CN103483445A - Preparation methods and applications of benzylpenicilloic acid artificial antigen and benzylpenicilloic acid artificial antibody - Google Patents
Preparation methods and applications of benzylpenicilloic acid artificial antigen and benzylpenicilloic acid artificial antibody Download PDFInfo
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- CN103483445A CN103483445A CN201310460758.9A CN201310460758A CN103483445A CN 103483445 A CN103483445 A CN 103483445A CN 201310460758 A CN201310460758 A CN 201310460758A CN 103483445 A CN103483445 A CN 103483445A
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- 239000000427 antigen Substances 0.000 title claims abstract description 51
- 102000036639 antigens Human genes 0.000 title claims abstract description 51
- 108091007433 antigens Proteins 0.000 title claims abstract description 51
- HCYWNSXLUZRKJX-RWMBFGLXSA-N benzylpenicilloic acid Chemical compound N1[C@@H](C(O)=O)C(C)(C)S[C@@H]1[C@@H](C(O)=O)NC(=O)CC1=CC=CC=C1 HCYWNSXLUZRKJX-RWMBFGLXSA-N 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims abstract description 66
- 238000000034 method Methods 0.000 claims abstract description 25
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- 238000006243 chemical reaction Methods 0.000 claims description 19
- 238000000502 dialysis Methods 0.000 claims description 14
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- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 12
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- CGIHPACLZJDCBQ-UHFFFAOYSA-N acibenzolar Chemical compound SC(=O)C1=CC=CC2=C1SN=N2 CGIHPACLZJDCBQ-UHFFFAOYSA-N 0.000 claims description 5
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- MSWZFWKMSRAUBD-UHFFFAOYSA-N 2-Amino-2-Deoxy-Hexose Chemical compound NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 1
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- SCLZRKVZRBKZCR-SLINCCQESA-M cloxacillin sodium Chemical compound [Na+].N([C@@H]1C(N2[C@H](C(C)(C)S[C@@H]21)C([O-])=O)=O)C(=O)C1=C(C)ON=C1C1=CC=CC=C1Cl SCLZRKVZRBKZCR-SLINCCQESA-M 0.000 description 1
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- YFAGHNZHGGCZAX-JKIFEVAISA-N dicloxacillin Chemical compound N([C@@H]1C(N2[C@H](C(C)(C)S[C@@H]21)C(O)=O)=O)C(=O)C1=C(C)ON=C1C1=C(Cl)C=CC=C1Cl YFAGHNZHGGCZAX-JKIFEVAISA-N 0.000 description 1
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- BDAGIHXWWSANSR-UHFFFAOYSA-N formic acid Substances OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
Abstract
The invention discloses preparation methods and applications of a benzylpenicilloic acid artificial antigen and a benzylpenicilloic acid artificial antibody, and relates to a method for simply, quickly and effectively detecting penicillin G and a degradation product of penicillin G, namely benzylpenicilloic acid. The specific antibody to penicillin G and the degradation product of penicillin G, namely benzylpenicilloic acid, is prepared by designing and synthesizing a hapten and the artificial antigen of a penicillin veterinary drug penicillin G with a 6-aminocaproic acid molecular structure and a beta-lactam ring molecular structure, and the degradation product of penicillin G, namely benzylpenicilloic acid; a molecular specific antigenic determinant of the veterinary drug is highlighted; the chemosynthesis difficulty is overcome; and an immune animal induces to generate the specific antibody with very high affinity.
Description
Technical field
The invention belongs to technical field of chemistry, relate to preparation method and the application of a kind of benzyl penicilloic acid artificial antigen and antibody thereof.
Background technology
Penicillin G (also claiming penicillin G) is a kind of of Penicillin antibiotics, and Penicillin antibiotics is to enter a clinical application class microbiotic the earliest, is also the microbiotic that China's turnout is maximum, usage quantity is maximum.Penicillin antibiotics has become treatment milk cow and the most frequently used medicine of other edibility Animal diseases, but improper due to the lack of standardization and milk sample collection of medication, causes in animal derived food the Detection Of Penicillin Residues phenomenon serious.Penicillin medicine is residual in vivo can produce serious anaphylaxis, and the anaphylaxis rate is in first of various kinds of drug.Because penicillin easily is degraded to penicilloic acid under the β-lactamase effect, make penicillin lose anti-microbial activity, this makes existing detection method can't detect Penicillin Residues, more encouraged the abuse of β-lactam antibitics in the milk-producing, accelerated the propagation of resistant organism, serious threat the long-run development of dairy industry in China.For this reason, to the restriction of Penicillin Residues also so more and more stricter, and, to analyzing the aspects such as determination object, kind, quantity, scope, index, new requirement and higher standard are all proposed.The animal food herbal medicine maximum residue limit(MRL) of the Ministry of Agriculture's issue, regulation to penicillin G: muscle≤50 μ g/kg, fat≤50 μ g/kg, liver≤50 μ g/kg, kidney≤50 μ g/kg, milk≤4 μ g/kg, although the benzyl penicilloic acid is not made to respective specified, its harm caused can not be ignored.
