CN100418982C - Lomefloxacin conjugate and its preparing method and use - Google Patents
Lomefloxacin conjugate and its preparing method and use Download PDFInfo
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- CN100418982C CN100418982C CNB2006100432091A CN200610043209A CN100418982C CN 100418982 C CN100418982 C CN 100418982C CN B2006100432091 A CNB2006100432091 A CN B2006100432091A CN 200610043209 A CN200610043209 A CN 200610043209A CN 100418982 C CN100418982 C CN 100418982C
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Abstract
The present invention discloses a Lomefloxacin conjugate in general formula (I). The Lomefloxacin conjugate is formed by coupling Lomefloxacin semiantigen, and bovine serum albumin or egg albumin as carrier substances which can generate immunogenicity, wherein n stands for molecular number of Lomefloxacin combined with bovine serum albumin molecule, the n is an integer between one and twenty; BSA is the bovine serum albumin, and the molecular weight range is between 6.6KDa and 6.9KDa. The present invention also discloses a method for preparing the Lomefloxacin conjugate; in the preparation method, Lomefloxacin is coupled with the carrier substance which can generate the immunogenicity to be combined into the conjugate which can induce an animal immunization system to generate antibodies. Through immune white rabbits in New Zealand, the Lomefloxacin conjugate is prepared into antiserum with the potency ratio of 1: 6400, and the minimum detection limit of the antiserum is 1 ppb. The present invention has the characteristics of simple and convenient method, rapid speed, specificity and accuracy, and provides a foundation for preparing enzyme immunoassay agent boxes of Lomefloxacin.
Description
Technical field
The present invention relates to a kind of conjugate and preparation method thereof and application of quinolone antibiotic, relate in particular to the conjugate and preparation method thereof and application of a kind of lomefloxacin (lomefloxacin).Belong to the antibiolics field of immunodetection.
Background technology
The following title that the present invention relates to is applicable to whole specification sheets and claims:
BSA: bovine serum albumin (Bovine Serum Albumin), Sigma company product
PBS: phosphoric acid buffer (Phosphate Buffered Saline) (0.01 M, pH=7.40)
Sephadex-G75: dextrane gel, Sigma company product
CBSA: through ethylene diamine-modified bovine serum albumin
Dialysis membrane: U.S.'s associating charing (United Carbide) company's product
Lomefloxacin: China Veterinery Drug Inspection Office
EDC: ethyl [3-(dimethylin) propyl group] carbodiimide, be called for short EDC, Sigma company product
Lomefloxacin (lomefloxacin) belongs to quinolone antibiotic.This class microbiotic is because its has a broad antifungal spectrum, and sterilizing ability is strong, be continue terramycin after in the beasts, birds and aquatic products breed medicine of widespread use.This class medicine can effectively prevent and treat poultry bacterial and Mycoplasma disease.Lomefloxacin is wherein the most frequently used one of the most effective medicine.But people find gradually by research, quinolone antibiotic residual toxic to human body in animal-derived food.As the shared medicine of a kind of people and animals, quinolone antibiotic residual very big to human health damage in food by food chain.The K of New England Minnesota State Department of Health
IRKE S
MITHHeaded by research group find that poultry uses quinolones to make Campylobacter produce resistance.According to the investigation of U.S. CDC, a kind of Campylobacter that causes disease by food to the resistance of quinolones by 1998 13.6% rise to 1999 17.6%.And China does not issue the detection method and the respective standard of quinolones residual in the animal-derived food as yet.The U.S. has begun to forbid quinolone antibiotic owing to consider the cross infection of this class medicine in livestock and poultry breeding industry.Though China does not forbid quinolone antibiotic as yet in the beasts, birds and aquatic products aquaculture, strict withdrawal time regulation is arranged.Along with the raising and the China joined WTO of living standards of the people, also more and more higher to the quality requirements of animal-derived food, the residual detection of food Chinese traditional medicine will be legal, routinizes.Therefore, research quinolones residual in food limited the quantity of and detection method is very important.
