CN101915840A - Enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibody - Google Patents
Enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibody Download PDFInfo
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- CN101915840A CN101915840A CN2010102317022A CN201010231702A CN101915840A CN 101915840 A CN101915840 A CN 101915840A CN 2010102317022 A CN2010102317022 A CN 2010102317022A CN 201010231702 A CN201010231702 A CN 201010231702A CN 101915840 A CN101915840 A CN 101915840A
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- chloramphenicol
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- linked immunosorbent
- chloromycetin
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- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical group ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 title claims abstract description 96
- 241000283973 Oryctolagus cuniculus Species 0.000 title claims abstract description 38
- 229960005091 chloramphenicol Drugs 0.000 title claims abstract description 36
- 238000008157 ELISA kit Methods 0.000 title claims description 17
- 239000000758 substrate Substances 0.000 claims abstract description 30
- 238000002965 ELISA Methods 0.000 claims abstract description 28
- 239000000243 solution Substances 0.000 claims abstract description 24
- 239000000427 antigen Substances 0.000 claims abstract description 23
- 102000036639 antigens Human genes 0.000 claims abstract description 23
- 108091007433 antigens Proteins 0.000 claims abstract description 23
- 238000005406 washing Methods 0.000 claims abstract description 12
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- 239000012086 standard solution Substances 0.000 claims abstract description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 10
- 238000006243 chemical reaction Methods 0.000 claims abstract description 8
- 241000283707 Capra Species 0.000 claims abstract description 5
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims abstract description 5
- 229940097572 chloromycetin Drugs 0.000 claims description 60
- 239000007788 liquid Substances 0.000 claims description 28
- 238000010790 dilution Methods 0.000 claims description 24
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- 238000000034 method Methods 0.000 claims description 20
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 12
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 12
- 235000019800 disodium phosphate Nutrition 0.000 claims description 12
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- ZBQCCTCQUCOXBO-UHFFFAOYSA-N 4-(4-aminophenyl)-2,2,6,6-tetramethylcyclohex-3-en-1-amine Chemical compound CC1(C)C(N)C(C)(C)CC(C=2C=CC(N)=CC=2)=C1 ZBQCCTCQUCOXBO-UHFFFAOYSA-N 0.000 claims description 3
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- AYIRNRDRBQJXIF-NXEZZACHSA-N (-)-Florfenicol Chemical compound CS(=O)(=O)C1=CC=C([C@@H](O)[C@@H](CF)NC(=O)C(Cl)Cl)C=C1 AYIRNRDRBQJXIF-NXEZZACHSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- LIRCDOVJWUGTMW-ZWNOBZJWSA-N Chloramphenicol succinate Chemical compound OC(=O)CCC(=O)OC[C@@H](NC(=O)C(Cl)Cl)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 LIRCDOVJWUGTMW-ZWNOBZJWSA-N 0.000 description 2
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- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
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- PFKDFLVWKSDYJO-UHFFFAOYSA-N n-[3-chloro-4-[[4-(4-methoxyphenyl)oxan-4-yl]methylcarbamoyl]phenyl]furan-2-carboxamide Chemical compound C1=CC(OC)=CC=C1C1(CNC(=O)C=2C(=CC(NC(=O)C=3OC=CC=3)=CC=2)Cl)CCOCC1 PFKDFLVWKSDYJO-UHFFFAOYSA-N 0.000 description 2
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- OTVAEFIXJLOWRX-NXEZZACHSA-N thiamphenicol Chemical compound CS(=O)(=O)C1=CC=C([C@@H](O)[C@@H](CO)NC(=O)C(Cl)Cl)C=C1 OTVAEFIXJLOWRX-NXEZZACHSA-N 0.000 description 2
- 229960003053 thiamphenicol Drugs 0.000 description 2
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 1
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- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 206010052804 Drug tolerance Diseases 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
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- 229960002579 chloramphenicol sodium succinate Drugs 0.000 description 1
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- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses an enzyme-linked immunosorbent assay (ELISA) kit for analyzing chloramphenicol residues based on a rabbit monoclonal antibody. The kit comprises a body and a 96-well ELISA plate and reagents arranged in the body, wherein a chloramphenicol envelop antigen is enveloped in the plate; and the reagents include (1) washing concentrate; (2) diluting concentrate; (3) chloramphenicol standard solution; (4) a chloramphenicol antibody; (5) a horseradish peroxidase labeled goat anti-rabbit antibody; (6) substrate diluent; (7) a substrate; (8) substrate development solution; and (9) reaction termination solution. The kit is applicable to detection of chloramphenicol residues in such samples as milk, swine urine, etc. The kit can simultaneously detect mass samples, is convenient and quick, is of great practical significance to field monitoring trace analysis, simultaneously greatly lowers the detection cost and has potential economic value.