Molecular weight is less than 1000 daltonian small molecules noxious chemicals, and as agricultural chemicals, veterinary drug and meta-bolites thereof, its traditional retention analysis is mainly to rely on the analysis means such as gas-chromatography, liquid chromatography or chromatogram one mass spectrometry.These physico-chemical analysis methods, very complicated, cost is higher, analysis speed is slow, is difficult to meet the needs of actual analysis, therefore in the urgent need to developing easy, quick, sensitive analytical technology.
But different from macromole, the micromolecular compound immunoassay has own characteristic:
(1) micromolecular compound (MW≤1000dolton) does not generally have immunogenicity, can not produce specific antibody, must synthesize the haptens at outstanding molecule stereo structure specificity position by the direct immunization animal, and connect and compose joiner with macromolecular carrier, could produce the specific antibody for this target micromolecular compound by immune animal.The binding substances of this haptens and macromolecular carrier is called artificial antigen.The preparation of artificial antigen is not arbitrarily, comprise the factors of any structural difference of binding site, combination, kind of carrier and haptens and target analytes as size, shape, composition, configuration, conformation, polarity, cloud density etc., all may greatly affect the character of corresponding antibodies, so they are the keys that determine to produce its specific antibody and set up immune analysis method.
(2) although micromolecular compound does not have immunogenicity, there is reactionogenicity, there is the ability with corresponding antibodies generation immunological response, and can externally quantitatively carry out, follow the law of mass action.
(3) detect the analytical technology of small molecules platform thing based on the antigen-antibody immune response, at present many employing enzyme immunoassays (ELISA).Utilize the quantitative combination of enzymatic reaction demonstration antigen-antibody, simple to operate, there is again suitable sensitivity, development in recent years is very fast.The veterinary drug ELISA grown up since the eighties and simple and easy enzyme immunity EIA technology, make the residue of veterinary drug analysis obtain larger vitality on method; Too complicated to using real-time analysis sample matrix, be difficult to the residue of veterinary drug of analyzing with common physico-chemical method, there is suitable using value.
Immuno analytical method is introduced into residue of veterinary drug and divides the folding field, becomes one of a kind of quantitative analysis tech that development and application potential are arranged most, is paid attention to widely.The key of this technical study is the preparation of haptenic molecular designing, synthetic and artificial holoantigen and antibody.Therefore, target analytes molecular immunology characteristic, and how by chemistry or biochemical technology, to give prominence to and to utilize these characteristics, be the important research contents in this field.This technology has become a brand-new field of trace analysis research at present, can be with traditional analysis side by side as a new analysis approach.
Micromolecular compound (molecular weight is less than 1000 dalton), rely on immunology, immunochemistry ultimate principle and animal nutrition.The key of this technical study is the preparation of haptenic molecular designing, synthetic and artificial holoantigen and antibody.Therefore, target analytes molecular immunology characteristic, and how by chemistry or biochemical technology, to give prominence to and to utilize these characteristics, be the important research contents in this field.This technology has become a brand-new field of trace analysis research at present, can be with traditional analysis side by side as a new analysis approach.Penicillin G and degradation product benzyl penicilloic acid artificial antigen thereof there is not yet report at present.
Summary of the invention
The objective of the invention is to overcome the defect of prior art, a kind of benzyl penicilloic acid artificial antigen and preparation method for antibody and application are provided, is a kind of method that simple and fast detects penicillin G degradation product benzyl penicilloic acid effectively.The objective of the invention is by designing synthetic haptens and artificial antigen with penicillins veterinary drug penicillin G degradation product benzyl penicilloic acid of 6-aminocaprolc acid molecular structure and beta-lactam nucleus molecular structure, preparation is for the specific antibody of penicillin G degradation product benzyl penicilloic acid; Unique distinction is to have given prominence to this class veterinary drug molecular specificity antigenic determinant, has overcome again the difficulty of chemosynthesis, and immune animal is induced and produces the very high specific antibody of affinity.
