CN1429845A - Mouse antichloromycetin monoclonal antibody and its use - Google Patents
Mouse antichloromycetin monoclonal antibody and its use Download PDFInfo
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- CN1429845A CN1429845A CN 01145286 CN01145286A CN1429845A CN 1429845 A CN1429845 A CN 1429845A CN 01145286 CN01145286 CN 01145286 CN 01145286 A CN01145286 A CN 01145286A CN 1429845 A CN1429845 A CN 1429845A
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Abstract
A monoclonal antibody GA3 of chloromycetin for mouse is disclosed, which has strong specificity for binding with chloromycetin. The immunoglobulin 6A3, its fragment and immune conjugate, and a reagent kit for detecting the residue of chloromycetin are also disclosed.
Description
Technical field
The present invention relates to detection range.More specifically, the present invention relates to the monoclonal antibody specific and the application thereof of chloramphenicol resistance.
Background technology
Paraxin is a kind of broad-spectrum antibiotics, is widely used in bacteriosis control in clinical.Because it is to the toxic side effect of human hemopoietic system, especially to children's bone marrow depression, strict control is used for clinical treatment.
Because the validity of paraxin, cheapness is so still be used to control to various Animal diseases.But it can remain in the tissue and juice of animal (for example: meat, egg, honey, milk), causes people to pass through food chain and absorbs paraxin indirectly, is detrimental to health.(Domenici.C.et?a1.Anti-cap?antibodies?for?appliccation?to?a?tirf?immunosensor.Lifechemistry?Reports.1994，Vol?11?P?409；Everest，S.J.et?al.Development?of?an?ELISA?for?the?detection?of?chlortetracycline，oxytetracyline?and?tetracycline?residues.Food?&AgriculturalImmunology(1994)6，55-61.)
In recent years, all there was strict control criterion various countries to the residual quantity of paraxin in the imported food.Chloramphenicol residue is below 10ng/g in the European and American countries requirement food, traditional detection means has not reached necessary requirement, enzymoimmunoassay can satisfy this requirement substantially, and China all depends on import, and cost an arm and a leg, limit the use of enterprise, influenced the outlet of the beastly bird food of China.(Thacker，D.J.et?al.Immunoassays(ELISA)?for?Rapid，quantitative?analysis?in?the?food-processing?industry.J.Agric?Food?chem，1996，44，2680-2685)。
Because more and more countries is more and more stricter to the detection of left drug in the import and export food, exploitation sensitive detection method has become the task of top priority.Present China detection technique residual to CAP, the technology from having announced has cylinder plate method, three kinds of vapor-phase chromatography and liquid phase chromatography.It is measured lower bound and is respectively 100ppb and 10ppb, does not all reach requirement.In recent years the application of polyclonal antibody enzyme-linked immunological technique can measure below the 10ng/ml, detects lower bound and can reach 1ng/ml, has satisfied requirement substantially.But polyclonal antibody enzyme-linked immunological technique sensitivity, specificity is relatively poor, and therefore, this area presses for the monoclonal antibody of exploitation specificity chloramphenicol resistance, and the effective ways of chlorine detection mycin.
Summary of the invention
Purpose of the present invention provides the monoclonal antibody of a specific specificity chloramphenicol resistance.
Another object of the present invention provides a kind of test kit of specific detection paraxin.
In a first aspect of the present invention, a kind of immunoglobulin (Ig) is provided, it is characterized in that it is the monoclonal antibody that is incorporated into paraxin specifically.
In an example of the present invention, described immunoglobulin (Ig) is from mouse.
In an example of the present invention, described immunoglobulin (Ig) is monoclonal antibody 6A3 and 1D1.
In another example of the present invention, described immunoglobulin (Ig) is 6A3 by mouse hybridoma cell, and CCTCC No.C200118 produces.
In a second aspect of the present invention, a kind of hybridoma that produces monoclonal antibody is provided, it is that mouse hybridoma cell is 6A3, CCTCC No.:C200118.
In a third aspect of the present invention, a kind of test kit of chlorine detection mycin is provided, it contains above-mentioned immunoglobulin (Ig) or immune conjugate, or its active fragments.Preferably, described test kit also contains the conjugate of paraxin and following material: protein, enzyme, natural or synthetic macromolecule polymer, this conjugate can combine specifically with immunoglobulin (Ig) of the present invention.
