CN105372419A - Preparing method for chloramphenicol hapten and antigen and application of chloramphenicol hapten and antigen in quantum dot immunofluorescence reagent kit - Google Patents
Preparing method for chloramphenicol hapten and antigen and application of chloramphenicol hapten and antigen in quantum dot immunofluorescence reagent kit Download PDFInfo
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Abstract
The invention discloses chloramphenicol hapten, corresponding artificial antigen and a monoclonal antibody marked by a quantum dot and further discloses a preparing method and application of the chloramphenicol hapten, the corresponding artificial antigen and the monoclonal antibody marked by the quantum dot. The chloramphenicol antigen can be obtained when the chloramphenicol hapten and carrier protein are connected. The chloramphenicol antigen can be applied to preparing a chloramphenicol specific antibody. The preparing method is easy and convenient to implement and low in cost, and the yield of the hapten is high. The artificial antigen and the chloramphenicol specific antibody marked by the quantum dot can be used for preparing a quantum dot immunofluorescence reagent kit for detecting chloramphenicol residues and have the advantages of being simple, fast, large in quantity of treated samples, high in sensitivity, high in specificity and the like.
Description
Technical field
The present invention relates to a kind of haptens, antigen and preparation method thereof, be specifically related to the preparation method of a kind of chloromycetin haptens and antigen and the application in quantum dot immune fluorescent kit thereof.
Background technology
Chloromycetin (Chloramphenicol, CAP) is that one has good inhibiting Cheap highly effective broad-spectrum antibiotic to Gram-positive and negative bacteria, is once being widely used in farming and animal husbandry.But chloromycetin residual in animal derived food is taken in by human body for a long time along with food chain, can cause various diseases.The lighter destroys the equilibrium state of normal flora in human body, flora imbalance, makes human body produce drug-fast bacteria pearl, brings harmful effect to ill use antibiotic therapy from now on; The people of microbiotic allergic constitution there will be allergic reaction, jeopardizes health.The synthesis of bone marrow cell protein can be disturbed time serious, and suppress juvenile cell DNA to synthesize, cause granulocyte to reduce, cause the malignant diseases such as alpastic anemia, haemolysis, purpura.
In view of the toxic and side effect of chloromycetin, international food educational circles is classified as banning drugs, European Union, the U.S. etc. all specify in regulation that residual chloromycetin limit standard is for " zero tolerance " (Zerotolerance), namely must not detect, according to European Union " 2002/657/EC " standard regulation, in animal derived food, the maximum of chloromycetin requires that detection limit is 0.3 μ g/kg.Soon U.S. FDA also makes corresponding regulation.The Ministry of Agriculture of China defines CAP and must not detect in the edible tissues of all food animals, and it is deleted from " Chinese veterinary pharmacopoeia ", is classified as banning drugs.And correspondingly formulated SN0219-93 and SCT3018-2004 industry standard, in line with international standards.
The detection method of chloromycetin is divided into physical-chemical process and immuno-chemical method, the former has liquid phase chromatography (LC), high performance liquid chromatography (HPLC), mass spectroscopy (MS) etc., the latter mainly enzyme exempts from method (ELISA), and the sensitivity of detection all reaches ppb (μ g/kg) rank.In recent years, Chinese scholars develops again the physical detection new methods such as liquid chromatography tandem mass spectrometry (LC-MS/MS), liquid chromatography electrospray ionization mass spectrometry (LC-EITMS), microbiological analysis, there has also been further research to the application of ELISA method.
Quantum dot immune fluorescent detection method have high specificity, stable fast, high, the stable reagent of wide, the simple to operate automaticity of sensing range and the advantages such as the term of validity long (6 ~ 18 months), its detectability than ELISA and Physico-chemical tests method high.
Summary of the invention
The object of this invention is to provide a kind of chloromycetin haptens, the preparation method of antigen and the application in quantum dot immune fluorescent kit thereof.
Chloromycetin haptens provided by the invention is the compound shown in formula I;
The invention also discloses the preparation method of compound shown in formula I, comprise the steps:
1. chloromycetin bulk drug 323mg is dropped in 10ml single port bottle, 5ml pyridinium dissolution;
2. ice bath is cooled to 0-5 degree, adds carboxylphenylsulphonyl chloride 230mg in batches, is warming up to outdoor after 1 hour, stirring reaction 18 hours;
3. TLC detects, and bulk drug chloromycetin reacts completely aftertreatment, is poured into by reactant liquor in 25ml frozen water, separates out white precipitate, filters, washing filter cake, dry that chloromycetin haptens 450mg, mass detect.
