CN103344758A - Chemiluminescence enzyme immunoassay detection method and kit of sulfamethazine - Google Patents
Chemiluminescence enzyme immunoassay detection method and kit of sulfamethazine Download PDFInfo
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- CN103344758A CN103344758A CN2013102777424A CN201310277742A CN103344758A CN 103344758 A CN103344758 A CN 103344758A CN 2013102777424 A CN2013102777424 A CN 2013102777424A CN 201310277742 A CN201310277742 A CN 201310277742A CN 103344758 A CN103344758 A CN 103344758A
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Abstract
The invention discloses a chemiluminescence enzyme immunoassay detection method and a kit of sulfamethazine. The method combines indirect enzyme immunoassay with a chemiluminescence technology and is used for detecting residual sulfamethazine in animal derived food, and the method is specific, rapid, sensitive and accurate. The IC50 of the kit is 4.006mug/L, the recovery rate is 94.42-102.73%, the agreement rate of the kit with the existing detection method is 100%, and meanwhile, the kit is wide in linear detection range, low in lowest detectable limit, suitable for trace analysis and batch detection of sulfamethazine and broad in application prospect.
Description
Technical field
The invention belongs to the chemiluminescence enzyme immunoassay technical field, relate in particular to a kind of sulfadimidine chemiluminescence enzyme immunoassay detection method and kit.
Background technology
Sulfadimidine (Sulfadimidine, SM
2) claim sulfamethazine, arnosulfan pyrimidine again, it is one of sulfa drugs most widely used in the livestock breeding industry, the medicine of doing feed addictive and bacteriosis commonly used, interpolation for a long time or abuse can cause the medicament residue in the animal food, and human health is threatened.Discover that human body is taken in and can be accumulated in vivo by any approach, influences the immune system of body, destroy tissues such as muscle, kidney, thyroid gland, can bring out thyroid cancer simultaneously; And, this medicine also can by placenta and milk from parent to fetus or the neonate shift, the thyroxine level in its blood plasma and the brain tissue is reduced, this brain growth to the human and animal has profound influence.On the other hand, the residual drug-resistance of bacteria of inducing easily of sulfadimidine makes medicine lose the value for the treatment of disease.Therefore, regulations such as Codex Committee on Food of the United Nations and European Union, the single 100 μ g/kg that all must not surpass of sulfa drugs in food and the feed; The maximum residue limit(MRL) of Japan's regulation is 10 μ g/kg; In Dec, 2002, China Ministry of Agriculture announced files specify No. 235, and in muscle, fat, liver and the kidney of all food animals, the sulfamido maximum residue limit(MRL) is 100 μ g/kg, and classified it as residue of veterinary drug monitoring emphasis.
At present, the method for detection 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, is fit to basic unit's field screening, but is subject to other biotic influence, and sensitivity is low, loss is high; Physico-chemical method precision height, accuracy height, but the instrument costliness, strongly professional, detection time is long, can be used for the conclusive evidence of doubtful sample; Immunoassay is easy and simple to handle, quick, special, highly sensitive, has been applied to the detection of multiple medicament residue in recent years, but the residual chemiluminescence enzyme immunoassay method of related detection sulfadimidine is not appeared in the newspapers as yet.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of economy, easy, quick, special, sensitive, sulfadimidine chemiluminescence enzyme immunoassay detection method and kit and the application in the sulfadimidine residue detection in animal derived food of this kit accurately.
Another technical matters that the present invention will solve provides 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 may further comprise the steps:
＜1〉handles testing sample;
＜2〉with the conjugate (SM of haptens sulfadimidine and carrier protein ovalbumin
2-OVA) as antigen coated in luminous solid phase carrier, after the sealing, add the series standard sample respectively or through the testing sample of pre-treatment, add the sulfadimidine monoclonal antibody again and react;
＜3〉through step＜1〉after, add ELIAS secondary antibody and react, and then add chemical luminescence for liquid, measure the luminous value of series standard sample and testing sample;
＜4〉with step＜3〉luminous value that records calculates inhibiting rate, the drawing standard curve, and calculate the content of sulfadimidine in the testing sample according to the inhibiting rate of the regression equation of typical curve and testing sample.