Its technical scheme is:
A kind of benzyl penicilloic acid artificial antigen, molecular formula is:
The preparation method of a kind of benzyl penicilloic acid artificial antigen of the present invention comprises the following steps:
(1) get (2S, 5R, 6R)-3,3-dimethyl-6-(2-phenylacetylamino)-7-oxo-4-thia-1-azabicyclo [3,2,0] heptane-2-formic acid sylvite 1.500g, add 4.0ml1M hydrochloric acid, obtain haptens (2S, 5R, 6R)-3,3-dimethyl-6-(2-phenylacetylamino)-7-oxo-4-thia-1-azabicyclo [3,2,0] heptane-2-formic acid white solid, distilled water washing drying can obtain haptens;
(2) getting gained white solid 0.5000g is dissolved in the 2ml tetrahydrofuran (THF), add 0.1500gN-N-Hydroxysuccinimide and 0.5000gN, the N-dicyclohexylcarbodiimide, under 22-25 ℃, stirring reaction spends the night, then be put in 4 ℃ of refrigerators interior 18 hours, the centrifugal precipitation of removing, upper strata Acibenzolar liquid is joined in the 2mlPBS solution that pH7.0 is dissolved with the 10mg keyhole limpet hemocyanin, after stirring, under 4 ℃, react 5 hours, by the reaction solution dialysis tubing of packing into, under 4 ℃, in the PBS of pH7.4, dialysis can obtain artificial antigen in three days;
(3) the haptens 1.9mg of synthesized is dissolved in 2ml dimethyl formamide wiring solution-forming A, 0.54mg horseradish peroxidase is dissolved in wiring solution-forming B in the 0.2mol/L phosphoric acid buffer of 2ml pH7.4, then under ice bath, solution A being added to solution B slowly, react and spend the night under 4 ℃ after stirring, finally reaction solution is inserted in dialysis tubing, the 0.2mol/L phosphoric acid buffer dialysis with pH7.4 under 4 ℃ can obtain enzyme-labelled antigen in three days.
A kind of benzyl penicilloic acid artificial antibody's preparation method comprises the following steps:
(1) initial immunity: get the 1mg artificial antigen synthetic according to the method for the invention and be dissolved in the solution that 0.9% NaCl solution and Freund's complete adjuvant equal-volume prepare, carry out animal immune;
(2) booster immunization: be dissolved in the above-mentioned artificial antigen of 0.5mg the solution that 0.9% NaCl solution and Freund's incomplete adjuvant equal-volume are prepared, carry out animal immune;
(3) antibody purification: the periodic monitor animal's antibody is tired, when antibody reaches suitable tiring to certain envelope antigen, gather blood, and centrifugal acquisition antiserum(antisera), use G-Sepharose albumen affinity column antagonistic Serum to carry out purifying, obtain the specific antibody of anti-benzyl penicilloic acid.
The foundation of competitive ELISA detection method:
Add antibody-solutions (1 μ g/100 μ L/ hole) on 96 hole enzyme plates, incubated at room is spent the night; PBST washes plate 3 times, under room temperature, with 1%BSA, seals (200 μ L/ hole) 1h; The penicillin G standard substance are mixed with to six concentration of 20,5,1.25,0.32,0.08,0.02 μ g/L, and after PBST washes plate 4 times, every hole adds 50 μ L mark liquid and 50 μ L enzyme-labelled antigens, incubated at room 1h; PBST washes plate 5 times, and every hole adds 100 μ L substrate solutions, 37 ℃ of colour developing 15min; Add 50 μ L stop buffers in every hole, termination reaction, dual wavelength mode (450～650nm) reads the OD value.
The application of benzyl penicilloic acid artificial antibody of the present invention in the benzyl penicilloic acid detects.
The application of benzyl penicilloic acid artificial antigen of the present invention in the benzyl penicilloic acid detects.
Compared with prior art, beneficial effect of the present invention: the present invention has the characteristics such as special, sensitive, accurate, quick, convenient, cheap.This design, synthetic haptens and target determinand similarity degree are high, the feature structure of determinand are retained complete, for preparing the good antibody of specificity, lay a good foundation.