Chloromycetin antigen provided by the invention is conjugate compound shown in formula I and carrier protein couplet obtained.
Common carrier albumen all can adopt, as bovine serum albumin(BSA) (BSA), ovalbumin (OVA), human serum albumins (HSA), mouse serum albumin (MSA), thyroprotein (TG) or hemocyanin (KLH) etc.
Shown in described formula I, the structure of the chloromycetin antigen that compound and BSA coupling obtain is shown in Fig. 1.
The invention also discloses method prepared by described chloromycetin antigen, comprise the steps:
1. BSA50mg is taken, be dissolved in 3.5mlCB liquid, take haptens 50/67000*60*507=22.7mg and be dissolved in 1.5mlDMF, add NHS50/67000*120*115.09=10.3mg, EDC50/67000*120*191.7=17.2mg, room temperature activates 2 hours;
2. be added drop-wise to by activating solution under ice bath in the CB solution of BSA, 4 degree of reactions are spent the night, and PBS dialyses.
Described chloromycetin antigen can prepare chloromycetin specific antibody as immunogene, also can prepare quantum dot immune fluorescent microwell plate as coating antigen.
The specific antibody that application chloromycetin antigen prepares specifically can be monoclonal antibody or polyclonal antibody.
Described chloromycetin antigen, described specific antibody all can be applicable to chlorine detection mycin.
The invention also discloses application chloromycetin antigen and the quantum dot immune fluorescent enzyme linked immunological kit for preparing of chloromycetin specific antibody, comprise quantum dot-labeled described antibody.
The preparation method of " quantum dot-labeled described antibody " is specific as follows:
1. get 2.5mg quantum dot, with the MES buffer solution of pH4.7,0.1M, then use the MES damping fluid of 1mlpH4.7,0.1M resuspended, add 0.96mgEDC and 1.15mgNHS, 37 DEG C are reacted 30 minutes, the centrifugal 5min of 20000rpm, collecting precipitation, is the quantum dot after activation;
2. the quantum dot after the activation that 1. step obtain is got, wash with the borate buffer solution of pH8.5,50mM, then by borate buffer solution mixing (cumulative volume is 0.8ml) of quantum dot, described antibody (protein content is 0.15mg) and pH8.5,50mM after 2.5mg activation, 25 DEG C are reacted 3.5 hours;
3. get completing steps liquid phase 2., add BSA and make its mass percentage be 5%, 37 DEG C of reactions 30 minutes, the centrifugal 5min of 20000rpm, collecting precipitation, with the PBS buffer solution of pH7.4,0.02M, resuspended with the PBS damping fluid of 1mlpH7.4,0.02M;
4. get the liquid phase that 3. step obtains, be diluted to 64000 times of volumes with the PBS damping fluid of pH7.4,0.02M.
Whether the present invention also protects above arbitrary described kit detecting in sample to be tested containing the application in chloromycetin.
The present invention relies on immunology, immunochemistry ultimate principle and retention analysis technological means, design, synthesized micromolecule target analytes haptens, and and carrier protein couplet, prepare effective artificial antigen, immune animal preparation is for the specific antibody of small molecule analysis thing.The specificity immunology of antigen-antibody is utilized to react, micro-Small molecular target analytes in quantitative detection sample.Preparation method of the present invention is simple and feasible, cost is lower, and yield of hapten is higher.Instant invention overcomes complicated to chloromycetin sample pretreatment in existing detection technique, consuming time and need a large amount of organic solvent extraction, and accurate expensive detecting instrument will be used and be unsuitable for shortcomings such as promoting the use of in testing process.Chloromycetin antigen of the present invention, can create the specific antibody for chloromycetin by immune animal, for detecting the residual chloromycetin in food fast, has simple to operate, quick, and processing sample amount is large, highly sensitive, the plurality of advantages such as high specificity.
Accompanying drawing explanation
Fig. 1 is the structural representation of chloromycetin antigen.
