of 4.1 reagent
A cleansing solution: use after the concentrated cleaning solution ultrapure water provided in kit is diluted 10 times.
B chemical luminescence for liquid: before interpolation chemical luminescence for liquid, chemical luminescence for liquid A liquid, B liquid are mixed according to 1:1.
The pre-treatment of 4.2 tissue samples
Carry out with reference to No. 1025, Ministry of Agriculture bulletin-24-2008 standard:
A gets in 3g animal muscle tissue to mortar to grind to pasty state and forwards in 50mL centrifuge tube;
B adds the centrifugal 10min of second fine 9mL mixing concussion 10min, 4000r/min of 84%;
C gets supernatant 3mL and adds 2mol/L sodium chloride 2mL, ethyl acetate 7mL, the centrifugal 5min of concussion mixing 10min, 4000r/min;
D gets upper liquid and dries up in nitrogen, adds the phosphate buffer 1 mL of 0.02mol/L, normal hexane 1mL and dissolves, concussion mixing 3min, in the centrifugal 5min of 4000r/min, takes off layer liquid for detecting.
4.3 testing process
Chemiluminescent plate takes out by A from 4 DEG C, balances to room temperature;
Series standard sample solution and testing sample are added Chemiluminescent plate according to every hole 50 μ L by B, respectively establish 3 holes to repeat, and then every hole adds 50 μ L sulfadimidine monoclonal antibodies, and mixing, hatches 30min for 37 DEG C;
C inclines liquid, washing 3 times, patting dry with washing trigger;
The every hole of D adds the HRP-goat anti-mouse IgG that 50 μ L1:2500 dilute, and hatches 45min for 37 DEG C;
E inclines liquid, washing 5 times, patting dry with washing trigger; Add 100 μ L chemical luminescence for liquid in every hole, mixing, measures each hole luminous value as early as possible;
F is according to formula, and according to obtained sample luminous value, the inhibiting rate of calculation sample, and inhibiting rate is substituted into standard regressive method, calculates residual quantity; As sample once diluted, be then multiplied by extension rate, be the residual quantity of sulfadimidine in sample.
The detection of 4.4 samples
Gather totally 141 parts, commercially available shrimp, fish and pork sample, carry out the pre-treatment of sample with reference to above-mentioned bulletin standard, then detect with kit of the present invention, specify according to the Ministry of Agriculture, when residual quantity is judged to defective more than 100 μ g/kg, wherein have 129 parts qualified, sample passes rate is 91.49%.
Embodiment 5 chemiluminescence Enzymoimmune reagent kit of the present invention compares with commercial ELISA Assay kit
Select 36 parts of tissue samples, detect the residual quantity of wherein sulfadimidine with kit of the present invention and commercial ELISA Assay kit simultaneously, the results are shown in Table 6.The yin and yang attribute coincidence rate of the two is 100%, statistical study is carried out to the testing result of two kinds of methods, checks known t=0.410, P=0.648 > 0.05 through t, between two kinds of method testing results, difference is not remarkable, illustrates that kit of the present invention has good effect.
In addition, sensing range of the present invention is wider, lowest detectable limit reduces 10 times than commercial ELISA Assay kit, and more meet trace analysis and batch detection, application prospect is good.
Table 6 kit results compares
| Sample number into spectrum | Commercial ELISA Assay kit testing result (μ g/kg) | This kit testing result (μ g/kg) |
| Shrimp 1 | 317.3386221 | 248.0751806 |
| Shrimp 2 | 335.6700716 | 266.10262 |
| Shrimp 15 | 1.001767113 | 2.309985198 |
| Shrimp 17 | 1.523619251 | 2.3584488 |
| Shrimp 19 | 1.301923065 | 2.961149759 |
| Shrimp 20 | 1.451238264 | 2.598121808 |
| Fish 1 | 335.6700716 | 378.3196916 |
| Fish 2 | 364.4887814 | 334.2108086 |
| Fish 11 | 2.194873187 | 1.890214447 |
| Fish 12 | 1.673115544 | 1.809148814 |
| Fish 14 | 2.900981043 | 3.53177296 |
| Fish 15 | 2.055676615 | 3.188808856 |
| Fish 17 | 1.743458869 | 1.218988793 |
| Fish 18 | 1.413698722 | 1.80391665 |
| Fish 20 | 1.150609629 | 2.922276658 |
| Pig 1 | 158.1578608 | 172.2441963 |
| Pig 2 | 410.8804742 | 445.0008146 |
| Pig 3 | 397.2662671 | 420.5712173 |
| Pig 4 | 407.8153277 | 443.0162229 |
| Pig 5 | 353.7336141 | 385.9277567 |
| Pig 10 | 8.559588927 | 18.16834143 |
| Pig 11 | 1.352423994 | 1.363395653 |
| Pig 15 | 353.7336141 | 340.2694691 |
| Pig 17 | 1.397909086 | 1.447940964 |
| Pig 19 | 5.15386114 | 4.40341437 |
| Pig 24 | 1.08168136 | 1.139793386 |
| Pig 25 | 1.377130226 | 2.182899973 |
| Pig 44 | 323.3350819 | 271.2961433 |
| Pig 61 | 360.417797 | 348.4369918 |
| Pig 83 | 296.6574807 | 347.6356024 |
| Pig 64 | 357.7290994 | 334.2100464 |
| Pig 65 | 318.5289507 | 241.6193998 |
| Pig 88 | 3.973078555 | 14.07213281 |
| Pig 86 | 28.3639221 | 32.23077022 |
| Pig 111 | 4.078580205 | 18.16573141 |
| Pig 125 | 4.078580205 | 1.799760481 |
Claims (5)
1. a sulfadimidine chemiluminescence enzyme immunoassay detection method, is characterized in that comprising the following steps:
<1> process testing sample, carries out with reference to No. 1025, Ministry of Agriculture bulletin-24-2008 standard;