Description
Technical field
The present invention relates to kit, relate in particular to a kind of enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibodies.
Background technology
Chloromycetin (Chloramphenicol, CAP), chemical name D-Su Shi-(-)-N-[α-(hydroxymethyl)-beta-hydroxy-p-nitrophenyl ethyl]-2, the 2-dichloro acetamide.It belongs to broad-spectrum antibiotic, and gram-positive bacteria, negative bacterium are all had good inhibition effect, thereby is widely used in herding, culture fishery.But residual CAP has potential harm to the health of human body in animal food.For this reason, many in the world countries have all formulated the high residue standard of chloromycetin, and strictness limits its use.In order to adapt to international competition and the challenge after the accession to WTO, must set up a kind of method for quick with independent intellectual property right.Therefore develop a kind of fast simply, be applicable to that the trace analysis of residual chloromycetin on-site supervision is of great practical significance.
The main method that residual chloromycetin is detected has chromatography, microbial method etc. at present.Though wherein methods such as HPLC method, gas chromatography and liquid chromatography-tandem mass spectrometry have sensitivity, characteristics such as accurate, this class methods pre-treatment is complicated, required instrument costliness, and need those skilled in the art, middle penetration and promotion is difficult to work in unit of primary level.Microorganism detection method is simple to operate, and expense is low, but length consuming time, susceptibility and selectivity are low, and easily omission also false positive can occur, causes erroneous judgement, and it is easy generally to be used for qualitative detection.And immunoassay has certain sensitivity and specificity, and does not need expensive instrument and equipment, for chloromycetin provides a simple and practical analyzing and testing approach.Be applicable to the fast detecting of great amount of samples, the ELISA detection method certainly will become the development trend that detects residual chloromycetin.
The conventional mouse monoclonal antibody of rabbit monoclonal antibodies has more advantages.At first, rabbit can discern the epi-position of more immunizing antigens than mouse.Secondly,, can carry out more fusion experiment, make the high flux screening fused cell become possibility because the rabbit spleen is bigger.This kit is used hybridoma technology and the recombinant antibodies technology obtains the chloromycetin rabbit monoclonal antibodies, develops the chloromycetin indirect enzyme-linked immunosorbent kit based on rabbit monoclonal antibodies, there is no report both at home and abroad.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of have high specific, highly sensitive enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibodies are provided.
Enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibodies comprises box body and 96 hole ELISA Plate and the reagent that are located in the box body, and 96 hole ELISA Plate endoperidiums have the chloromycetin envelope antigen, and the reagent in the box is as follows:
(1) concentrated solution for washing;
(2) dilution concentrate;
(3) chloromycetin standard solution;
(4) chloramphenicol antibody;
(5) horseradish peroxidase mark goat anti-rabbit antibody;
(6) substrate dilution;
(7) substrate;
(8) substrate colour developing liquid;
(9) reaction terminating liquid.
Described chloromycetin envelope antigen is the coupled product of chloromycetin and ovalbumin, and the chloromycetin envelope antigen can combine with the chloramphenicol resistance antibody specificity.