Fig. 2 is chloromycetin quantum dot immune fluorescent detection kit canonical plotting.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.
The haptenic preparation of embodiment 1, chloromycetin
The haptenic preparation method of chloromycetin, concrete operation step comprises:
Drop into chloromycetin bulk drug 323mg in 10ml single port bottle, 5ml pyridinium dissolution, ice bath is cooled to 0-5 degree, add carboxylphenylsulphonyl chloride 230mg in batches, after 1 hour, be warming up to outdoor, stirring reaction 18 hours, TLC detects, bulk drug chloromycetin reacts completely aftertreatment, is poured into by reactant liquor in 25ml frozen water, separates out white precipitate, filter, washing filter cake, dry that haptens 450mg, mass detect.
Haptenic structural formula is shown in formula I:
The preparation of embodiment 2, chloromycetin artificial antigen
One, the synthesis of chloromycetin immunizing antigen (CAP-BSA)
1. BSA50mg is taken, be dissolved in 3.5mlCB liquid, take haptens 50/67000*60*507=22.7mg and be dissolved in 1.5mlDMF, add NHS50/67000*120*115.09=10.3mg, EDC50/67000*120*191.7=17.2mg, room temperature activates 2 hours;
2. be added drop-wise to by activating solution under ice bath in the CB solution of BSA, 4 DEG C of reactions are spent the night, and PBS dialyses.
Immunogenic structural formula is as follows.
Two, the synthesis of chloromycetin envelope antigen (CAP-OVA)
1. OVA33.5mg is taken, be dissolved in 3.5mlCB liquid, take haptens 33.5/45000*60*507=22.5mg and be dissolved in 1.5mlDMF, add NHS33.5/45000*120*115.09=10.3mg, EDC33.5/45000*120*191.7=17.17mg, room temperature activates 2 hours;
2. be added drop-wise to by activating solution under ice bath in the CB solution of OVA, 4 DEG C of reactions are spent the night, and PBS dialyses.
The structural formula of coating antigen is shown in formula II.
Prepared by embodiment 3, chloromycetin specific antibody
One, the preparation of chloramphenicol
Immunogene (CAP-BSA) solution prepared by Example 1, dilutes with the PBS damping fluid of pH7.4,0.01M, obtains immunogene dilution, for the preparation of polyclonal antibody.Adopt new zealand white rabbit as immune animal.
Immunologic process is as follows:
First immunisation: immunogene dilution and isopyknic Freund's complete adjuvant are mixed and made into emulsifying agent, neck dorsal sc multi-point injection, immunizing dose is 2.5mg/;
Booster immunization: after first immunisation 4 weeks, after 8 weeks and after 12 weeks, respectively carry out a booster immunization, by immunogene dilution and isopyknic incomplete Freund's adjuvant mixing and emulsifying, neck dorsal sc multi-point injection, single immunization dosage is 2.5mg/;
Final immunization: first immunisation carried out final immunization after 16 weeks, direct neck dorsal sc multi-point injection immunogene dilution, immunizing dose is 2.5mg/.
Final immunization is after 1 week, and blood sampling is separation of serum also, is the polyclonal antibody that immunogene is corresponding.
Two, the preparation of chloromycetin monoclonal antibody
1. immunogene (CAP-BSA) solution of Example 1 preparation, by 100 μ g/ only, mix with physiological saline solution immunogene and Freund's complete adjuvant equal-volume, the female mouse of neck dorsal sc injection immunity Balb/c in 6 ~ 8 week age, within after initial immunity the 7th, 14,28 day, mix with immunogene and incomplete Freund's adjuvant equal-volume, each supplementary immunization once, with immune complex 100 μ g/ only merges first 3 days, and supplementary immunization is once more not add Freund's adjuvant.
2. carry out according to a conventional method, the splenocyte getting immune mouse mixes with the murine myeloma cell being in exponential phase (SP2/0), then the fusion agent (PEG4000) slowly adding preheating in 45s merges, suspend evenly with HAT nutrient culture media, add appropriate feeder cells again, be incubated at 96 well culture plates, in 37 DEG C, 5%CO
2cultivate in incubator, within 5 days, partly change liquid with HT nutrient culture media afterwards, when 9 days, entirely change liquid.