Through verification experimental verification, above-mentioned haptens, its simple synthetic method, and main raw material used (2S, 5R, 6R)-3,3-dimethyl-6-(2-phenylacetylamino)-7-oxo-4-thia-1-azabicyclo [3,2,0] heptane-2-formic acid sylvite, comparatively cheap, the easy acquisition of 6 one hexosamine price, all can buy in general chemical reagents corporation.Because combined coefficient is high, reactions steps is few, haptens only needs single step reaction to synthesize, thereby has improved the controllability of reaction, and reaction yield has reached respectively 96% and 86%.In addition, extraction, the purification process of synthetic product are easy, only need to carry out acid precipitation to synthetic product, can obtain highly purified purpose product.Therefore, the synthetic haptenic method of the present invention is compared with additive method, more is easy to popularize.
The accompanying drawing explanation
Fig. 1 is the penicillin G mass spectrum;
Fig. 2 is the typical curve that penicilloic acid ELISA detects;
Fig. 3 is the effect that milk substrate is eliminated;
Fig. 4 is the effect that milk powder matrix is eliminated;
Fig. 5 is the stability of lyophilized antibodies.
Embodiment
Describe technical scheme of the present invention in detail below in conjunction with the accompanying drawing specific embodiment.
The present invention design, synthesized the haptens of small molecules target analytes, and with the carrier protein coupling, prepare effective artificial antigen, the immune animal preparation is for the specific antibody of small molecule analysis thing.Its selectivity is decided by the specificity of immunological response, can be to set up fast and accurately penicillin G and the residual quantitative analysis method of degradation product benzyl penicilloic acid lays the foundation.The key of this technical study is the preparation of haptenic molecular designing, synthetic and artificial holoantigen.
Benzyl penicilloic acid structure is as shown below:
The molecular structure of benzyl penicilloic acid can be divided into two structural units: a part is penicilloyl, and a part is the benzyl side chain.The space structure complexity of benzyl side chain and penicilloyl, molecular weight is larger, can be used as antigenic determinant, and the carboxyl on the hydrogenation thiazole ring can connect by carrier proteins.Therefore when synthetic haptens, must retain base side chain and penicilloyl structure, utilize the carboxyl on the hydrogenation thiazole ring to be connected therefore with carrier proteins, it is 334 structures (2S as shown below that the present invention selects molecular weight MW, 5R, 6R)-3,3-dimethyl-6-(2-phenylacetylamino)-7-oxo-4-thia-1-azabicyclo [3,2,0] heptane-2-formic acid is haptens
The synthetic molecules formula that is connected with keyhole limpet hemocyanin is
Compound be artificial antigen, connect synthetic enzyme-labelled antigen with horseradish peroxidase.Artificial antigen obtains antibody through immune animal.
Specific practice is:
Get (2S, 5R, 6R)-3,3-dimethyl-6-(2-phenylacetylamino)-7-oxo-4-thia-1-azabicyclo [3,2,0] heptane-2-formic acid sylvite 1.500g, add 4.0ml1M hydrochloric acid, obtain haptens (2S, 5R, 6R)-3,3-dimethyl-6-(2-phenylacetylamino)-7-oxo-4-thia-1-azabicyclo [3,2,0] heptane-2-formic acid white solid, distilled water washing drying can obtain haptens.
Getting gained white solid 0.5000g is dissolved in the 2ml tetrahydrofuran (THF), add 0.1500gN-N-Hydroxysuccinimide and 0.5000gN, the N-dicyclohexylcarbodiimide, under 22-25 ℃, stirring reaction spends the night, then be put in 4 ℃ of refrigerators interior 18 hours, the centrifugal precipitation of removing, upper strata Acibenzolar liquid is joined in the 2mlPBS solution that pH7.0 is dissolved with the 10mg keyhole limpet hemocyanin, after stirring, under 4 ℃, react 5 hours, by the reaction solution dialysis tubing of packing into, under 4 ℃, in the PBS of pH7.4, dialysis can obtain artificial antigen in three days.
The haptens 1.9mg that gets synthesized is dissolved in 2ml dimethyl formamide wiring solution-forming A, 0.54mg horseradish peroxidase is dissolved in wiring solution-forming B in the 0.2mol/L phosphoric acid buffer of 2ml pH7.4, then under ice bath, solution A being added to solution B slowly, react and spend the night under 4 ℃ after stirring, finally reaction solution is inserted in dialysis tubing, 0.2mol/L phosphoric acid buffer dialysis with pH7.4 under 4 ℃ can obtain enzyme-labelled antigen in three days, for color reaction.