In the residual detection of antibiotic medicine, instrumental method such as liquid chromatography and mass spectrum and liquid phase mass spectrometry are most widely used methods.These methods are accurate, and are stable, reliable, can be used as standard method.But instrumental method costs an arm and a leg, and is time-consuming longer, and causes organic solvent to pollute, and needs large-scale instrument and equipment, needs special technician, so be difficult to be used for execute-in-place.Enzyme linked immunosorbent detection method (ELISA) provides a kind of fabulous scanning means.This method has fast, and is accurately simple and easy, do not need advantages such as the technician operates, and this makes the ELISA method become a kind of ideal, can be used for the detection method of conventional sweep.The core of enzyme linked immunosorbent detection method is to need high-quality antibody.Most antibiotics comprises that quinolones all is the small molecules organic compound, does not have immunogenicity, is referred to as haptens.So, must be transformed into the immunogen (being referred to as complete antigen again) that can cause animal immuning system generated antibody to these compounds.By retrieval, now in the world as yet not relevant for the report of the immunogenic synthetic and Antibody Preparation of lomefloxacin, and the ELISA test kit of present domestic detection lomefloxacin drug residue is mostly purchased in abroad, storage life is short, the price height, far can not satisfy the detection needs, the immunogenic synthetic and Antibody Preparation of therefore studying lomefloxacin just seems very necessary.
Summary of the invention
At above-mentioned the deficiencies in the prior art, the problem to be solved in the present invention is: provide a kind of and can cause animal immune system produces the antibody of specific reaction at lomefloxacin immunogen, i.e. conjugate of lomefloxacin (1omefloxacin) and preparation method thereof.Simultaneously, the present invention also provides the conjugate of described lomefloxacin as the application of immunogen in preparation lomefloxacin specific reaction antibody.
The conjugate of lomefloxacin of the present invention is made of lomefloxacin haptens and antigenic carrier substance bovine serum albumin of generation or ovalbumin coupling.
Wherein: the immunogenic carrier substance of above-mentioned generation is bovine serum albumin preferably.
The conjugate of lomefloxacin of the present invention, its general structure is as (I)
Wherein: n: for the molecule number of a bovine serum albumin molecule bonded lomefloxacin, described n is an integer 1～20, BSA is bovine serum albumin (Bovine Serum Albumin), molecular weight ranges is 6.6KDa～6.9Kda;
Above-mentioned conjugate demonstrates following physical chemical characteristics:
(1) outward appearance: white powder solid;
(2) ultra-violet absorption spectrum: 288nm, 322nm.
The conjugate of above-mentioned lomefloxacin, wherein n is preferably integer 5～15, and the BSA molecular weight ranges is 6.7KDa～6.8KDa.
The preparation method of the conjugate of above-mentioned lomefloxacin is: lomefloxacin is coupled together with producing immunogenic carrier substance, be combined into and have the conjugate that brings out animal immuning system generated antibody, and keep the biological activity of this conjugate constant.