Step＜1〉carry out with reference to No. 1025 bulletin-24-2008 standards of the Ministry of Agriculture; Step＜2〉undertaken by following operation: press 1:100000 dilution envelope antigen with coating buffer, every hole adds 100 μ L, 4 ℃ hatch 10h after, the coating buffer that inclines is machine-washed and is washed the chemiluminescence plate 3 times with washing plate, pats dry; Then, every hole adds 350 μ L confining liquids, hatches 1h for 37 ℃, and the deblocking liquid that inclines is washed 3 times with washing the plate machine washing, pats dry; After 37 ℃ of oven dry, use masking foil vacuum seal, 4 ℃ of preservations; The chemiluminescence plate is taken out from 4 ℃, and balance adds chemiluminescence plate according to every hole 50 μ L with series standard sample solution and testing sample to room temperature, respectively establishing 3 holes repeats, every hole adds 50 μ L sulfadimidine monoclonal antibodies then, and mixing is hatched 30min for 37 ℃; The liquid that inclines is washed 3 times with washing the plate machine washing, pats dry; Step＜3〉to be undertaken by following operation: every hole adds the HRP-goat anti-mouse IgG of 50 μ L1:2500 dilution, hatches 45min for 37 ℃; The liquid that inclines is washed 5 times with washing the plate machine washing, pats dry; Add 100 μ L chemical luminescence for liquid in every hole, mixing is measured each hole luminous value as early as possible.
Confining liquid is 5% skimmed milk power; 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 being coated with envelope antigen (SM
2-chemiluminescence plate OVA), 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 respectively 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; Concentrated cleaning solution is by NaCl8.00g, KH
2PO
40.20g, Na
2HPO
412H
2O2.90g, KCl0.20g, Tween-20 0.50mL add water and are settled to 100mL and make; Chemical luminescence for liquid is from super quick ECL chemical luminescence reagent kit P0018--BeyoECL Plus.
The mentioned reagent box is the application in the sulfadimidine residue detection in animal derived food.
The sulfadimidine monoclonal antibody prepares by following operation: with the conjugate (SM of haptens sulfadimidine and carrier protein bovine serum albumin(BSA)
2-BSA) as immunogene, immunity female BALB/c mouse in 6 age in week, carry out the screening of Fusion of Cells and positive strain according to conventional method, the positive cell that filters out is involved in a criminal case continuous clone 3 times, through evaluation, frozen and build strain after, carry out the preparation of ascites, then by purifying ascites, obtain the monoclonal antibody of energy specific recognition sulfadimidine.
Above-mentioned sulfadimidine monoclonal antibody prepares by following operation:
＜1〉animal immune
With health 6 ages in week female BALB/c mouse by fundamental immunity and 5 booster immunizations after, the strongest the highest to tiring, competitive mouse is impacted immunity;
＜2〉Fusion of Cells and colony screening
Impact immunity after 3 days, put to death after extracing the eyeball of mouse bloodletting, collect blood, the aseptic spleen of getting, utilize cell-fusion techniques that mouse immune splenocyte and Sp2/0 cell are merged, cell suspension after the fusion adds in 96 orifice plates that are covered with feeder cells in advance, adopts HAT to select nutrient culture media screening fused cell;
When treating that cell grows to culture hole area 1/10, aseptic accurate absorption 100 μ L supernatants add to envelope antigen SM
2In the ELISA Plate of-OVA; Surveying cells and supernatant with indirect elisa method tires, adopt limiting dilution assay that positive cell is cloned 3 times continuously, it is 100% o'clock until positive rate, cell line is enlarged cultivation, evaluation, frozen and build strain, to identifying that the back cell line carries out Continuous Cultivation and goes down to posterity more than 2 months, detect its stability and antibody-secreting ability every 5 generations;
＜3〉cell cryopreservation and recovery
The hybridoma that will be in exponential phase with cryopreserving liquid is made cell suspension, is sub-packed in frozen pipe, places liquid nitrogen to preserve; Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, centrifugal removal cryopreserving liquid moves in the cell bottle and cultivates;
＜4〉preparation of ascites and purifying
Adopt in the body and induce method, the paraffin oil of will sterilizing injection Balb/c mouse peritoneal in 8 age in week injected hybridoma after 7-14 days, collected ascites after 7-10 days; The ascites of collecting namely gets the sulfadimidine monoclonal antibody after the first pure and mild affinity chromatography of saturated ammonium sulfate.