In a fourth aspect of the present invention, the purposes of mouse antichloromycetin monoclonal antibody of the present invention is provided, it is used to detect chloramphenicol residue.
Description of drawings
Fig. 1 has shown the typical curve of paraxin.
Embodiment
The inventor after years of research and found that, become CAP-HS-BSA with Paraxin succinate (CAP-HS) and BSA are coupled, and with this as immunogen, immune Balb/c mouse, its splenocyte and SP2/0 merge, and obtain the two strain cell strains (1D1,6A3) of the anti-CAP antibody of secretion.Ascites is through the Separose-4B-CAP affinitive layer purification.By polystyrene board, make competitive ELISA with CAP-HRP and CAP or testing sample with the antibody purified bag, the result shows that detection sensitivity is 0.001ng/ml; With 4 kinds of common antibiotics cross reacting rate＜0.01%, with paraxin derivative thiamphenicol cross reacting rate＜0.03%; Paraxin adds recovery test and shows that repeatability, accuracy are good.
The present invention includes the corresponding aminoacid sequence with chloramphenicol resistance monoclonal antibody (as 6A3) monoclonal antibody, have the monoclonal antibody of chloramphenicol resistance monoclonal antibody 6A3 variable region chain, and other protein or protein conjugate and fusion expressed product with these chains.Particularly, the present invention includes and have the hypervariable region of containing (complementary determining region, any protein of light chain CDR) and heavy chain or protein conjugate and fusion expressed product (being immune conjugate and fusion expressed product), as long as identical or at least 90% homology of hypervariable region of this hypervariable region and light chain of the present invention and heavy chain, preferably at least 95% homology.As is known to the person skilled in the art, immune conjugate and fusion expressed product comprise: medicine, toxin, cytokine (cytokine), radionuclide, enzyme and other diagnosis or treatment molecule and chloramphenicol resistance monoclonal antibody or its fragment bonded and the conjugate that forms.The present invention also comprises and chloramphenicol resistance monoclonal antibody or its fragment bonded cell surface marker thing or antigen.
For chloramphenicol resistance monoclonal antibody heavy chain of the present invention and sequence of light chain, can measure with ordinary method.The hypervariable region of chloramphenicol resistance monoclonal antibody V chain or complementary determining region (complementarity determiningregion, CDR) interesting especially, because relate to conjugated antigen to small part in them.Therefore, the present invention includes those the light chain immunoglobulins and the molecule of weight chain variable chain, as long as its CDR and chloramphenicol resistance monoclonal antibody CDR have the homology of (preferably more than 95%) more than 90% with band CDR.
The present invention not only comprises complete monoclonal antibody, also comprises having immunocompetent antibody fragment, as Fab or (Fab ')
2Fragment; Heavy chain of antibody; Light chain of antibody.
The present invention also provides above-mentioned immunoglobulin (Ig) or its segmental dna molecular.The sequence of these dna moleculars can as above be used routine techniques, and utilizing mouse hybridoma cell is that 6A3 (CCTCC No.:C200118) obtains.In addition, also the encoding sequence of light chain and heavy chain can be merged, form single-chain antibody.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
The invention still further relates to the carrier that comprises above-mentioned suitable dna sequence dna and suitable promotor or control sequence.These carriers can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, handle the combination of (salt analysis method), centrifugal, the broken bacterium of infiltration, supersound process, super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods with protein precipitant.
In addition, the present invention also provides a kind of test kit of chlorine detection mycin, and it contains above-mentioned immunoglobulin (Ig) or immune conjugate, or its active fragments.A kind of paraxin diagnostic kit detects lower bound and can reach 0.01ng/g.
The invention has the advantages that: highly sensitive, high specificity, simple and efficient.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment
Material:
(A) reagent: Paraxin succinate (CAP-HS), BSA, N-hydroxy-succinamide, N., N.-two hexamethylene carbodiimides, dimethyl formamide, goat anti-mouse igg-HRP, PEG all are the SIGMA product, HAT is the GIBCO product.