Consisting of of described concentrated solution for washing: sodium chloride 7～9g, potassium dihydrogen phosphate 0.1～0.3g, sodium hydrogen phosphate 3～5g, potassium chloride 0.1～0.3g, polysorbas20 0.5～1ml and deionized water.Consisting of of described dilution concentrate: sodium chloride 7～9g, potassium dihydrogen phosphate 0.1～0.3g, sodium hydrogen phosphate 3～5g, potassium chloride 0.1～0.3g and deionized water.Described chloramphenicol antibody is a rabbit monoclonal antibodies, is to be immune animal with the rabbit, and process hybridoma technology and recombinant antibodies technology finally obtain.Described substrate colour developing liquid is 3,3,5,5-tetramethyl benzidine preparation liquid.Described substrate dilution is the pH5.0 citrate buffer solution, the consisting of of pH5.0 citrate buffer solution: 3～6g citric acid, 6～9g sodium hydrogen phosphate and deionized water.The concentration of described chloromycetin standard solution is: 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.625ng/mL or 0.3125ng/mL.Described substrate is hydrogen peroxide or urea peroxide.Described reaction terminating liquid is 2M sulfuric acid or hydrochloric acid.
The present invention in chloromycetin detects, IC
50Value is 1.06ng/mL, inhibiting rate curve linear fit equation: y=39.705x+48.946, and sensing range is between 0.19-6.05ng/mL, and lowest detectable limit is 0.10ng/mL.Chloramphenicol resistance antibody has higher specificity in the kit, except with Chloramphenicol Succinate acid sodium, Thiamphenicol, the Florfenicol cross reacting rate is respectively 2.09%, outside 18.10%, 12.45% and other quinolones and the equal no cross reaction of sulfa drugs.
The present invention is applicable to the detection of residual chloromycetin in the samples such as milk, honey.This kit can detect sample in enormous quantities simultaneously, and is convenient quick again, and the on-site supervision trace analysis is of great practical significance; The detection cost is reduced greatly, have potential economic worth.
Description of drawings
Fig. 1 is to be the chloromycetin monoclonal antibody indirect competitive ELISA typical curve of matrix with the PBS damping fluid;
Fig. 2 is to be the chloromycetin monoclonal antibody indirect competitive ELISA typical curve of matrix with milk.
Concrete embodiment
The present invention is based on the indirect enzyme-linked immunosorbent absorption kit of rabbit monoclonal antibodies exploitation analyzing chloramphenicol residues, this kit is based on immune response and enzymatic reaction, can detect the residual of chloromycetin in milk and the honey equal samples.Enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibodies comprises box body and 96 hole ELISA Plate and the reagent that are located in the box body.96 hole ELISA Plate endoperidiums have the chloromycetin envelope antigen, and the reagent in the box is as follows: (1) concentrated solution for washing; (2) dilution concentrate; (3) chloromycetin standard solution; (4) chloramphenicol antibody; (5) horseradish peroxidase mark goat anti-rabbit antibody; (6) substrate dilution; (7) substrate; (8) substrate colour developing liquid; (9) reaction terminating liquid.
Described chloromycetin envelope antigen is the coupled product of chloromycetin and ovalbumin, and the chloromycetin envelope antigen can combine with the chloramphenicol resistance antibody specificity; Consisting of of described concentrated solution for washing: sodium chloride 7～9g, potassium dihydrogen phosphate 0.1～0.3g, sodium hydrogen phosphate 3～5g, potassium chloride 0.1～0.3g, polysorbas20 0.5～1ml and deionized water.Consisting of of described dilution concentrate: sodium chloride 7～9g, potassium dihydrogen phosphate 0.1～0.3g, sodium hydrogen phosphate 3～5g, potassium chloride 0.1～0.3g and deionized water.Described chloramphenicol antibody is a rabbit monoclonal antibodies, is to be immune animal with the rabbit, and process hybridoma technology and recombinant antibodies technology finally obtain.Described substrate colour developing liquid is 3,3,5,5-tetramethyl benzidine preparation liquid.Described substrate dilution is the pH5.0 citrate buffer solution, the consisting of of pH5.0 citrate buffer solution: 3～6g citric acid, 6～9g sodium hydrogen phosphate and deionized water.The concentration of described chloromycetin standard solution is: 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.625ng/mL or 0.3125ng/mL.Described substrate is hydrogen peroxide or urea peroxide.Described reaction terminating liquid is 2M sulfuric acid or hydrochloric acid.