The preparation method of the conjugate of above-mentioned lomefloxacin, finished by following steps:
(1) preparation of solution A: with lomefloxacin, N-Hydroxysuccinimide, ethyl [3-(dimethylin) propyl group] carbodiimide (EDC), be dissolved in the dimethyl formamide with mol ratio 1: 2～8: 5～15, reaction generates the active intermediate of lomefloxacin and ethyl [3-(dimethylin) propyl group] carbodiimide, and is standby;
(2) preparation of cBSA: under 0～4 ℃ of condition, earlier quadrol being dissolved in pH is 7.38～7.56, and concentration is in 0.01M～0.02M phosphate buffer solution, and transferring pH with concentrated hydrochloric acid is 7.38～7.56; Mol ratio with quadrol: BSA: EDC is 15～25: 1: 15～25 amount takes by weighing BSA and EDC, adds then in the ethylenediamine solution, and stirring reaction is 2～4 hours under 20 ℃ ± 5 ℃ conditions; The reaction soln of quadrol and BSA under 0～4 ℃ of condition, is stirred dialysis 70～80 hours with above-mentioned phosphoric acid buffer, used distill water dialysis then instead 20～30 hours, changed a dialyzate in per 6 hours; At 0～4 ℃,, get supernatant liquor with the solution after 13000 rev/mins of centrifugal above-mentioned dialysis 15 minutes; The freeze-drying supernatant liquor obtains white powder solid cBSA, and is standby;
(3) preparation of solution B: it is 7.38～7.56 that cBSA is dissolved in pH, and concentration is in the phosphate buffer solution of 0.01M～0.02 M, is made into the solution of 10.0 ± 5.0mg/ml, and is standby;
(4) be that 15～50: 1 amount is got solution A and solution B respectively by the mol ratio of lomefloxacin and cBSA, under 20 ℃ ± 5 ℃ temperature, solution A dropwise joined in the solution B under the continuous whipped state, reacted 4～6 hours, obtain solution C;
(5) solution C stirs dialysis 70～80 hours with above-mentioned phosphoric acid buffer, uses distill water dialysis again instead 20～30 hours, dialyzate of replacing in per 6 hours; Then under 0～4 ℃,, get supernatant liquor with the solution after 13000 rev/mins of centrifugal above-mentioned dialysis 15 minutes;
(6) freeze-drying supernatant liquor obtains the conjugate of white lomefloxacin.
In the preparation method of the conjugate of above-mentioned lomefloxacin: the mol ratio of described lomefloxacin of step (1) and N-Hydroxysuccinimide, EDC is preferably 1: 5: 10.
In the preparation method of the conjugate of above-mentioned lomefloxacin: the mol ratio of described quadrol of step (2) and bovine serum albumin, EDC is preferably 20: 1: 20.
In the preparation method of the conjugate of above-mentioned lomefloxacin: the described phosphoric acid buffer pH of step (2) (3) is preferably 7.40, and concentration is preferably 0.01M.
In the preparation method of the conjugate of above-mentioned lomefloxacin: the mole ratio of described lomefloxacin of step (4) and bovine serum albumin is preferably 30: 1.
The conjugate of lomefloxacin of the present invention is as the application of immunogen in preparation lomefloxacin specific reaction antibody.
Utilize the technical scheme of the present invention can be successfully haptens lomefloxacin and particularly bovine serum albumin BSA coupling of carrier proteins, can cause immune response in animal body thereby synthesized, produce complete immunogen---the conjugate of lomefloxacin of antibody.
The conjugate that utilizes lomefloxacin of the present invention has successfully obtained the haptens lomefloxacin is had the antibody of specific reaction as the immunogen immune new zealand white rabbit.Through the ELISA experimental identification, utilize the lomefloxacin specific reaction antibody of the conjugate of lomefloxacin of the present invention as immunogen preparing, its antiserum titre reaches 1: 6400, and its lowest detection is limited to 1ppb.
The preparation success of the conjugate of above-mentioned lomefloxacin and high lomefloxacin specific reaction antibody of tiring is for the enzyme-linked immunologic detecting kit for preparing lomefloxacin provides the foundation.In actual applications, the lomefloxacin specific reaction antibody of described preparation is plated in the micropore dish, just can be used for checking the residual of lomefloxacin in the animal-derived food.Because it is simple and easy that the method for the invention has, special fast, characteristic of accurate is so can be used for the usefulness of the preliminary scanning detection of food test quarantine.So not only can save a large amount of proving times, can also be used for execute-in-place, thereby it is time-consuming longer to have remedied instrumental method, need the large-scale instrument and equipment support, need special technician's operation, difficulty is used for on-the-spot deficiency.So haptens lomefloxacin and carrier proteins be bovine serum albumin BSA conjugate synthetic and sero-fastly successfully be prepared as this quick test method and lay a good foundation particularly.