3., after Fusion of Cells, when cell grows to 1/4 of culture hole area, a point step screening method screening hybridoma is adopted.Primary election adopts indirect ELISA method, with envelope antigen (wrapping by concentration and positive serum dilutability by its best of square formation method conventional titration in advance) package amount point immunofluorescence microwell plate, add measured hole culture supernatant, hatch, sheep anti-mouse igg-HRP is added and IgM-HRP, OPD carry out chromogenic reaction after cleaning.The positive Kong Zaiyong indirect competitive ELISA method screening filtered out, first mixes the chloromycetin equal-volume of cell conditioned medium with 100 μ g/mL, 37 DEG C of water-bath effect 30min, then joins bag by good quantum dot immune fluorescent microwell plate.Replace chloromycetin with PBS to compare, all the other steps are the same simultaneously.If the OD after chloromycetin blocks
450nm value drops to less than 50% of control wells, be then judged to the positive, and detecting through 2 ~ 3 times is all positive hole, carries out subcloning immediately with limiting dilution assay.
4. 2 ~ 3 subclones are built the hybridoma after strain and expand cultivation, collect supernatant indirect ELISA mensuration and tires, frozen; And get 8 ~ 10 week age Balb/c mouse peritoneal injecting fluid paraffin 0.5mL/, lumbar injection hybridoma 1 ~ 2 × 10 after 7 ~ 10 days
6/ only, extract mouse ascites after 7 ~ 10 days, centrifuging and taking supernatant, mensuration is tired, and frozen for subsequent use.
The preparation of three, quantum dot-labeled specific antibody
1. get 2.5mg quantum dot, with the MES buffer solution of pH4.7,0.1M, then use the MES damping fluid of 1mlpH4.7,0.1M resuspended, add 0.96mgEDC and 1.15mgNHS, 37 DEG C are reacted 30 minutes, the centrifugal 5min of 20000rpm, collecting precipitation, is the quantum dot after activation.
2. the quantum dot after the activation that 1. step obtain is got, wash with the borate buffer solution of pH8.5,50mM, then by borate buffer solution mixing (cumulative volume is 0.8ml) of quantum dot, monoclonal antibody (protein content is 0.15mg) and pH8.5,50mM after 2.5mg activation, 25 DEG C of reactions 3.5 hours (monoclonal antibody and quantum dot form stable covalent peptide bonds combination).
3. completing steps liquid phase is 2. got, add BSA and make its mass percentage be 5% (object closes residual activity amino sites), 37 DEG C are reacted 30 minutes, the centrifugal 5min of 20000rpm, collecting precipitation, with the PBS buffer solution of pH7.4,0.02M, resuspended with the PBS damping fluid of 1mlpH7.4,0.02M, 4 DEG C of preservations are stand-by.
4. get the liquid phase that 3. step obtains, be diluted to 64000 times of volumes with the PBS damping fluid of pH7.4,0.02M, obtain primary antibodie working fluid.
Four, the mensuration that quantum dot-labeled chloramphenicol antibody is tired
Chloromycetin standard items available from Sigma.
With the working concentration of the chloromycetin specific antibody of square formation titrimetry determination chloromycetin envelope antigen and preparation, the working concentration of chloromycetin envelope antigen is 2 μ g/L, the working concentration of quantum dot-labeled polyclonal antibody is 1:4000, and the working concentration of quantum dot-labeled monoclonal antibody is 1:64000.
Quantum dot immune fluorescent kit of embodiment 4, chlorine detection mycin and preparation method thereof
Quantum dot is purchased from TELUS Science and Technology Ltd. of Shenzhen, and catalog number is
lumiQDTM20.The sign of this quantum dot is as follows: particle diameter is 20nm, the CV of particle diameter is 15%, and quantum yield is 60%, and surface-bound carboxylic content is 5 × 10-3mmol/mg, water-soluble, CdSe/ZnS nucleocapsid structure, and excitation wavelength is 345nm, and emission wavelength is 620nm; Red fluorescence quantum dot.Carboxyl on quantum dot and the amino on protein form peptide bond and couple together.