The present invention has inquired into the chemiluminescence enzyme immune mechanism that detects sulfadimidine first, the inventor combines indirect enzyme immune reaction and chemiluminescence, sulfadimidine chemiluminescence enzyme immunoassay detection method and kit have successfully been set up, this method is applied in the animal derived food in the sulfadimidine residue detection, have special, quick, sensitive, characteristic of accurate, the IC of kit
50Be 4.006 μ g/L, the recovery is 94.42～102.73%, with the identical rate of existing detection method be 100%, be suitable for trace analysis and the batch detection of sulfadimidine.(0.1～1000 μ g/L) is wideer than ELISA for linear detection range of the present invention, when detecting the sample of sulfadimidine severe overweight (content is lower than 1000 μ g/L above the ELISA sensing range), need not sample is further diluted and duplicate detection, reduced workload, saved cost and the time detected; Simultaneously, lowest detectable limit of the present invention (0.17 μ g/L) has reduced by 10 times than ELISA, meets trace analysis and batch detection more, has application promise in clinical practice.
Description of drawings
Fig. 1 is the typical curve of embodiment 2 sulfadimidine chemiluminescence enzyme immunoassay detection methods.
Among the figure: X-axis is the logarithm value of sulfadimidine standard model concentration, and 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
1.1 animal immune
Conjugate (SM with haptens sulfadimidine and carrier protein bovine serum albumin(BSA)
2-BSA) as immunogene, with 10 health 6 the week ages female BALB/c mouse be divided into 2 groups at random, wherein 1 group is control group; Behind fundamental immunity and 5 booster immunizations, serum titer is measured in blood sampling, and the strongest the highest to tiring, competitive mouse is impacted immunity;
1.2 Fusion of Cells and colony screening
Impact immunity after 3 days, put to death after extracing the eyeball of mouse bloodletting, collect blood, the aseptic spleen of getting, utilize cell-fusion techniques that No. 5 mouse immune splenocytes and Sp2/0 cell are merged (fusion rate is 71.4%), cell suspension after the fusion adds in 96 orifice plates that are covered with feeder cells in advance, adopts HAT to select nutrient culture media screening fused cell;
When treating that cell grows to culture hole area 1/10, aseptic accurate absorption 100 μ L supernatants add to envelope antigen SM
2In the ELISA Plate of-OVA; Surveying cells and supernatant with indirect elisa method tires, adopt limiting dilution assay that positive cell is cloned 3 times continuously, it is 100% o'clock until positive rate, cell line is enlarged cultivation, evaluation, frozen and build strain, to identifying that the back cell line carries out Continuous Cultivation and goes down to posterity more than 2 months, detect its stability and antibody-secreting ability every 5 generations;
1.3 cell cryopreservation and recovery
The hybridoma that will be in exponential phase with cryopreserving liquid is made cell suspension, is sub-packed in frozen pipe, places liquid nitrogen to preserve; Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, centrifugal removal cryopreserving liquid moves in the cell bottle and cultivates;
1.4 the preparation of ascites and purifying
Adopt in the body and induce method, the paraffin oil of will sterilizing injection Balb/c mouse peritoneal in 8 age in week injected hybridoma after 7-14 days, collected ascites after 7-10 days; The ascites of collecting identifies through the SDS-PAGE electrophoresis that after the first pure and mild affinity chromatography of saturated ammonium sulfate it is pure to confirm that the sulfadimidine monoclonal antibody 1E7 that obtains reaches electrophoresis.
1.5 the evaluation of monoclonal antibody
1.5.1 the mensuration of protein content
The protein content of 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 for ordinate, with SM
2The concentration of-McAb is horizontal ordinate, the drawing standard curve, and with the affinity costant that formula 1 calculates under the different antigen coated concentration, the affinity costant (Ka) that draws the sulfadimidine monoclonal antibody of averaging is 0.12 * 10
7L/mol.