Reagent setting in the box:
(1) concentrated solution for washing is 1 bottle, and 25～35mL/ bottle contains sodium chloride 7～9g, potassium dihydrogen phosphate 0.1～0.3g, sodium hydrogen phosphate 3～5g, potassium chloride 0.1～0.3g, polysorbas20 0.5～1ml, is 30～40 times of normal working concentration; (2) 1 bottle of concentrate of dilution, 25～35mL/ bottle contains sodium chloride 7～9g, potassium dihydrogen phosphate 0.1～0.3g, sodium hydrogen phosphate 3～5g, potassium chloride 0.1～0.3g, is 30～40 times of normal working concentration; (3) chloromycetin standard solution (10ng/mL, 5ng/mL, 1ng/mL, 0.5ng/mL, 0.1ng/mL, 0.05ng/mL) is each 1 bottle, the 1mL/ bottle; (4) chloramphenicol antibody 1 pipe, 25～50 μ l/ pipe, 2000～3000 times of dilutions during use; (5) horseradish peroxidase mark goat anti-rabbit antibody (ELIAS secondary antibody) 1 pipe, 50-100 μ l/ pipe, 1000～2000 times of dilutions during use; (6) the substrate dilution is 1 bottle, and 25～35mL/ bottle includes 3～6g citric acid, 6～9g sodium hydrogen phosphate, for the 30-40 of normal use amount doubly; (7) substrate is 1 bottle, 1～5mL/ bottle, and it is hydrogen peroxide or urea peroxide; (8) substrate colour developing liquid is 1 bottle of tetramethyl benzidine preparation liquid, 2～5mL/ bottle, and compound method is that 10mgTMB is dissolved among the 2mLDMSO; (9) reaction terminating liquid is 1 bottle, 15～20mL/ bottle, and it is 2mol/L sulfuric acid or hydrochloric acid.
Embodiment 1: chloromycetin immunizing antigen and envelope antigen synthetic
The concrete operations step is as follows: get CAP micromolecule 80mg, be dissolved in the 1.5mL absolute ethyl alcohol, add 12mL 1mol/L hydrochloric acid.Add zinc powder 65mg, 70 ℃ of water-bath heating 50min.After the cooling,, dropwise add 0.5mol/L NaNO under the pH 1-2 condition at 4 ℃
2Several, lucifuge stirs, and is dusty blue (solution I) until starch potassium iodide paper; Take by weighing BSA 150mg, be dissolved in the 12mL phosphate buffer (PBS, pH 7.4), cooling.(solution II).Then I liquid is slowly added in the II liquid while stirring, add the NaOH solution of 0.2mol/L simultaneously, pH is remained at about 7-8.4 ℃ are stirred 12h, and reaction product is with the PBS 5d that dialyses.Envelope antigen OVA-CPFX prepares with method, is used for coated elisa plate.
The coupling principle of chloromycetin and carrier protein is as follows:
Embodiment 2: the generation of chloromycetin rabbit monoclonal antibodies
1, the immunity of rabbit
Just exempt from the immunizing antigen of 500 μ g is mixed with the equivalent Freund's complete adjuvant, after the emulsification, adopt subcutaneous 5 the injection white rabbits in back (1mL/ only) fully; Three all back immunizing antigens with 200 μ g mix the back and carry out the immunity second time with method with incomplete Freund; Per two all immunity later on once, altogether after the immunity 3～7 times, serum titer reaches 64000 when above, injects with the immunizing antigen auricular vein of 500 μ g, rear neck artery was adopted whole blood in 4 days, the aseptic spleen of getting, preparation Fusion of Cells.