Embodiment
Embodiment 1
(1) preparation of liquid A: take by weighing lomefloxacin 10.00mg, N-Hydroxysuccinimide 13.12mg, EDC 27.31mg is dissolved in the 3ml dimethyl formamide, and reaction generates the active intermediate of lomefloxacin and EDC, and is standby;
(2) preparation of cBSA: under 0～4 ℃ of condition, earlier quadrol 18.03mg being dissolved in 20ml pH is 7.40, and concentration is in the 0.01M phosphate buffer solution, and transferring pH with concentrated hydrochloric acid is 7.40; Take by weighing 1000.00mg BSA (molecular weight 68,000) and 57.51mg EDC respectively, add then in the ethylenediamine solution, stirring reaction is 2 hours under 20 ℃ of conditions; The reaction soln of quadrol and BSA under 0～4 ℃ of condition, is stirred dialysis 70 hours with above-mentioned phosphoric acid buffer, used distill water dialysis then instead 24 hours, changed a dialyzate in per 6 hours; At 0～4 ℃,, get supernatant liquor with the solution after 13000 rev/mins of centrifugal above-mentioned dialysis 15 minutes; The freeze-drying supernatant liquor obtains white powder solid cBSA, and is standby;
(3) preparation of solution B: it is 7.40 that cBSA is dissolved in pH, and concentration is in the phosphate buffer solution of 0.01M, is made into the solution of 10.0mg/ml, and is standby;
(4) be that 20: 1 amounts are got solution A and solution B respectively by the mol ratio of lomefloxacin and cBSA, under 20 ℃ of temperature, solution A dropwise joined in the solution B under the continuous whipped state, reacted 6 hours, obtain solution C;
(5) solution C stirs dialysis 70 hours with above-mentioned phosphoric acid buffer, uses distill water dialysis again instead 24 hours, dialyzate of replacing in per 6 hours; Then under 0～4 ℃,, get supernatant liquor with the solution after 13000 rev/mins of centrifugal above-mentioned dialysis 15 minutes;
(6) freeze-drying supernatant liquor obtains the conjugate of white lomefloxacin.
Embodiment 2
(1) preparation of A: take by weighing lomefloxacin 10.00mg, N-Hydroxysuccinimide 16.40mg, EDC 54.63mg is dissolved in the 4.6ml dimethyl formamide, and reaction generates the active intermediate of lomefloxacin and EDC, and is standby;
(2) preparation of cBSA: under 0～4 ℃ of condition, earlier quadrol 18.03mg being dissolved in 20ml pH is 7.40, and concentration is in the 0.01M phosphate buffer solution, and transferring pH with concentrated hydrochloric acid is 7.40; Take by weighing 1000.00mgBSA (molecular weight 68,000) and 57.51mg EDC respectively, add then in the ethylenediamine solution, stirring reaction is 4 hours under 25 ℃ of conditions; The reaction soln of quadrol and BSA under 0～4 ℃ of condition, is stirred dialysis 72 hours with above-mentioned phosphoric acid buffer, used distill water dialysis then instead 30 hours, changed a dialyzate in per 6 hours; At 0～4 ℃,, get supernatant liquor with the solution after 13000 rev/mins of centrifugal above-mentioned dialysis 15 minutes; The freeze-drying supernatant liquor obtains white powder solid cBSA, and is standby;
(3) preparation of solution B: it is 7.40 that cBSA is dissolved in pH, and concentration is in the phosphate buffer solution of 0.01M, is made into the solution of 10.0mg/ml, and is standby;
(4) be that 30: 1 amounts are got solution A and solution B respectively by the mol ratio of lomefloxacin and cBSA, under 25 ℃ of temperature, solution A dropwise joined in the solution B under the continuous whipped state, reacted 4 hours, obtain solution C;
(5) solution C stirs dialysis 72 hours with above-mentioned phosphoric acid buffer, uses distill water dialysis again instead 30 hours, dialyzate of replacing in per 6 hours; Then under 0～4 ℃,, get supernatant liquor with the solution after 13000 rev/mins of centrifugal above-mentioned dialysis 15 minutes;
(6) freeze-drying supernatant liquor obtains the conjugate of white lomefloxacin.