Quantum dot immune fluorescent detection kit comprises following assembly:
One, bag is by the microwell plate of coating antigen
Coating antigen solution prepared by Example 2, is buffered liquid dilution (bag is buffered the carbonate buffer solution of liquid and pH9.6,0.05M) with bag, obtains the coating antigen dilution that protein concentration is 5ng/mL.The phosphate buffer of 10g bovine serum albumin(BSA), 0.1mLproclin300 and 1000mLpH7.4,0.01M is mixed, obtains confining liquid.
(1) coating antigen dilution is added microwell plate (100 μ L/ hole), hatch 16 hours for 37 DEG C.
(2), after completing steps (1), the liquid inclined in hole, washing, pats dry.
(3), after completing steps (2), every hole adds 200 μ L confining liquids, 37 DEG C of incubation 2h.
(4), after completing steps (3), the liquid inclined in hole, preserves with the vacuum seal of aluminium film after dry.
Two, quantum dot-labeled antibody working fluid
Quantum dot-labeled polyclonal antibody antibody diluent embodiment 3 prepared dilutes 4000 times or the monoclonal antibody antibody diluent embodiment 3 prepared dilutes 64000 times, obtains chloramphenicol antibody working fluid.
Antibody diluent: get BSA10mg, is settled to 1000mL with the PBS buffer solution of pH7.4,0.02M, obtains antibody diluent.Three, standard solution
Chloromycetin standard solution: phosphate buffer chloromycetin being dissolved in pH7.4,0.05M, obtains the standard solution that concentration is 0.02 μ g/L, 0.06 μ g/L, 0.18 μ g/L, 0.54 μ g/L and 1.62 μ g/L respectively.Using the negative control solution of the phosphate buffer of pH7.4,0.05M as standard solution, be called 0 solution.
Four, dilution
The PBS damping fluid of pH7.4,0.02M.
Five, concentrated cleaning solution: 10mL Tween-20,5g sodium azide and 990mL phosphate buffer are mixed, obtains described cleansing solution; The concentration of described phosphate buffer is 0.01MpH value is 7.4;
The quantum dot immune fluorescent kit using method of embodiment 5, chlorine detection mycin
One, (pork, chicken, the flesh of fish, shrimp) pre-treating method is organized
1. the sample after 3 ± 0.03g homogeneous is accurately taken in 50mL centrifuge tube;
2. 6mL ethyl acetate is added, abundant whirling motion 1min;
3. more than 4000r/min, centrifugal 10min;
4. 4mL upper strata ethyl acetate is got in new centrifuge tube;
5., in 50 ~ 60 DEG C of water-baths, nitrogen dries up;
6. add 2mL normal hexane, abundant whirling motion 10s, then add 1mL redissolution liquid, abundant whirling motion 30s;
7. more than 4000r/min, centrifugal 5min, discard upper strata normal hexane and middle layer impurity completely;
8. get 50 μ L to detect.
Tissue (pork, chicken, the flesh of fish, shrimp) Sample Dilution coefficient: 0.5
Two, apply quantum dot immunofluorescent reagent box to detect
Standard solution or sample to be tested solution 50 μ L is added by the microwell plate of coating antigen to bag, primary antibodie working fluid 50 μ L, room temperature reaction 30min, abandon supernatant, (each washing process is all as follows: add 250 μ L cleansing solutions in every hole to wash 3 times, supernatant is abandoned after 30 seconds), pat dry with thieving paper, detect its florescent intensity value with fluorescence microplate reader.Fluorescence microplate reader is set to excitation wavelength 345nm, emission wavelength 620nm.The mean value (B) of the luminous intensity of the standard solution of each concentration divided by the luminous intensity values (B0) of 0 solution, then is multiplied by 100%, i.e. Percentage bound.Computing formula: Percentage bound (%)=B/B0 × 100%.With the semilog value of the chloramphenicol concentration (ng/mL) in standard solution for X-axis, B/B0 is Y-axis, drawing standard curve map (see Fig. 2).The concentration of sample to be tested Chlorine in Solution mycin can be obtained according to the regression equation of typical curve.In the present invention, the analysis of testing result can utilize professional software, can realize the express-analysis of great amount of samples, and whole testing process only needs just can complete for 60 minutes.Chloramphenicol concentration in the standard solution that Percentage bound (B/B0) is corresponding when being 50% is IC50 value.According to canonical plotting, IC50=0.060ng/mL.