1.5.3 the evaluation of subclass
Mouse monoclonal antibody Ig class/subgroup identification according to one hundred logical experiment material center difficult to understand, Luoyang is IgG2b with ELISA kit measurement 1E7 subclass.
The foundation of the chemiluminescence enzyme immunoassay detection method that embodiment 2 sulfadimidines are residual
2.1 the preparation of related reagent
Carbonate buffer solution (pH9.6, i.e. coating buffer): accurately take by weighing Na
2CO
31.59g, NaHCO
32.93g, after the ultrapure water dissolving, regulate pH to 9.6, be settled to 1000mL.
Cleansing solution (pH7.4): accurately take by weighing NaCl8.00g, KH
2PO
40.20g, Na
2HPO
412H
2O2.90g, KCl0.20g after the ultrapure water dissolving, regulate pH to 7.4, add Tween-20 0.50mL, are settled to 1000mL.
Phosphate buffer (PBS) is (pH7.4): accurately take by weighing NaCl8.00g, KH
2PO
40.20g, Na
2HPO
412H
2O2.90g, KCl0.20g after the ultrapure water dissolving, regulate pH to 7.4, are settled to 1000mL.
Confining liquid: accurately take by weighing skimmed milk power 1.0g, add the 20mL phosphate buffer, stir to dissolving fully.
The super quick ECL chemical luminescence reagent kit of BeyoECL Plus(, P0018,100mL): available from green skies biotechnology research institute.
2.2 determining of antigen coated condition
Use the conjugate (SM of haptens sulfadimidine and carrier protein ovalbumin
2-OVA) as envelope antigen, this antigen is wrapped quilt respectively after by 1:25000,1:50000,1:75000,1:100000 and 1:125000 dilution under 37 ℃ of 10h, 25 ℃ of 10h, 4 ℃ of 10h and 37 ℃ of 3h, 25 ℃ of 3h and 4 ℃ of 3h conditions, select reaction normal curve IC
50The condition of (drug concentrations during 50% inhibiting rate, i.e. Fan Ying sensitivity) minimum is as the antigen coated condition of the best.Determine that by table 1 result envelope antigen dilutes by 1:100000, hatch 10h for best at 4 ℃.
The optimization result of the antigen coated condition of table 1
2.3 determining of sealing condition
By the chemiluminescence plate, as closed material, select reaction normal curve IC with the PBS of 20mM, 5% skimmed milk power, 1% BSA, 5% hyclone with the antigen coated condition bag of the best
50Minimum, as reaction sealed liquid; Then, the skimmed milk power with 5% is as confining liquid, and setting off-period is 37 ℃ of 45min, 37 ℃ of 1h, 37 ℃ of 1.5h, 37 ℃ of 2h, calculates the IC under the different condition
50, select reaction normal curve IC
50Minimum, as reaction sealed condition, determined that by table 2 result 37 ℃ of 1h of skimmed milk power of 5% are best.
The optimization result of table 2 sealing condition
2.4 determining of antibody dilution multiple
Under the best bag quilt, sealing condition, the sulfadimidine monoclonal antibody is diluted according to 1:1000,1:1500,1:2000 and 1:2500 respectively, with reaction normal curve IC
50As judging index, determine reaction optimum antibody extension rate.Determine that by table 3 result 1:1500 is the antibody optimum diluting multiple.
2.5 determining of competitive reaction time
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 by table 3 result 30min is the best competitive reaction time.
The optimization result of table 3 antibody dilution multiple, competitive reaction time
2.6HRP-determining of goat anti-mouse IgG extension rate
Under above optimal conditions, respectively the 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.The result shows that when pressing the 1:1000 dilution, values of chemiluminescence is too high, and error is bigger; When pressing the 1:6000 dilution, values of chemiluminescence is too little, and the result is difficult for judging; And during 1:2000 and 1:3000 dilution, luminous numerical values recited is light-emitting appearance optimum detection scope.Take all factors into consideration, determine that 1:2500 is the best dilute concentration of HRP-goat anti-mouse IgG.