Embodiment 3
(1) preparation of liquid A: take by weighing lomefloxacin 10.00mg, N-Hydroxysuccinimide 26.24mg, EDC 81.95mg is dissolved in the 10ml dimethyl formamide, and reaction generates the active intermediate of lomefloxacin and EDC, and is standby;
(2) preparation of cBSA: under 0～4 ℃ of condition, earlier quadrol 18.52mg being dissolved in 20ml pH is 7.56, and concentration is in the 0.02 M phosphate buffer solution, and transferring pH with concentrated hydrochloric acid is 7.56; Take by weighing 1000.00mg BSA (molecular weight 67,000) and 57.51mg EDC respectively, add then in the ethylenediamine solution, stirring reaction is 3 hours under 22 ℃ of conditions; The reaction soln of quadrol and BSA under 0～4 ℃ of condition, is stirred dialysis 80 hours with above-mentioned phosphoric acid buffer, used distill water dialysis then instead 20 hours, changed a dialyzate in per 6 hours; At 0～4 ℃,, get supernatant liquor with the solution after 13000 rev/mins of centrifugal above-mentioned dialysis 15 minutes; The freeze-drying supernatant liquor obtains white powder solid cBSA, and is standby;
(3) preparation of solution B: it is 7.56 that cBSA is dissolved in pH, and concentration is in the phosphate buffer solution of 0.02 M, is made into the solution of 12.0mg/ml, and is standby;
(4) be that 50: 1 amounts are got solution A and solution B respectively by the mol ratio of lomefloxacin and cBSA, under 22 ℃ of temperature, solution A dropwise joined in the solution B under the continuous whipped state, reacted 5 hours, obtain solution C;
(5) solution C stirs dialysis 80 hours with above-mentioned phosphoric acid buffer, uses distill water dialysis again instead 20 hours, dialyzate of replacing in per 6 hours; Then under 0～4 ℃,, get supernatant liquor with the solution after 13000 rev/mins of centrifugal above-mentioned dialysis 15 minutes;
(6) freeze-drying supernatant liquor obtains the conjugate of white lomefloxacin.
(7) carry out purifies and separates with Sephadex-G75 (Sigma), (0.01M pH=7.40) is elutriant with PBS.
Collect the pure product of conjugate of lomefloxacin.The conjugate that freeze-drying obtains lomefloxacin is a lomefloxacin immunogen pressed powder.
Embodiment 4
The preparation purifying and the detection of antibody
1. the preparation of antibody
Select the conjugate of the prepared lomefloxacin of the foregoing description 2 to carry out the animal immune experiment with preparation antibody as immunogen.
Get the solution 1ml of conjugate of the lomefloxacin of 1mg/ml, add isopyknic Freund's complete adjuvant, after fully emulsified, give the male and healthy new zealand white rabbit of four body weight 2kg through subcutaneous multi-point injection, 1ml/ only exempts to carry out two after fully emulsified with amount antigen and Freund's incomplete adjuvant after 15 days, two exempt from after, every 15 days booster immunizations once, the antigen amount reduces by half, and immunity is 5 times altogether.Last immunity is after 7 days, the heart blood sampling, room temperature left standstill 1 hour, 0-4 ℃ is spent the night, 13000 rev/mins centrifugal 15 minutes, collect serum ,-20 ℃ of preservations, standby.
2. purifying antibody
Adding saturated ammonium sulphate to the final concentration of ammoniumsulphate soln under the whipped state in the antiserum(antisera) of above-mentioned preparation is that volume percent is 50%, and 0-4 ℃ of placement spent the night, and has throw out to separate out; Centrifugal 15 minutes with 13000 rev/mins, abandon supernatant liquor, the PBS that adds 0.01M, pH7.4 in throw out is to resolution of precipitate, and adding saturated ammonium sulphate solution to the final concentration of ammoniumsulphate soln then is that volume percent is 33%, 0-4 ℃ of placement spent the night, and has throw out to separate out; With 13000 rev/mins centrifugal 15 minutes, abandon supernatant liquor, the PBS that adds 0.01M, pH7.4 in throw out is to resolution of precipitate.With the above-mentioned purifying thing PBS with 0.01M, pH7.4,0-4 ℃ of dialysis changed dialyzate 3 times, adds the quality volume percent then and be 0.02% sodiumazide, and-20 ℃ of preservations are standby.