The preparation method of benzyl penicilloic acid antibody:
Immune animal is selected female new zealand white rabbit, immunization method adopts subcutaneous and intramuscular injection, carry out booster immunization after just exempting from four times, booster immunization respectively at initial immunity after immunity three times behind 2 weeks, 4 weeks and 6 weeks, after this interval is one month, carries out the 5th immunity, after 9 days, by the auricular vein of rabbit, gets blood, carry out bioactivity, specific practice is:
Initial immunity: get 1mg and be dissolved in the solution that 0.9% NaCl solution and Freund's complete adjuvant equal-volume prepare according to the synthetic artificial antigen of the described method of claim 3, carry out animal immune;
Booster immunization: be dissolved in the above-mentioned artificial antigen of 0.5mg the solution that 0.9% NaCl solution and Freund's incomplete adjuvant equal-volume are prepared, carry out animal immune;
Antibody purification: the periodic monitor animal's antibody is tired, when antibody reaches suitable tiring to certain envelope antigen, gather blood, and centrifugal acquisition antiserum(antisera), use G-Sepharose albumen affinity column antagonistic Serum to carry out purifying, obtain the specific antibody of anti-benzyl penicilloic acid.
The foundation of competitive ELISA detection method:
Add antibody-solutions (1 μ g/100 μ L/ hole) on 96 hole enzyme plates, incubated at room is spent the night; PBST washes plate 3 times, under room temperature, with 1%BSA, seals (200 μ L/ hole) 1h; The penicillin G standard substance are mixed with to six concentration of 20,5,1.25,0.32,0.08,0.02 μ g/L, and after PBST washes plate 4 times, every hole adds 50 μ L mark liquid and 50 μ L enzyme-labelled antigens, incubated at room 1h; PBST washes plate 5 times, and every hole adds 100 μ L substrate solutions, 37 ℃ of colour developing 15min; Add 50 μ L stop buffers in every hole, termination reaction, dual wavelength mode (450～650nm) reads the OD value.
Embodiment 1:
The preparation method of benzyl penicilloic acid artificial antigen
With (2S, 5R, 6R)-3,3 dimethyl-6-(2-phenylacetylamino)-7-oxo-4-thia-1-azabicyclo [3,2,0] heptane-2-formic acid, take amide group CO=NH as bridge, connect hemocyanin (keyhole limpet hemocyanin, KLH) by active ester method upper, synthetic artificial antigen; Specific practice is:
Get 1.5mg (2S, 5R, 6R) 3,3-dimethyl-6-(2-phenylacetylamino)-7-oxo-4-thia-1-azabicyclo [3,2,0] heptane-2-formic acid sylvite adds 4.0ml l M hydrochloric acid, obtains haptens (2S, 5R, 6R)-3,3-dimethyl-6-(2-phenylacetylamino)-7-oxo-4-thia-1-azabicyclo [3,2,0] heptane-2-formic acid white solid, distilled water washing drying can obtain haptens.
Getting gained white solid 0.5000g is dissolved in the 2ml tetrahydrochysene furan food in one's mouth, add 0.1500gN-N-Hydroxysuccinimide and 0.5000gN, the N-dicyclohexylcarbodiimide, under 22-25 ℃, stirring reaction spends the night, then be put in 4 ℃ of refrigerators interior 18 hours, the centrifugal precipitation of removing, upper strata Acibenzolar liquid is joined in the 2mlPBS solution that pH7.0 is dissolved with the 10mg keyhole limpet hemocyanin, after stirring, under 4 ℃, react 5 hours, by the reaction solution dialysis tubing of packing into, under 4 ℃, in the PBS of pH7.4, dialysis can obtain artificial antigen in three days.
Embodiment 2:
The preparation method of benzyl penicilloic acid antibody
Immune animal is selected female new zealand white rabbit, immunization method adopts subcutaneous and intramuscular injection, carry out booster immunization after just exempting from four times, booster immunization respectively at initial immunity after immunity three times behind 2 weeks, 4 weeks and 6 weeks, after this interval is one month, carries out the 5th immunity, after 9 days, by the auricular vein of rabbit, gets blood, carry out bioactivity, specific practice is:
Initial immunity: get 1mg and be dissolved in the solution that 0.9% NaCl solution and Freund's complete adjuvant equal-volume prepare according to the synthetic artificial antigen of the described method of claim 3, carry out animal immune;
Booster immunization: be dissolved in the above-mentioned artificial antigen of 0.5mg the solution that 0.9% NaCl solution and Freund's incomplete adjuvant equal-volume are prepared, carry out animal immune;
Antibody purification: the periodic monitor animal's antibody is tired, when antibody reaches suitable tiring to certain envelope antigen, gather blood, and centrifugal acquisition antiserum(antisera), use G-Sepharose albumen affinity column antagonistic Serum to carry out purifying, obtain the specific antibody of anti-benzyl penicilloic acid.