(B) animal: female Balb/c mouse, 6-8 week order is about body weight 20 grams, available from the Shanghai Experimental Animal Center.
Embodiment 1
The preparation of chloramphenicol resistance monoclonal antibody and purifying
(A) immunizing antigen, detect antigen, the preparation of paraxin enzyme labelling thing:
According to Domenici.C.et al.Anti-cap antibodies forappliccation to a tirf immunosensor.Life chemistry Reports.1994, method described in Vol 11 P 409, with Paraxin succinate respectively with BSA, rabbit igg, HRP carries out coupling by carbodiimide activation fat method, thereby make CAP-HS-BSA as immunizing antigen, CAP-HS-IgG is as detecting antigen, and CAP-HS-HRP is as paraxin enzyme labelling thing.
(B) foundation of anti-CAP hybridoma cell strain
Every mouse is to inject with nape in the antigen emulsification venter posterior of 50 μ g amount, and immunity is 8 times altogether, and splenocyte and the SP2/0 with mouse merges then.Immunity BALB/c mouse serum titer reaches 1: 15000, and cell confluency is more than 95%, filters out 46 positive colonies altogether, and positive rate is 8.2%.After the cloning, select two strains secretions high-titer antibody cell (1D1,6A3).Through 4 months go down to posterity, the abdominal cavity inoculation, liquid nitrogen cryopreservation, recovery, two strain of hybridoma all can stably excreting mAb.Secrete this two strains cell strain difference called after 1D1 and the 6A3 of anti-CAP antibody.
(C) MONOCLONAL ANTIBODIES SPECIFIC FOR
Hybridoma technology prepares ascites routinely, and ascites obtains monoclonal antibody 1D1 and 6A3 behind the Separose-4B-CAP affinitive layer purification.
Embodiment 2
The CHARACTERISTICS IDENTIFICATION of monoclonal antibody
(A) hypotype is identified
Adopt conventional double fastener heart ELISA method to carry out the hypotype Screening and Identification.
(B) mensuration of affinity costant
Press Friguet B, et al:Measurement of the true affinity constantin solution of antigen-antibody by enzyme-linked immunosorbent assay.J immunol Meth 1985; The described method of 77:305 is carried out affinity costant and is measured.
Ig hypotype and affinity constant qualification result are listed in table 1.
Table 1 Ig subclass and affinity costant measurement result
| mAb | The Ig subclass | Affinity costant (M -1) |
| 1D1 | IgG 1 | 8.0×10 9 |
| 6A3 | IgG 1 | 7.8×10 8 |
(C) foundation of competitive ELISA and recovery test
The foundation of competitive ELISA: get respectively 50 μ l CAP reference liquids (55,25,10,5,1,0.1ng/ml) and 50ul CAP-HRP enzyme labelling thing (1: 10000) with the bag be at war with TMB-H by the anti-CAP monoclonal antibody on the polystyrene batten
2O
2Colour developing is surveyed the 450nm absorption value, the drawing standard curve.
Recovery test: (55,25,10,5,2.5,1ng/ml) 1ml adds in the 10g chicken after the homogenate, mixing to get CAP standard substance liquid.The access method of quenching by Britain Randox company test kit specification sheets extracts CAP, with the amount of competition law mensuration CAP, looks into its yield on synchronous typical curve.
The result:
Typical curve and sensitivity: adopting the CAP standard substance to set sensing range is 0.1-55ng/ml, and the result shows that increasing progressively optical density(OD) with concentration tapers off, and typical curve is seen Fig. 1.Its detectability can reach 0.001ng/ml. and see Table 2.
Table 2 paraxin detects minimum concentration (ng/ml)
| CAPng/ml | 1D1O.D | 6A3O.D |
| 55 | 0.482 | 0.499 |
| 25 | 0.812 | 0.944 |
| 10 | 1.228 | 1.254 |
| 5 | 1.566 | 1.558 |
| 1 | 1.962 | 1.784 |
| 0.1 | 2.154 | 2.081 |
| 0.01 | 2.304 | 2.285 |
| 0.001 | 2.512 | 2.313 |
| 0.0001 | 2.735 | 2.472 |
| 0.01MPBS | 2.785 | 2.506 |
Replica test
Repeat typical curve 4 times, each every standard point 3 multiple holes.CV is 4.5-8.9% in must criticizing as calculated, and is average 6.7%, is 5.6-10.7% between batch, average 8.2%.