3. the enzyme linked immunosorbent detection of antibody
(1) titration: method adopts conventional indirect enzyme-linked immunosorbent absorption detection method:
On the enzyme plate in 96 holes, with the lomefloxacin in 100 μ l/ holes and the conjugate of ovalbumin (10 μ g/ml) bag quilt, 0-4 ℃ of placement spent the night, and uses PBST (the PBS+ volume percent of 1000mlpH7.4, concentration 0.01M is 0.05%Tween20) to wash plate four times then; With the sealing of 250 μ l/ hole confining liquids (1000mlPBST+ quality volume percent is 1% ovalbumin), room temperature was placed 3 hours, washed plate; Behind the flush away confining liquid, add the antiserum(antisera) in 100ul/ hole, room temperature was placed 2 hours, washed plate; After the flush away antiserum(antisera), every hole adds the goat anti-rabbit igg 100ul of 1: 1000 horseradish peroxidase-labeled, and room temperature was placed 1 hour, washed plate; Add the colour developing of substrate O-Phenylene Diamine, room temperature is placed 10min, adds 2MHCl again and stops.Microplate reader A492nm detects.
After measured: the conjugate antibody titer of lomefloxacin of the present invention reaches 1: 6400.
The judgement of tiring is tired for the enzyme linked immunosorbent detection of this antibody greater than the highly diluted multiple of 2: 1 serum with P/N.
Wherein: above-mentioned P surveys the absorbance that serum is measured at a certain extension rate in generation, and above-mentioned N is negative to impinging upon the absorbance that corresponding extension rate is measured.
(2) specific assay:
Determination step and titration are similar, under the envelope antigen and antibody concentration condition of above-mentioned the best, add lomefloxacin solution (from 100ppm-1ppt) when adding antibody, combine limited antibody with the envelope antigen competition, the concentration of lomefloxacin medicine is high more, antibody just combines fewly more with envelope antigen, thereby colour developing is shallow more, and absorbance is low more.Compare with blank (only add antibody, do not add the absorbance of lomefloxacin medicine) again, to determine antibodies specific.
Better by measuring antibodies specific, its lowest detectable limit can reach 1ppb, and detection sensitivity is higher, and less with the cross reaction of quinolones other medicines.
Embodiment 5
The conjugate of preparation lomefloxacin and ovalbumin:
(1) preparation of A: take by weighing lomefloxacin 10.00mg, N-Hydroxysuccinimide 16.40mg, EDC 54.63mg is dissolved in the 4.6ml dimethyl formamide, and reaction generates the active intermediate of lomefloxacin and EDC, and is standby;
(2) preparation of solution B: it is 7.40 that ovalbumin is dissolved in pH, and concentration is in the phosphate buffer solution of 0.01M, is made into the solution of 10.0mg/ml, and is standby;
(3) be that 30: 1 amounts are got solution A and solution B respectively by the mol ratio of lomefloxacin and ovalbumin, under 25 ℃ of temperature, solution A dropwise joined in the solution B under the continuous whipped state, reacted 4 hours, obtain solution C;
(4) solution C stirs dialysis 72 hours with above-mentioned phosphoric acid buffer, uses distill water dialysis again instead 24 hours, dialyzate of replacing in per 6 hours; Then under 0～4 ℃,, get supernatant liquor with the solution after 13000 rev/mins of centrifugal above-mentioned dialysis 15 minutes;
(5) freeze-drying supernatant liquor obtains the conjugate of white lomefloxacin and ovalbumin.