In order to further illustrate beneficial effect of the present invention, carried out following experiment.
1, penicilloic acid is haptenic synthetic
Adopt active ester method to prepare artificial antigen, dried product has been carried out to Mass Spectrometric Identification.
Result is as shown in the figure: 333.57[M-H]
-, the molecular weight of penicillin G is 334, data conform to compound molecular weight, and purity is higher, are applicable to carrying out the Acibenzolar reaction.
2, the foundation of competitive ELISA detection method
By optimization experiment, determined that the antibody package amount is 1 μ gwell
-1, the enzyme-labelled antigen Dilution ratio is 1:1200, confining liquid is 1%BSA, with the phosphate buffer soln of pH5.7, does standard sample dilution liquid, from 16 μ gL
-1dilute 6 concentration point downwards through 4 times of gradients, with direct competitive ELISA method, drawing standard curve.From typical curve: IC
50=0.320 ± 0.012 μ g L
-1, IC
15=0.031 ± 0.002 μ g L
-1.
3, the elimination of sample substrate impact
1) the milk substrate impact is eliminated
Experiment is found by careful comparison, only with PBS, does not add any sequestering agent dilute sample, although that absorbancy differs is larger, and the coincidence of inhibiting rate curve.So, adopt the shade method that adds a certain amount of skim-milk to adjust typical curve, make absorbancy reach consistent.Attempted following concentration: 0.1% skim-milk, 0.2% skim-milk, 0.3% skim-milk, 0.4% skim-milk, 0.5% skim-milk, and, by 20,40,60,80,100 times of diluted samples, compare the absorbance deviation (table 1,2) under each condition.
The absorbance of table 1 skim-milk and diluted milk
The absorbancy deviation of table 2 skim-milk and diluted milk
Data presentation, skim-milk concentration is 0.In the time of 1%, 40 times of milk samples, the absorbancy of the two is the most approaching, and deviation is 0.63%, so select 0.1% skim-milk and 40 times of dilutions of milk sample to eliminate the matrix impact, result is as Fig. 3.Milk is after centrifugal grease removal, and 40 times of dilutions, can make typical curve and matrix curve overlap.In milk sample, the IC of ELISA method
50=12.8 μ gL
-1, IC
15=1.2 μ gL
-1.
2) impact of milk powder matrix is eliminated
Milk powder is the enriched material of milk, and experiment adds a certain amount of PBS to be made into reconstituted milk, and after sample pre-treatments, to the concentration of skim-milk, and the extension rate of reconstituted milk is optimized.The absorbance that compares each concentration of skim-milk and each extension rate of reconstituted milk, calculation deviation, select the condition of deviation minimum to eliminate matrix impact (table 4,5).
Table 4 skim-milk and the absorbancy of diluting milk powder
Table 5 skim-milk and the absorbancy deviation of diluting milk powder
Data in analytical table, when skim-milk concentration is 0.4%, 80 times of powdered milk samples, the absorbancy of the two is the most approaching, and deviation is 0.54%.So select 80 times of dilutions of 0.4% skim-milk and milk powder to eliminate the matrix impact, milk powder redissolves through centrifugal grease removal, after 80 times of dilutions, can make typical curve and matrix curve overlap, in powdered milk sample, the IC of ELISA method
50=102.4 μ gkg
-1, IC
15=9.6 μ gkg
-1.
4, the specificity of penicilloic acid CD-ELISA method
Select the analog of penicillin thiazole acid: penicillin G, penbritin, amoxycilline Trihydrate bp, Cloxacillin Sodium, dicloxacillin, Prostaphlin, Cephalexin Monohydrate Micro/Compacted, analog enzymolysis product, paraxin, gentamicin are done cross reaction.The results are shown in Table 6.In analytical table, data can be found out, penicilloic acid and penicillin G have faint cross reaction, because the artificial antigen of penicilloic acid is to be prepared by penicillin G, structure and penicillin G are very similar, so show certain cross reaction, but because the preparation of enzyme target is to utilize penicilloic acid, so result shows that cross reaction is very faint.The cross reacting rate of other penicillin medicines and degradation product thereof and antibody all is less than 0.01%, and common antibiotics and antibody is no cross reaction also, and this illustrates that prepared antibody has very high specificity to penicilloic acid.