2.7 the foundation of typical curve, sensitivity and lowest detectable limit
Under optimal conditions, sulfadimidine is made into 0.1～1000 μ g/L series concentration, repeat in each concentration 3 hole, detects.Logarithm value with the standard model solution concentration is horizontal ordinate, is ordinate with the luminous value of each concentration sulfadimidine standard items divided by " 0 " concentration hole luminous value (RLU/RUL0%), the 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 specificity of the present invention
With analytical review method of the present invention some common microbiotic (sulphoamidine, sulphadiazine, sulfanilamide (SN) five first pyrimidines, penicillin, Ofloxacin, chloromycetin, diethylstilbestrol) are detected, measure IC
50, according to formula 2, calculate the cross reacting rate of each medicine.By table 4 as seen, the method that the present invention sets up and common antibiotic cross reacting rate show that all less than 0.01% 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
Carry out recovery experiment with basic, normal, high three concentration (1 μ g/L, 10 μ g/L, 100 μ g/L), each sample is set 3 holes and is repeated, and measures its values of chemiluminescence, with the regression equation of the mean value substitution typical curve that obtains, obtain respective standard sample concentration value, calculate recovery rate.Determine the recovery of the present invention 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 the residual chemiluminescence enzyme immunoassay detection kit of embodiment 3 sulfadimidines
According to the result of study of embodiment 1 and 2, the residual chemiluminescence enzyme immunoassay detection kit of assembling sulfadimidine.
3.1 kit is formed
A is coated with envelope antigen (SM
2-OVA) chemiluminescence 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;
D HRP-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 are settled to 100mL;
F chemical luminescence for liquid A liquid, B liquid (super quick ECL chemical luminescence reagent kit P0018--BeyoECL Plus);
3.2 the bag quilt of chemiluminescence plate
Press 1:100000 dilution envelope antigen with coating buffer, every hole adds 100 μ L, 4 ℃ hatch 10h after, the coating buffer that inclines is machine-washed and is washed the chemiluminescence plate 3 times with washing plate, pats dry; Every hole adds 350 μ L confining liquids then, hatches 1h for 37 ℃, and the deblocking liquid that inclines is washed 3 times with washing the plate machine washing, pats dry; After 37 ℃ of oven dry, use masking foil vacuum seal, 4 ℃ of preservations.
The application of the residual chemiluminescence enzyme immunoassay detection kit of embodiment 4 sulfadimidines
4.1 the preparation of reagent
A cleansing solution: the concentrated cleaning solution that provides in the kit is used with after 10 times of the ultrapure water dilutions.
B chemical luminescence for liquid: before adding chemical luminescence for liquid, chemical luminescence for liquid A liquid, B liquid are mixed according to 1:1.
4.2 the pre-treatment of tissue sample
Carry out with reference to No. 1025 bulletin-24-2008 standards of the Ministry of Agriculture:
A gets 3g animal muscle tissue and grinds in the mortar to pasty state and forward in the 50mL centrifuge tube;
B adds 84% the fine 9mL mixing of second concussion 10min, the centrifugal 10min of 4000r/min;
C gets supernatant 3mL and adds 2mol/L sodium chloride 2mL, ethyl acetate 7mL, concussion mixing 10min, the centrifugal 5min of 4000r/min;
D gets upper strata liquid and dries up in nitrogen, adds phosphate buffer 1 mL, the normal hexane 1mL dissolving of 0.02mol/L, concussion mixing 3min, in the centrifugal 5min of 4000r/min, take off layer liquid for detection of.
4.3 testing process
A takes out the chemiluminescence plate from 4 ℃, balance is to room temperature;
B adds the chemiluminescence plate according to every hole 50 μ L with series standard sample solution and testing sample, respectively establishes 3 holes and repeats, and every hole adds 50 μ L sulfadimidine monoclonal antibodies then, and mixing is hatched 30min for 37 ℃;
The C liquid that inclines is washed 3 times with washing the plate machine washing, pats dry;
The every hole of D adds the HRP-goat anti-mouse IgG of 50 μ L1:2500 dilution, hatches 45min for 37 ℃;
The E liquid that inclines is washed 5 times with washing the plate machine washing, pats dry; Add 100 μ L chemical luminescence for liquid in every hole, mixing is measured each hole luminous value as early as possible;
F is according to formula, according to acquisition sample luminous value, and the inhibiting rate of calculation sample, and with inhibiting rate substitution standard regression equation, calculate residual quantity; Once diluted as sample, then multiply by extension rate, be the residual quantity of sulfadimidine in the sample.