Claims (7)
1. the conjugate of a lomefloxacin, its general structure is as (I)
(I)
Wherein: n: for the molecule number of a bovine serum albumin molecule bonded lomefloxacin, described n is an integer 1～20, BSA is a bovine serum albumin, molecular weight ranges is 6.6KDa～6.9KDa;
Above-mentioned conjugate demonstrates following physical chemical characteristics:
(1) outward appearance: white powder solid;
(2) ultra-violet absorption spectrum: 288nm, 322nm.
2. the conjugate of lomefloxacin as claimed in claim 1, it is characterized in that: described n is an integer 5～15, and the BSA molecular weight ranges is 6.7KDa～6.8KDa.
3. the preparation method of the conjugate of the described lomefloxacin of claim 1, finished by following steps:
(1) preparation of solution A: with lomefloxacin, N-Hydroxysuccinimide, ethyl [base in the 3-(dimethylin)] carbodiimide, be dissolved in the dimethyl formamide with mol ratio 1: 2～8: 5～15, reaction generates the active intermediate of lomefloxacin and ethyl [3-(dimethylin) propyl group] carbodiimide, and is standby;
(2) preparation of cBSA: under 0～4 ℃ of condition, earlier quadrol being dissolved in pH is 7.38～7.56, and concentration is in 0.01M～0.02M phosphate buffer solution, and transferring pH with concentrated hydrochloric acid is 7.38～7.56; Mol ratio with quadrol: BSA: EDC is 15～25: 1: 15～25 amount takes by weighing BSA and EDC, adds then in the ethylenediamine solution, and stirring reaction is 2～4 hours under 20 ℃ ± 5 ℃ conditions; The reaction soln of quadrol and BSA under 0～4 ℃ of condition, is stirred dialysis 70～80 hours with above-mentioned phosphoric acid buffer, used distill water dialysis then instead 20～30 hours, changed a dialyzate in per 6 hours; At 0～4 ℃,, get supernatant liquor with the solution after 13000 rev/mins of centrifugal above-mentioned dialysis 15 minutes; The freeze-drying supernatant liquor obtains white powder solid cBSA, and is standby;
(3) preparation of solution B: it is 7.38～7.56 that cBSA is dissolved in pH, and concentration is in the phosphate buffer solution of 0.01M～0.02M, is made into the solution of 10.0 ± 5.0mg/ml, and is standby;
(4) be that 15～50: 1 amount is got solution A and solution B respectively by the mol ratio of lomefloxacin and cBSA, under 20 ℃ ± 5 ℃ temperature, solution A dropwise joined in the solution B under the continuous whipped state, reacted 4～6 hours, obtain solution C;
(5) solution C stirs dialysis 70～80 hours with above-mentioned phosphoric acid buffer, uses distill water dialysis again instead 20～30 hours, dialyzate of replacing in per 6 hours; Then under 0～4 ℃,, get supernatant liquor with the solution after 13000 rev/mins of centrifugal above-mentioned dialysis 15 minutes;
(6) freeze-drying supernatant liquor obtains the conjugate of white lomefloxacin.
4. the preparation method of the conjugate of lomefloxacin as claimed in claim 3, it is characterized in that: described lomefloxacin of step (1) and N-Hydroxysuccinimide, EDC mol ratio are 1: 5: 10.
5. the preparation method of the conjugate of lomefloxacin as claimed in claim 3, it is characterized in that: the mol ratio of described quadrol of step (2) and bovine serum albumin, EDC is 20: 1: 20.
6. the preparation method of the conjugate of lomefloxacin as claimed in claim 3, it is characterized in that: the mole ratio of described lomefloxacin of step (4) and bovine serum albumin is 30: 1.
In the claim 1～2 conjugate of each described lomefloxacin as the application of immunogen in preparation lomefloxacin specific reaction antibody.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US5284776A (en) * | 1993-08-17 | 1994-02-08 | Abbott Laboratories | Aqueous quinolone concentration assay |
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| US5284776A (en) * | 1993-08-17 | 1994-02-08 | Abbott Laboratories | Aqueous quinolone concentration assay |
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