Table 6 penicilloic acid antibodies specific
5 accuracy
Add respectively the penicilloic acid of 3 different content levels in milk and milk powder, detect the calculation sample recovery of standard addition by the direct competitive ELISA method of setting up.The rate of recovery of ELISA method is at 72.75%-93.25%, and the variation coefficient is less than 15%, thus think the method error within the acceptable range, the detection method of foundation meets the accuracy in detection requirement.
Table 7ELISA adds recovery test (n=3)
6 doses of box stability studies and improvement
The technical key of quick detection kit is in transportation and preservation process, the stability of biotechnological formulation (antibody, enzyme-labelled antigen etc.).In order to study, integrate with practical application, exploitation penicilloic acid direct competitive ELISA quick detection kit, experimental study antibody used and enzyme target stability, for the exploitation of quick detection kit lays the foundation.
1) stability of body
Antibody has certain negative impact to activity after lyophilize, in the process by dilution antibody, adds protective material, can make still to keep biological activity after the antibody freeze-drying.But protectant interpolation may cause certain influence to detection sensitivity; Simultaneously, test kit more or less all can be subject to the impact of temperature variation in transportation and preservation process, and the length of lower shelf time of differing temps also may affect the biological activity of antibody, typical temperature is higher, time is longer, and antibody is influenced larger, and activity decreased will be faster.Therefore, need to weigh the impact of these preservation condition antagonist activity, unaffected to guarantee detecting performance.
The stability of lyophilized antibodies
Take existing coated antibody as contrast, and comparative analysis adds the impact of antibody protective agent postlyophilization antagonist stability, as shown in Figure 5.After the antibody freeze-drying, each control of the concentration rate changes and the absorbance loss all is less than 10%, within can accepting scope.
The stability of antibody to temperature
Relatively antibody is under 4 ℃ and 37 ℃ of conditions, places respectively the IC of direct competitive ELISA of the correspondence of 1,3,5,7 day
50value and absorbancy change.If antibody can be stablized and deposit 7 days under 37 ℃, remarkable change does not occur in the absorbance of direct competitive ELISA and detection sensitivity, and this antibody can be stablized and deposit 6 to 12 months under 4 ℃.The results are shown in Table 8.
Table 8 antibody stability experiment result (n=3)
Result shows, under 4 ℃ of conditions, preserves antibody, and F is less than threshold value 21.19, there was no significant difference, detection sensitivity ripple disable; Preserve antibody under 37 ℃ of conditions, F is less than threshold value 21.19, there was no significant difference, detection sensitivity and ripple disable.Therefore can guarantee to place and still can keep antibody activity by 1 year in six months under 4 ℃ of conditions, detect performance substantially constant.
2) stability of mark antigen
Under 4 ℃ and 37 ℃ conditions of comparison, absorbancy and IC that competitive ELISA corresponding to enzyme-labelled antigen of placing respectively 1,3,5,7 day detects
50.If enzyme-labelled antigen can be stablized and deposit 7 days under 37 ℃, this enzyme-labelled antigen can be stablized and deposit 6 to 12 months under 4 ℃.The results are shown in Table 9.
Table 9 enzyme labelling thing stability experiment result (n=3)
The above; it is only preferably embodiment of the present invention; protection scope of the present invention is not limited to this; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses, the simple change of the technical scheme that can obtain apparently or equivalence are replaced and are all fallen within the scope of protection of the present invention.
Claims (5)
1. a benzyl penicilloic acid artificial antigen, is characterized in that, molecular formula is:
2. the preparation method of the described benzyl penicilloic acid of claim 1 artificial antigen, is characterized in that, comprises the following steps:
(1) get (2S, 5R, 6R)-3,3-dimethyl-6-(2-phenylacetylamino)-7-oxo-4-thia-1-azabicyclo [3,2,0] heptane-2-formic acid sylvite 1.500g, add 4.0ml1M hydrochloric acid, obtain haptens (2S, 5R, 6R)-3,3-dimethyl-6-(2-phenylacetylamino)-7-oxo-4-thia-1-azabicyclo [3,2,0] heptane-2-formic acid white solid, distilled water washing drying can obtain haptens;
(2) getting gained white solid 0.5000g is dissolved in the 2ml tetrahydrofuran (THF), add 0.1500gN-N-Hydroxysuccinimide and 0.5000gN, the N-dicyclohexylcarbodiimide, under 22-25 ℃, stirring reaction spends the night, then be put in 4 ℃ of refrigerators interior 18 hours, the centrifugal precipitation of removing, upper strata Acibenzolar liquid is joined in the 2mlPBS solution that pH7.0 is dissolved with the 10mg keyhole limpet hemocyanin, after stirring, under 4 ℃, react 5 hours, by the reaction solution dialysis tubing of packing into, under 4 ℃, in the PBS of pH7.4, dialysis can obtain artificial antigen in three days;
(3) the haptens 1.9mg of synthesized is dissolved in 2ml dimethyl formamide wiring solution-forming A, 0.54mg horseradish peroxidase is dissolved in wiring solution-forming B in the 0.2mol/L phosphoric acid buffer of 2ml pH7.4, then under ice bath, solution A being added to solution B slowly, react and spend the night under 4 ℃ after stirring, finally reaction solution is inserted in dialysis tubing, the 0.2mol/L phosphoric acid buffer dialysis with pH7.4 under 4 ℃ can obtain enzyme-labelled antigen in three days.