4.4 the detection of sample
Gather totally 141 parts in commercially available shrimp, fish and pork sample, carry out the pre-treatment of sample with reference to above-mentioned bulletin standard, detect with kit of the present invention then, stipulate according to the Ministry of Agriculture, when residual quantity surpass 100 μ g/kg be judged to defective, wherein have 129 parts qualified, the sample qualification rate is 91.49%.
The comparison of embodiment 5 chemiluminescence Enzymoimmune reagent kits of the present invention and commercially available ELISA kit
Select 36 parts of tissue samples, detect the wherein residual quantity of sulfadimidine simultaneously with kit of the present invention and commercially available ELISA kit, the results are shown in Table 6.The yin and yang attribute coincidence rate of the two is 100%, testing result to two kinds of methods is carried out statistical study, checks t=0.410 as can be known through t, P=0.648＞0.05, difference is not remarkable between two kinds of method testing results, illustrates that kit of the present invention has effect preferably.
In addition, sensing range of the present invention is wideer, lowest detectable limit has reduced by 10 times than commercially available ELISA kit, meets trace analysis and batch detection more, and application prospect is good.
Table 6 kit result relatively
| Sample number into spectrum | Commercially available ELISA kit testing result (μ g/kg) | This kit testing result (μ g/kg) |
| |
317.3386221 | 248.0751806 |
| |
335.6700716 | 266.10262 |
| |
1.001767113 | 2.309985198 |
| Shrimp 17 | 1.523619251 | 2.3584488 |
| Shrimp 19 | 1.301923065 | 2.961149759 |
| |
1.451238264 | 2.598121808 |
| |
335.6700716 | 378.3196916 |
| |
364.4887814 | 334.2108086 |
| Fish 11 | 2.194873187 | 1.890214447 |
| Fish 12 | 1.673115544 | 1.809148814 |
| Fish 14 | 2.900981043 | 3.53177296 |
| |
2.055676615 | 3.188808856 |
| Fish 17 | 1.743458869 | 1.218988793 |
| Fish 18 | 1.413698722 | 1.80391665 |
| |
1.150609629 | 2.922276658 |
| |
158.1578608 | 172.2441963 |
| |
410.8804742 | 445.0008146 |
| |
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 |
| |
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 (7)
1. sulfadimidine chemiluminescence enzyme immunoassay detection method is characterized in that may further comprise the steps:
＜1〉handles testing sample;
＜2〉with the conjugate of haptens sulfadimidine and carrier protein ovalbumin as antigen coated in luminous solid phase carrier, after the sealing, add the series standard sample respectively or through the testing sample of pre-treatment, add the sulfadimidine monoclonal antibody again and react;
＜3〉through step＜1〉after, add ELIAS secondary antibody and react, and then add chemical luminescence for liquid, measure the luminous value of series standard sample and testing sample;
＜4〉with step＜3〉luminous value that records calculates inhibiting rate, the drawing standard curve, and calculate the content of sulfadimidine in the testing sample according to the inhibiting rate of the regression equation of typical curve and testing sample.