Recovery test
CAP that extracts and CAP-HRP competition the results are shown in Table 3.The 1D1 rate of recovery is at 83.3-94.4%, average out to 88.9%, and the 6A3 rate of recovery is at 84.8-97.9%, and average out to 91.4% actually records CAP concentration and to add concentration approaching, shows that the accuracy of method is fine.
The table 3 paraxin rate of recovery (%) CAP 1D1 6A3 (ng/ml) the observed value rate of recovery observed value rate of recovery
(ng/ml)???(％)??????(ng/ml)???(％)55?????????49.5??????90.2??????46.65?????84.825?????????20.83?????83.3??????22.83?????91.310?????????9.03??????90.3??????9.79??????97.95??????????4.52??????90.4??????4.68??????93.62.5????????2.27??????90.8??????2.37??????94.81??????????0.944?????94.4??????0.96??????95.6
(D) specificity analyses
With the derivative of 4 kinds of common antibiotics and paraxin (first sulfone-CAP, HS-CAP) respectively with the CAP-HRP inhibition test that is at war with, observe monoclonal antibody and its cross reacting rate.
The result shows, the derivative of mAb of the present invention and CAP and 4 kinds of common antibiotics (1mg/ml) and CAP-HRP competition inhibition test, the result show cross reacting rate all＜0.03 and＜0.01% (seeing Table 4), CAPELISA method high specificity is described.
The cross reacting rate of table 4.Mab and common antibiotics and CAP derivative
The microbiotic cross reacting rate
1D1?????????????????????6A3
HS-CAP???????????232.2???????????????????189.2
CAP??????????????100?????????????????????100
Thiamphenicol-CAP＜0.03＜0.03
Penicillin＜0.01＜0.01
Sulphadiazine Sodium＜0.01＜0.01
Streptomycin sulphate＜0.01＜0.01
Tsiklomitsin＜0.01＜0.01
The monoclonal antibody of the chloramphenicol resistance that the inventor develops first, can with the paraxin specificity combine, with paraxin derivative and common antibiotics no cross reaction, its cross reacting rate＜0.01%, highly sensitive, detect lower bound and can reach 0.01-0.001ng/g, add reclaim and batch in, batch between test show the repeatability of method, accuracy meets the monitoring standard of farming animals foods prods.
The residual concentration that monoclonal antibody ELISA not only detects CAP is feasible, and compares with polyclonal antibody, and the former is highly sensitive, good stability.The residual chloromycetin quantity measuring method of inventor's development can satisfy the demand that China's import and export food detects fully.
The preservation of biomaterial
A kind of positive hybridoma cell is hybridoma cell line 6A3, and this hybridoma lies in and is preserved in Chinese typical culture collection center (CCTCC, China December 20 calendar year 2001, the Wuhan City), preserving number is Chinese typical culture collection center (CCTCC, China, Wuhan City) No.C200118.
Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (8)
1. an immunoglobulin (Ig) is characterized in that, it is the monoclonal antibody that is incorporated into paraxin specifically.
2. immunoglobulin (Ig) as claimed in claim 1 is characterized in that described immunoglobulin (Ig) is from mouse.
3. immunoglobulin (Ig) as claimed in claim 1 is characterized in that, it is monoclonal antibody 6A3 and 1D1.
4. immunoglobulin (Ig) as claimed in claim 1 is characterized in that it is 6A3 by mouse hybridoma cell, and CCTCC No.C200118 produces.
5. a hybridoma that produces monoclonal antibody is characterized in that, it is that mouse hybridoma cell is 6A3, CCTCC No.:C200118.
6. a test kit that detects chloramphenicol residue is characterized in that, it contains the described immunoglobulin (Ig) of claim 1.
7. test kit as claimed in claim 6 is characterized in that, also contains the conjugate that paraxin and following material form: protein, enzyme, natural or synthetic macromolecule polymer.