2, the preparation of rabbit monoclonal antibodies
Rabbit splenic lymphocyte and the 240E cell that is in exponential phase are merged, and fusion agent is 50% PEG.Cell after the fusion, is sub-packed in 2 * 20 96 porocyte culture plates that are added with feeder cells as selective medium with HAT, puts 37 ℃, 5%CO
2Cultivate in the incubator.The background height is found in the back preliminary examination of 2 weeks, changes liquid.After one week, filter out 72 of positive colonies, the hybridoma of the initial survey positive is transferred in the 24 porocyte culture plates with indirect elisa method.After one week, it is strong to adopt indirect elisa method, indirect competitive ELISA method to filter out secretory antibody affinity, the hybridoma 9-3 that cell growth state is good.This hybridoma is preserved portion in liquid nitrogen, portion is used for lysis, extracts mRNA, makes up plasmid, produces antibody.Separating mRNA from the 9-3 hybridoma is cloned its heavy chain and light chain gene respectively.Gene is connected with plasmid after enzyme is cut, coated plate picking clone.The Escherichia coli incubated overnight is extracted plasmid, and heavy chain light chain plasmid pairing transfection to 293T cell is expressed.Aseptic collecting cell is got supernatant after centrifugal.6 strain CAP monoclonal antibody plasmids have been obtained after the little commentaries on classics.Determine that by indirect competitive ELISA test, ELISA sandwich method CAP 9-3H1/L2 plasmid enters big reincarnation and produces.Adopt Protein A purification process purifying also to collect the CAP monoclonal antibody, altogether 10mL CAP monoclonal antibody, IgG concentration is 1.23mg/mL.
Embodiment 3: the bag quilt of ELISA Plate
Chloromycetin envelope antigen pH9.6,0.05mol/L carbonate buffer solution (contain 2.93g sodium bicarbonate and 1.59g sodium carbonate, be dissolved in distilled water or ultrapure water 1L) is diluted to 0.2 μ g/mL, adds 50 μ L in every hole of ELISA Plate, 4 ℃ of bags by 12h or 37 ℃ of bags by 2h, the coating buffer body that inclines with PBST washing 3～5 times, pats dry, in every hole, add 250 μ L, 3% skimmed milk then, 37 ℃ were sealed 1 hour, took out back washing 3～5 times, and drying is preserved after sealing film.
Embodiment 4: the measuring principle of kit
At first chloromycetin and macromolecular carrier albumen coupling are made compound as envelope antigen, and make envelope antigen be attached to certain surface of solid phase carriers.Add chloromycetin standard solution or sample (containing diluted sample to be measured) and chloromycetin rabbit monoclonal antibodies to be measured then, chloromycetin standard solution or sample to be tested combine chloramphenicol antibody with the envelope antigen competitiveness of chloromycetin, the antibody that combines with envelope antigen can show color after adding ELIAS secondary antibody, the antibody that combines with the chloromycetin micromolecule that dissociates is removed when washing.Add the substrate colour developing at last, chloromycetin content is many more in chloromycetin mark liquid or the sample, and the antibody that combines with solid phase antigen is few more, last color development habituation just, and the resistance rate is just high more; Otherwise, color development increased response then, inhibiting rate lowers, thereby promptly obtains typical curve according to the standard solution acquisition inhibiting rate of known quantity chloromycetin and the semilog relation mapping between the chloromycetin content earlier, releases the concentration of sample to be tested again according to the inhibiting rate of sample.