Three, the mensuration of chloramphenicol concentration in sample
With the fluorescence intensity mean value of each detection sample solution luminous intensity mean value divided by 0 solution, then be multiplied by 100%, be inhibited rate.The inhibiting rate of each detection sample solution corresponding, then can read from typical curve the semilog value detecting sample solution, the semilog value of solution converses the residual quantity of chloromycetin in sample solution per sample again, the last extension rate being multiplied by each sample pretreatment process again, can calculate the concentration of chloromycetin in sample.
Embodiment 6, chloromycetin quantum dot immune fluorescent kit Detection results are evaluated
One, kit sensitivity
Sensitivity index using lowest detectable limit as kit of the present invention.Get 20 parts of blank samples to detect, calculate the mean value of blank sample fluorescence intensity, and this mean value is brought into the sample concentration that typical curve obtains correspondence, calculate the standard deviation (SD) of each corresponding concentration value, add by mean value the lowest detectable limit (LOD) that three times of standard deviations are this sample, the results are shown in Table 1.
The lowest detectable limit of table 1 kit in pork, chicken, the flesh of fish, shrimp
| Sample | Sample mean (μ g/kg, n=20) | SD(n＝20) | LOD(μg/kg) |
| Pork | 0.005 | 0.003 | 0.014 |
| Chicken | 0.004 | 0.003 | 0.013 |
| The flesh of fish | 0.011 | 0.004 | 0.023 |
| Shrimp | 0.007 | 0.005 | 0.022 |
Two, accuracy and precision test
In the pork not containing chloromycetin, chicken, the flesh of fish, shrimp sample, add chloromycetin standard items, make the final concentration of chloromycetin standard items in pork, chicken meat sample be respectively 0.015,0.03,0.06 μ g/kg; Final concentration in the flesh of fish, shrimp sample is respectively 0.025,0.05,0.1 μ g/kg, and the sample after adding is carried out pre-treatment according to described method respectively, obtains detecting sample solution.
From the kit of 3 different batches, each extraction 3 kits detect, and each sample repeats 5 times, calculate batch interior interassay coefficient of variation respectively.Result is respectively in table 2 ~ 5.
Result shows: the average TIANZHU XINGNAO Capsul of pork, chicken, the flesh of fish, shrimp sample, 75.0 ~ 100.0%, illustrates that the accuracy of kit is good; Variation within batch coefficient is 4.2 ~ 10.0%, and interassay coefficient of variation is 4.5 ~ 8.1%.The equal < 10% of interassay coefficient of variation in crowd, illustrates that the accuracy of kit is good.
Chloromycetin accuracy in detection and Precision test result in table 2 pork
Chloromycetin accuracy in detection and Precision test result in table 3 chicken
Chloromycetin accuracy in detection and Precision test result in table 4 flesh of fish
Chloromycetin accuracy in detection and Precision test result in table 5 shrimp
Three, kit storage life
Kit preservation condition is 2-8 DEG C, preserves after 6 months, measures the IC50 value of chloromycetin.Consider in transport and use procedure, have improper preservation condition and occur, kit is placed 6 days under 37 DEG C of preservation conditions, carries out hot Acceleration study.Result shows that this kit indices meets the requirements completely.Consider that the freezing situation of kit occurs, kit is put into-20 DEG C of refrigerator freezings 9 days, measurement result also shows that kit indices is completely normal.
Table 6 Cord blood experimental result
| Time (d) | 0 | 10 | 20 | 30 | 60 | 90 | 120 | 150 | 180 |
| IC50(ng/mL) | 0.052 | 0.066 | 0.055 | 0.057 | 0.073 | 0.060 | 0.077 | 0.063 | 0.057 |
The hot Acceleration study of table 7
| Time (d) | 1 | 2 | 3 | 4 | 5 | 6 |
| IC50(ng/mL) | 0.060 | 0.058 | 0.073 | 0.067 | 0.080 | 0.075 |
Four, cross reacting rate test
Select with CAP structure or intimate other drug according to described method preparing standard solution, and drawing standard curve, calculate its 50% inhibition concentration respectively by the typical curve of various medicine.With following formulae discovery kit to the cross reacting rate of other analog.Less with the cross reacting rate of other drug, illustrate that chloromycetin quantum dot immune fluorescent detection kit is better to the detection specificity of chloromycetin.The results are shown in Table 8.