8. the purposes of the described immunoglobulin (Ig) of claim 1 is characterized in that, it is used to detect chloramphenicol residue.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01145286 CN1234730C (en) | 2001-12-30 | 2001-12-30 | Mouse antichloromycetin monoclonal antibody and its use |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01145286 CN1234730C (en) | 2001-12-30 | 2001-12-30 | Mouse antichloromycetin monoclonal antibody and its use |
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| Publication Number | Publication Date |
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| CN1429845A true CN1429845A (en) | 2003-07-16 |
| CN1234730C CN1234730C (en) | 2006-01-04 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN 01145286 Expired - Fee Related CN1234730C (en) | 2001-12-30 | 2001-12-30 | Mouse antichloromycetin monoclonal antibody and its use |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1308687C (en) * | 2004-04-30 | 2007-04-04 | 中国农业大学 | Enzyme linked immuno kit for detecting chloromycetin |
| CN1308685C (en) * | 2004-04-30 | 2007-04-04 | 中国农业大学 | Kit for detecting chloromycetin |
| CN1308686C (en) * | 2004-04-30 | 2007-04-04 | 中国农业大学 | Kit for detecting chloromycetin |
| CN1308688C (en) * | 2004-04-30 | 2007-04-04 | 中国农业大学 | Enzyme linked immuno kit for detecting chloromycetin |
| CN101955539A (en) * | 2010-05-06 | 2011-01-26 | 北京维德维康生物技术有限公司 | Immunoassay kit for detecting chloramphenicol and dedicated antibody thereof |
| CN102146138A (en) * | 2011-04-06 | 2011-08-10 | 浙江工业大学 | Monoclonal antibody to chloramphenicol and application thereof |
| RU2513697C2 (en) * | 2007-11-02 | 2014-04-20 | Новартис Аг | Improved nogo-a-binding molecules and pharmaceutical application thereof |
| CN110658342A (en) * | 2019-10-12 | 2020-01-07 | 石家庄市畜产品质量监测中心 | Time-resolved fluorescence immunochromatographic assay quantitative detection method for chloramphenicol in aquatic products |
-
2001
- 2001-12-30 CN CN 01145286 patent/CN1234730C/en not_active Expired - Fee Related
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1308687C (en) * | 2004-04-30 | 2007-04-04 | 中国农业大学 | Enzyme linked immuno kit for detecting chloromycetin |
| CN1308685C (en) * | 2004-04-30 | 2007-04-04 | 中国农业大学 | Kit for detecting chloromycetin |
| CN1308686C (en) * | 2004-04-30 | 2007-04-04 | 中国农业大学 | Kit for detecting chloromycetin |
| CN1308688C (en) * | 2004-04-30 | 2007-04-04 | 中国农业大学 | Enzyme linked immuno kit for detecting chloromycetin |
| RU2513697C2 (en) * | 2007-11-02 | 2014-04-20 | Новартис Аг | Improved nogo-a-binding molecules and pharmaceutical application thereof |
| CN101955539A (en) * | 2010-05-06 | 2011-01-26 | 北京维德维康生物技术有限公司 | Immunoassay kit for detecting chloramphenicol and dedicated antibody thereof |
| CN101955539B (en) * | 2010-05-06 | 2012-10-24 | 北京维德维康生物技术有限公司 | Immunoassay kit for detecting chloramphenicol and dedicated antibody thereof |
| CN102146138A (en) * | 2011-04-06 | 2011-08-10 | 浙江工业大学 | Monoclonal antibody to chloramphenicol and application thereof |
| CN102146138B (en) * | 2011-04-06 | 2013-04-24 | 浙江工业大学 | Monoclonal antibody of chloramphenicol and application thereof |
| CN110658342A (en) * | 2019-10-12 | 2020-01-07 | 石家庄市畜产品质量监测中心 | Time-resolved fluorescence immunochromatographic assay quantitative detection method for chloramphenicol in aquatic products |
| CN110658342B (en) * | 2019-10-12 | 2023-06-02 | 石家庄市畜产品质量监测中心 | Method for quantitatively detecting chloramphenicol in aquatic products through time-resolved fluorescence immunochromatography |
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