Embodiment 5: the pre-treatment of sample to be tested
The pre-treatment of A, milk detecting sample:
Fresh milk in 4 ℃ of centrifugal 20min of following 5000r/min, discards upper strata fat, takes off a layer clear liquid, adds the potassium ferricyanide solution of 0.3 μ L 17%, adds the solution of zinc sulfate of 0.3 μ L 52% after shaking up again, shakes up rapidly.The centrifugal 15min of 5000r/min, getting supernatant, to do certain dilution with PBS stand-by.
The pre-treatment of B, pig urine samples:
Pig urine high speed centrifugation 10min gets the upper strata stillness of night, and 5 times of dilutions are stand-by.
Embodiment 6: the use of chlorine detection mycin kit
(1) reagent preparation:
A, dilution concentrate: the dilution concentrate uses with behind 30～40 times of ultrapure water or the distilled water dilutings in the kit.
B, concentrated solution for washing: concentrated solution for washing uses with behind 30～40 times of ultrapure water or the distilled water dilutings in the kit.
C, substrate dilution: the substrate dilution uses with behind 30～40 times of ultrapure water or the distilled water dilutings in the kit.
(2) the kit operating process is as follows:
A, kit need ambient-temp-stable more than half an hour before using.
B, take out ELISA Plate, add 50 μ L standard specimens or pending sample and join separately in the hole, standard specimen and sample are done 2-4 repetition simultaneously, add 50 μ L then and dilute the chloramphenicol antibody of getting well, and hatch 1 hour for 37 ℃.
C, pour out the liquid in the hole, microwell plate is inverted on the thieving paper pats,, wash 3～5 times, pat dry with 300 μ L cleansing solutions to guarantee to remove fully the liquid in the hole.
D, the good ELIAS secondary antibody 100 μ l of adding dilution reacted 50 minutes.
E, get substrate colour developing liquid 200 μ L and be added among the substrate buffer solution 10mL, add 0.02% substrate hydrogen peroxide again, after the vibration evenly, every hole adds the chromophoric solution for preparing more than the 100 μ L, slight vibration plate, and 15min is hatched in 37 ℃ of dark places.
F, adding 50 μ L stop buffers are measured OD
450The value result of determination.
G, calculate inhibiting rate, draw the ELISA typical curve according to the OD value of variable concentrations standard specimen.
H, the OD that measures according to sample
450Value and extension rate are by the mensuration content ng/mL (being ppb, is that 1g calculates with 1mL liquid) of chloromycetin in the typical curve calculating sample.Chloromycetin content=analytical concentration in the sample * extension rate (ppb).
Embodiment 7:ELISA kit measurement result
1, ELISA typical curve and sensitivity
With standard items concentration logarithm value is horizontal ordinate, relative B/B
0* 100% value is drawn linear standard curve (B for ordinate
0: standard items concentration is the pairing light absorption value of 0ng/mL; B: the pairing light absorption value of other each concentration).Record on the curve that the micromolecular concentration value of pairing chloromycetin is sensitivity when reaching 50% inhibiting rate.This curve has favorable linearity between 0.3125～10ng/mL as can be known from the typical curve of Fig. 1 chloromycetin ELISA average measurement, and the concentration value that reaches 10% inhibiting rate chloromycetin is 0.10ng/mL, comparatively ideal sensitivity is arranged, IC
50Value is 1.06ng/mL.
2, the specificity of ELISA
Adopt the indirect competitive ELISA method, will with the medicine of chloromycetin structural similarity, join simultaneously in the 96 hole ELISA Plate as chloramphenicol sodium succinate, Thiamphenicol, Florfenicol and QNS that some are common and sulfa drugs and chloromycetin rabbit monoclonal antibodies, with the inhibiting rate is ordinate, each sample concentration logarithm is a horizontal ordinate drawing standard curve, calculates IC separately respectively
50Value.Again according to the micromolecular IC of the relative chloromycetin of chloromycetin rabbit monoclonal antibodies
50The IC that is worth relative various competition things with the chloromycetin rabbit monoclonal antibodies
50The ratio of value promptly obtains cross reacting rate (CR%).Crossing-over rate to other medicines the results are shown in Table 1～table 3.