2. sulfadimidine chemiluminescence enzyme immunoassay detection method according to claim 1 is characterized in that:
Step＜1〉carry out with reference to No. 1025 bulletin-24-2008 standards of the Ministry of Agriculture;
Step＜2〉undertaken by following operation: press 1:100000 dilution envelope antigen with coating buffer, every hole adds 100 μ L, 4 ℃ hatch 10h after, the coating buffer that inclines is machine-washed and is washed the chemiluminescence plate 3 times with washing plate, pats dry; Then, every hole adds 350 μ L confining liquids, hatches 1h for 37 ℃, and the deblocking liquid that inclines is washed 3 times with washing the plate machine washing, pats dry; After 37 ℃ of oven dry, use masking foil vacuum seal, 4 ℃ of preservations; The chemiluminescence plate is taken out from 4 ℃, and balance adds chemiluminescence plate according to every hole 50 μ L with series standard sample solution and testing sample to room temperature, respectively establishing 3 holes repeats, every hole adds 50 μ L sulfadimidine monoclonal antibodies then, and mixing is hatched 30min for 37 ℃; The liquid that inclines is washed 3 times with washing the plate machine washing, pats dry;
Step＜3〉to be undertaken by following operation: every hole adds the HRP-goat anti-mouse IgG of 50 μ L1:2500 dilution, hatches 45min for 37 ℃; The liquid that inclines is washed 5 times with washing the plate machine washing, pats dry; Add 100 μ L chemical luminescence for liquid in every hole, mixing is measured each hole luminous value as early as possible.
3. sulfadimidine chemiluminescence enzyme immunoassay detection method according to claim 2, it is characterized in that: described confining liquid is 5% skimmed milk power; 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.
4. a sulfadimidine chemiluminescence enzyme immunoassay detection kit is characterized in that comprising the chemiluminescence 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 ovalbumin; Described sulfadimidine series standard sample solution is 6 concentration gradients, is respectively 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; Described concentrated cleaning solution is by NaCl8.00g, KH
2PO
40.20g, Na
2HPO
412H
2O2.90g, KCl0.20g, Tween-20 0.50mL add water and are settled to 100mL and make; Described chemical luminescence for liquid is from super quick ECL chemical luminescence reagent kit P0018--BeyoECL Plus.
5. according to the application in the sulfadimidine residue detection in animal derived food of the described kit of claim 4.
6. sulfadimidine monoclonal antibody, it is characterized in that by following operation preparation: with the conjugate of haptens sulfadimidine and carrier protein bovine serum albumin(BSA) as immunogene, immunity female BALB/c mouse in 6 age in week, carry out the screening of Fusion of Cells and positive strain according to conventional method, the positive cell that filters out is involved in a criminal case continuous clone 3 times, through evaluation, frozen and build strain after, carry out the preparation of ascites, by purifying ascites, obtain the monoclonal antibody of energy specific recognition sulfadimidine then.
7. sulfadimidine monoclonal antibody according to claim 6 is characterized in that by following operation preparation:
＜1〉animal immune
With health 6 ages in week female BALB/c mouse by fundamental immunity and 5 booster immunizations after, the strongest the highest to tiring, competitive mouse is impacted immunity;
＜2〉Fusion of Cells and colony screening
Impact immunity after 3 days, put to death after extracing the eyeball of mouse bloodletting, collect blood, the aseptic spleen of getting, utilize cell-fusion techniques that mouse immune splenocyte and Sp2/0 cell are merged, cell suspension after the fusion adds in 96 orifice plates that are covered with feeder cells in advance, adopts HAT to select nutrient culture media screening fused cell;
When treating that cell grows to culture hole area 1/10, aseptic accurate absorption 100 μ L supernatants add in the ELISA Plate of envelope antigen; Surveying cells and supernatant with indirect elisa method tires, adopt limiting dilution assay that positive cell is cloned 3 times continuously, it is 100% o'clock until positive rate, cell line is enlarged cultivation, evaluation, frozen and build strain, to identifying that the back cell line carries out Continuous Cultivation and goes down to posterity more than 2 months, detect its stability and antibody-secreting ability every 5 generations;
＜3〉cell cryopreservation and recovery
The hybridoma that will be in exponential phase with cryopreserving liquid is made cell suspension, is sub-packed in frozen pipe, places liquid nitrogen to preserve; Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, centrifugal removal cryopreserving liquid moves in the cell bottle and cultivates;
＜4〉preparation of ascites and purifying
Adopt in the body and induce method, the paraffin oil of will sterilizing injection Balb/c mouse peritoneal in 8 age in week injected hybridoma after 7-14 days, collected ascites after 7-10 days; The ascites of collecting namely gets the sulfadimidine monoclonal antibody after the first pure and mild affinity chromatography of saturated ammonium sulfate.
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