Cross reacting rate (%)=(IC of chloromycetin
50the IC of value/medicine to be measured
50value) × 100%
Test findings shows, kit of the present invention is better to chloromycetin, Chloramphenicol Succinate cross reacting rate, can be used for detecting chloromycetin and Chloramphenicol Succinate in sample simultaneously; All be less than 1% to the cross reacting rate of erythromycin, methyl chloride mycin, Florfenicol, gentamicin, so kit is good to the specificity of chloromycetin, namely kit of the present invention can chlorine detection mycin.
Table 8 chloromycetin kit cross reacting rate
| Medicine name | Cross reacting rate (%) |
| Chloromycetin | 100.0 |
| Chloramphenicol Succinate | 119 |
| Chloramphenicol Base | 3.6 |
| Methyl chloride mycin | <0.1 |
| Florfenicol | <0.1 |
| Erythromycin | <0.1 |
| Gentamicin | <0.1 |
Claims (10)
1. a chloromycetin haptens is compound shown in formula I:
formula I.
2. the preparation method of compound shown in formula I, comprises the steps:
1. chloromycetin bulk drug 323mg is dropped in 10ml single port bottle, 5ml pyridinium dissolution;
2. ice bath is cooled to 0-5 degree, adds carboxylphenylsulphonyl chloride 230mg in batches, is warming up to outdoor after 1 hour, stirring reaction 18 hours;
3. TLC detects, and bulk drug chloromycetin reacts completely aftertreatment, is poured into by reactant liquor in 25ml frozen water, separates out white precipitate, filters, washing filter cake, dry that chloromycetin haptens 450mg, mass detect.
3. a chloromycetin antigen is conjugate compound shown in formula I and carrier protein couplet obtained.
4. chloromycetin antigen according to claim 3, it is characterized in that, described carrier protein is human serum albumins, bovine serum albumin(BSA), ovalbumin, mouse haemocyanin or rabbit serum proteins.
5. the preparation method of the chloromycetin antigen described in claim 3 or 4, comprises the steps:
1. take BSA50mg, be dissolved in 3.5mlCB liquid, take haptens 50/67000*60*507=22.7mg and be dissolved in 1.5mlDMF, add NHS50/67000*120*115.09=10.3mg, EDC50/67000*120*191.7=17.2mg, room temperature activates 2 hours;
2. be added drop-wise to by activating solution under ice bath in the CB solution of BSA, 4 degree of reactions are spent the night, and PBS dialyses.
6. chloromycetin antigen described in claim 3 or 4 is preparing the application in chloromycetin specific antibody.
7. application rights requires the quantum dot-labeled specific antibody that described in 3 or 4, chloromycetin antigen prepares.
8. the application of specific antibody in chlorine detection mycin described in chloromycetin antigen, claim 7 described in claim 3 or 4.
9. application rights requires the quantum dot immune fluorescent detection kit that described in 3 or 4, described in chloromycetin antigen, claim 7, specific antibody prepares.
10. quantum dot immune fluorescent detection kit described in claim 9, it is characterized in that, it comprises: be coated with the microwell plate of chloromycetin antigen, quantum dot-labeled antibody working fluid, chloromycetin standard solution, concentrated cleaning solution.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110872344A (en) * | 2019-11-19 | 2020-03-10 | 中国农业大学 | Chloramphenicol complete antigen and preparation method and application thereof |
| CN114539171A (en) * | 2021-12-31 | 2022-05-27 | 华南农业大学 | MAC type multi-cluster tandem linear hapten, artificial antigen, preparation method and application thereof |
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| CN110872344A (en) * | 2019-11-19 | 2020-03-10 | 中国农业大学 | Chloramphenicol complete antigen and preparation method and application thereof |
| CN110872344B (en) * | 2019-11-19 | 2021-07-16 | 中国农业大学 | Chloramphenicol complete antigen and preparation method and application thereof |
| CN114539171A (en) * | 2021-12-31 | 2022-05-27 | 华南农业大学 | MAC type multi-cluster tandem linear hapten, artificial antigen, preparation method and application thereof |
| CN114539171B (en) * | 2021-12-31 | 2023-05-05 | 华南农业大学 | MAC type multi-cluster tandem linear hapten and artificial antigen as well as preparation methods and applications thereof |
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