CR%=IC
50Chloromycetin/IC
50Other compete micromolecule * 100%
The cross reactivity of table 1 and structural similarity medicine
The cross reactivity of table 2 and part QNS
Table 3 and part sulfa drugs cross reactivity
3, the degree of accuracy of ELISA and accuracy determination
Adding chloromycetin, to make its concentration in the milk be that 1ng/mL, 5ng/mL, each concentration of 10ng/mL, 15ng/mL are established four and repeated to measure; Adding chloromycetin in pig urine, to make its concentration be 2ng/mL, 4ng/mL, 8ng/mL, and each concentration is established four and repeated to measure respectively.
Because undesirable with typical curve recovery when detecting milk that the PBS buffer system is set up, thus this experiment to have set up with milk be matrix, add the typical curve of chloromycetin titer.As can be seen from Figure 2, micromolecule concentration is good at 0.5ng/mL-20ng/mL half interval contour linear fit, R
2=0.9883.With external standard method the accuracy of indirect elisa method being measured milk sample is detected.Result's (table 4) shows that the recovery is between 75.40%-117.00, and the coefficient of variation satisfies and detects needs between 5.57%-16.39%.
Table 4 milk ELISA accuracy testing result
Use external standard method, the typical curve of setting up with the PBS damping fluid is a foundation, indirect elisa method is measured the accuracy of pig urine samples and is tested.Result's (table 5) shows, with 5 times of pig urine dilutions and add the chloromycetin titer and make its concentration reach 8ng/mL, and 4ng/mL, the average recovery rate of 2ng/mL is respectively 79.38%, 89.50% and 114.50%, and the coefficient of variation is respectively 9.76%, 6.98%, and 1.75%.The recovery and Variation Lines number average satisfy ELISA and detect requirement.
ELISA accuracy testing result in the table 5 pig urine
Claims (10)
1. the enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibodies is characterized in that comprising box body and is located at 96 interior hole ELISA Plate and reagent of box body, and 96 hole ELISA Plate endoperidiums have the chloromycetin envelope antigen, and the reagent in the box is as follows:
(1) concentrated solution for washing;
(2) dilution concentrate;
(3) chloromycetin standard solution;
(4) chloramphenicol antibody;
(5) horseradish peroxidase mark goat anti-rabbit antibody;
(6) substrate dilution;
(7) substrate;
(8) substrate colour developing liquid;
(9) reaction terminating liquid.
2. the enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibodies according to claim 1, it is characterized in that described chloromycetin envelope antigen is the coupled product of chloromycetin and ovalbumin, the chloromycetin envelope antigen can combine with the chloramphenicol resistance antibody specificity.
3. the enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibodies according to claim 1 is characterized in that consisting of of described concentrated solution for washing: sodium chloride 7～9g, potassium dihydrogen phosphate 0.1～0.3g, sodium hydrogen phosphate 3～5g, potassium chloride 0.1～0.3g, polysorbas20 0.5～1ml and deionized water.
4. the enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibodies according to claim 1 is characterized in that consisting of of described dilution concentrate: sodium chloride 7～9g, potassium dihydrogen phosphate 0.1～0.3g, sodium hydrogen phosphate 3～5g, potassium chloride 0.1～0.3g and deionized water.
5. the enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibodies according to claim 1, it is characterized in that described chloramphenicol antibody is a rabbit monoclonal antibodies, be to be immune animal with the rabbit, process hybridoma technology and recombinant antibodies technology finally obtain.
6. the enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibodies according to claim 1 is characterized in that described substrate colour developing liquid is 3,3,5,5-tetramethyl benzidine preparation liquid.
7. the enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibodies according to claim 1, it is characterized in that described substrate dilution is the pH5.0 citrate buffer solution, the consisting of of pH5.0 citrate buffer solution: 3～6g citric acid, 6～9g sodium hydrogen phosphate and deionized water.