3. a benzyl penicilloic acid artificial antibody preparation method, is characterized in that, comprises the following steps:
(1) initial immunity: get 1mg and be dissolved in the solution that 0.9% NaCl solution and Freund's complete adjuvant equal-volume prepare according to the synthetic artificial antigen of the described method of claim 2, carry out animal immune;
(2) booster immunization: be dissolved in the above-mentioned artificial antigen of 0.5mg the solution that 0.9% NaCl solution and Freund's incomplete adjuvant equal-volume are prepared, carry out animal immune;
(3) antibody purification: the periodic monitor animal's antibody is tired, when antibody reaches suitable tiring to certain envelope antigen, gather blood, and centrifugal acquisition antiserum(antisera), use G-Sepharose albumen affinity column antagonistic Serum to carry out purifying, obtain the specific antibody of anti-benzyl penicilloic acid.
4. the application of the described benzyl penicilloic acid of claim 1 artificial antigen in the benzyl penicilloic acid detects.
5. the application of the described benzyl penicilloic acid of claim 3 artificial antibody in the benzyl penicilloic acid detects.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104614529A (en) * | 2015-01-14 | 2015-05-13 | 北京农学院 | Ampicillin penicilloic acid detection kit and detection method |
| CN104764878A (en) * | 2015-03-06 | 2015-07-08 | 天津科技大学 | An immunocolloidal gold test strip for detecting penicilloic acid and a preparing method thereof |
| WO2018030840A1 (en) * | 2016-08-12 | 2018-02-15 | 가톨릭대학교 산학협력단 | Composition for hardening soft tissue |
| CN107904210A (en) * | 2017-12-27 | 2018-04-13 | 江南大学 | One plant of procaine monoclonal antibody hybridoma cell strain 4G6 and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101429241A (en) * | 2008-12-04 | 2009-05-13 | 浙江大学 | Penicillin and carrier protein couplet product, method for producing beta-lactam penicillin antibody, and uses thereof |
| CN102040661A (en) * | 2009-10-19 | 2011-05-04 | 天津科技大学 | Artificial antigen and specific antibody of veterinary drug penicillin G degradation product benzylpenicilloic thiazole acid |
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- 2013-10-08 CN CN201310460758.9A patent/CN103483445A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101429241A (en) * | 2008-12-04 | 2009-05-13 | 浙江大学 | Penicillin and carrier protein couplet product, method for producing beta-lactam penicillin antibody, and uses thereof |
| CN102040661A (en) * | 2009-10-19 | 2011-05-04 | 天津科技大学 | Artificial antigen and specific antibody of veterinary drug penicillin G degradation product benzylpenicilloic thiazole acid |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104614529A (en) * | 2015-01-14 | 2015-05-13 | 北京农学院 | Ampicillin penicilloic acid detection kit and detection method |
| CN104764878A (en) * | 2015-03-06 | 2015-07-08 | 天津科技大学 | An immunocolloidal gold test strip for detecting penicilloic acid and a preparing method thereof |
| WO2018030840A1 (en) * | 2016-08-12 | 2018-02-15 | 가톨릭대학교 산학협력단 | Composition for hardening soft tissue |
| US10745417B2 (en) | 2016-08-12 | 2020-08-18 | The Catholic University Of Korea Industry-Academic Cooperation Foundation | Composition for hardening soft tissue |
| US11773109B2 (en) | 2016-08-12 | 2023-10-03 | The Catholic University Of Korea Industry-Academic Cooperation Foundation | Composition for hardening soft tissue |
| CN107904210A (en) * | 2017-12-27 | 2018-04-13 | 江南大学 | One plant of procaine monoclonal antibody hybridoma cell strain 4G6 and its application |
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