<2> using the conjugate of haptens sulfadimidine and carrier protein oralbumin as antigen coated in luminous solid phase carrier, after closing, add series standard sample solution or the testing sample through pre-treatment respectively, then add sulfadimidine monoclonal antibody and react; Specifically undertaken by following operation: with coating buffer by 1:100000 dilute envelope antigen, every hole adds 100 μ L, 4 DEG C hatch 10h after, incline coating buffer, with washing trigger washing chemistry luminous plaque 3 times, pats dry; Then, every hole adds 350 μ L confining liquids, hatches 1h for 37 DEG C, and incline deblocking liquid, washing 3 times, patting dry with washing trigger; After 37 DEG C of oven dry, use masking foil vacuum seal, 4 DEG C of preservations; Chemiluminescent plate is taken out from 4 DEG C, balances to room temperature, according to every hole 50 μ L, series standard sample solution and testing sample are added Chemiluminescent plate, 3 holes are respectively established to repeat, then every hole adds 50 μ L sulfadimidine monoclonal antibodies, and mixing, hatches 30min for 37 DEG C; Incline liquid, washing 3 times, patting dry with washing trigger;
<3> is after step <2>, add ELIAS secondary antibody to react, and then add chemical luminescence for liquid, measure the luminous value of series standard sample solution and testing sample, specifically undertaken by following operation: every hole adds the HRP-goat anti-mouse IgG that 50 μ L1:2500 dilute, and hatches 45min for 37 DEG C; Incline liquid, washing 5 times, patting dry with washing trigger; Add 100 μ L chemical luminescence for liquid in every hole, mixing, measures each hole luminous value as early as possible;
The luminous value that <4> records with step <3> calculates inhibiting rate, drawing standard curve, and the content calculating sulfadimidine in testing sample according to the regression equation of typical curve and the inhibiting rate of testing sample.
2. sulfadimidine chemiluminescence enzyme immunoassay detection method according to claim 1, is characterized in that: described confining liquid is the skimmed milk power of 5%; The extension rate of described sulfadimidine monoclonal antibody is 1:1500; The concentration of described series standard sample solution is 0.1 μ g/L, 1 μ g/L, 10 μ g/L, 100 μ g/L, 1000 μ g/L; Described coating buffer is the carbonate buffer solution of pH9.6.
3. a sulfadimidine chemiluminescence enzyme immunoassay detection kit, is characterized in that comprising the Chemiluminescent plate, sulfadimidine series standard sample solution, sulfadimidine monoclonal antibody working fluid, concentrated cleaning solution, HRP-goat anti-mouse IgG working fluid, the chemical luminescence for liquid that are coated with envelope antigen; Described envelope antigen is the conjugate of sulfadimidine and carrier protein oralbumin; Described sulfadimidine series standard sample solution is 6 concentration gradients, is 0 μ g/L, 0.1 μ g/L, 1 μ g/L, 10 μ g/L, 100 μ g/L, 1000 μ g/L respectively; Described concentrated cleaning solution is by NaCl8.00g, KH
2pO
40.20g, Na
2hPO
412H
2o2.90g, KCl0.20g, Tween-20 0.50mL, adding water is settled to 100mL and makes; Described chemical luminescence for liquid is from super quick ECL chemical luminescence reagent kit P0018--BeyoECLPlus.
4. sulfadimidine chemiluminescence enzyme immunoassay detection kit according to claim 3, is characterized in that sulfadimidine monoclonal antibody is prepared by following operation:
<1> animal immune
By 6 of health week age female BAl BIc/c mouse by after fundamental immunity and 5 booster immunizations, impacts is carried out to the strongest the highest, competitive mouse of tiring immune;
<2> Fusion of Cells and colony screening
Impact immunity after 3 days, put to death after extracing eyeball of mouse bloodletting, collect blood, asepticly get spleen, cell-fusion techniques is utilized mouse immune splenocyte and Sp2/0 cell to be merged, cell suspension after fusion adds to and is covered with in 96 orifice plates of feeder cells in advance, adopts HAT Selective agar medium screening fused cell;
When Growth of Cells is to culture hole area 1/10, aseptic accurate absorption 100 μ L supernatant, adds in the ELISA Plate of envelope antigen; Survey cells and supernatant with indirect elisa method to tire, limiting dilution assay is adopted to clone 3 times continuously to positive cell, until when positive rate is 100%, cell line is carried out that expansion is cultivated, qualification, frozen and build strain, carry out Continuous Cultivation to cell line after qualification to go down to posterity more than 2 months, detect its stability and antibody-secreting ability every 5 generations;
<3> cell cryopreservation and recovery
With cryopreserving liquid, the hybridoma being in exponential phase is made cell suspension, be sub-packed in cryopreservation tube, be placed in liquid nitrogen and preserve; Take out cryopreservation tube during recovery, put into 37 DEG C of water-bath middling speeds immediately and melt, centrifugal segregation cryopreserving liquid, move in cell bottle and cultivate;
The preparation and purification of <4> ascites
Adopt in body and induce method, sterilizing paraffin oil is injected Balb/c mouse peritoneal in 8 week age, injects hybridoma after 7-14 days, after 7-10 days, collect ascites; The ascites of collecting, after saturated ammonium sulfate just pure and mild affinity chromatography, obtains sulfadimidine monoclonal antibody.
5. the application of kit in animal derived food in Sulfamethazine Residues detection according to claim 4.
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