8. the enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibodies according to claim 1 is characterized in that the concentration of described chloromycetin standard solution is: 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.625ng/mL or 0.3125ng/mL.
9. the enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibodies according to claim 1 is characterized in that described substrate is hydrogen peroxide or urea peroxide.
10. the enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibodies according to claim 1 is characterized in that described reaction terminating liquid is 2M sulfuric acid or hydrochloric acid.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102288761A (en) * | 2011-05-09 | 2011-12-21 | 广州万孚生物技术有限公司 | Chloramphenicol testing kit and preparation method of same |
| CN102288765A (en) * | 2011-07-07 | 2011-12-21 | 清华大学深圳研究生院 | Immunofluorescence chloramphenicol detecting method based on quantum dots and special kit |
| CN102914653A (en) * | 2012-10-22 | 2013-02-06 | 大连海洋大学 | Chloramphenicol chemiluminiscence ELISA (Enzyme-Linked Immunosorbent Assay) kit |
| CN103018451A (en) * | 2011-09-20 | 2013-04-03 | 北京勤邦生物技术有限公司 | Enzyme-linked immunoassay kit for chloramphenicol detection, and applications thereof |
| CN105319369A (en) * | 2014-07-24 | 2016-02-10 | 江苏维赛科技生物发展有限公司 | Enzyme linked immunosorbent assay kit used for detecting chloramphenicol, and detection method thereof |
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| CN1690709A (en) * | 2004-04-30 | 2005-11-02 | 中国农业大学 | Enzyme linked immuno kit for detecting chloromycetin |
| CN1766629A (en) * | 2005-11-03 | 2006-05-03 | 北京望尔生物技术有限公司 | ELISA kit for detecting chloramphenicols in animal derived food |
| CN101526537A (en) * | 2009-01-12 | 2009-09-09 | 深圳市绿诗源生物技术有限公司 | Elisa reagent for detecting chloramphenicol and method thereof |
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| CN1766629A (en) * | 2005-11-03 | 2006-05-03 | 北京望尔生物技术有限公司 | ELISA kit for detecting chloramphenicols in animal derived food |
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| CN102288761A (en) * | 2011-05-09 | 2011-12-21 | 广州万孚生物技术有限公司 | Chloramphenicol testing kit and preparation method of same |
| CN102288765A (en) * | 2011-07-07 | 2011-12-21 | 清华大学深圳研究生院 | Immunofluorescence chloramphenicol detecting method based on quantum dots and special kit |
| CN102288765B (en) * | 2011-07-07 | 2013-12-04 | 清华大学深圳研究生院 | Immunofluorescence chloramphenicol detecting method based on quantum dots and special kit |
| CN103018451A (en) * | 2011-09-20 | 2013-04-03 | 北京勤邦生物技术有限公司 | Enzyme-linked immunoassay kit for chloramphenicol detection, and applications thereof |
| CN103018451B (en) * | 2011-09-20 | 2016-04-20 | 北京勤邦生物技术有限公司 | The enzyme linked immunological kit of chlorine detection mycin and application thereof |
| CN102914653A (en) * | 2012-10-22 | 2013-02-06 | 大连海洋大学 | Chloramphenicol chemiluminiscence ELISA (Enzyme-Linked Immunosorbent Assay) kit |
| CN105319369A (en) * | 2014-07-24 | 2016-02-10 | 江苏维赛科技生物发展有限公司 | Enzyme linked immunosorbent assay kit used for detecting chloramphenicol, and detection method thereof |
| CN106546741A (en) * | 2016-10-31 | 2017-03-29 | 浙江大学 | Progesterone residue analysis enzyme-linked immune adsorption kit based on single gram of drop antibody of rabbit |
| CN112649556A (en) * | 2020-11-13 | 2021-04-13 | 新疆大学 | Method for rapidly measuring concentration of hydrogen peroxide |
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