CN101182356A - Clenbuterol complete antigen and method for preparing monoclonal antibody thereof - Google Patents
Clenbuterol complete antigen and method for preparing monoclonal antibody thereof Download PDFInfo
- Publication number
- CN101182356A CN101182356A CNA2007100477148A CN200710047714A CN101182356A CN 101182356 A CN101182356 A CN 101182356A CN A2007100477148 A CNA2007100477148 A CN A2007100477148A CN 200710047714 A CN200710047714 A CN 200710047714A CN 101182356 A CN101182356 A CN 101182356A
- Authority
- CN
- China
- Prior art keywords
- clenbuterol
- clenbuterol hydrochloride
- complete antigen
- beaker
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses the preparation methods of a complete antigen of clenbuterol hydrochloride and a monoclonal antibody of the clenbuterol hydrochloride. Firstly, the clenbuterol hydrochloride reacts with sodium nitrite under the acid condition to obtain azo clenbuterol hydrochloride; then the azo clenbuterol hydrochloride coupled with bovine serum albumin under the alkaline condition to prepare for the complete antigen of the clenbuterol hydrochloride. A balb/c pure line rat is immunized, after IELISA test shows that the serum of the rat after immunity is eligible, the cell fusion is processed for preparing for the monoclonal antibody of the clenbuterol hydrochloride. The complete antigen of the clenbuterol hydrochloride prepared by the invention can be used for immunizing the animal. The prepared monoclonal antibody can be used for testing the residual quantity of the clenbuterol hydrochloride in meat, meat products, livestock feed and animal body before the animal is slaughtered. The coupling rate of the clenbuterol hydrochloride in the complete antigen of the clenbuterol hydrochloride obtained by the method of the invention with the bovine serum albumin is 17, and a molecular structure formula thereof is as above.
Description
Technical field
The present invention relates to a kind of clenbuterol complete antigen and anti-Clenbuterol hydrochloride MONOCLONAL ANTIBODIES SPECIFIC FOR method, this antibody can be used for detecting the residual quantity of Clenbuterol hydrochloride in meat product and livestock feed.
Background technology
Clenbuterol hydrochloride (CLB) has another name called clenbuterol hydrochloride, is a kind of β
2Adrenoreceptor agonists.Can quicken the conversion and the decomposition of its body fat behind the animal edible, and promote protein synthesis, thus by illegal retailer be used to raise pig, ox is improved lean ratio and reaps staggering profits.But the people has eaten symptoms such as can producing palpitaition, face neck and limb muscle vibration, dizzy, weak, irregular pulse after the meat product that contains CLB, has had a strong impact on the people's life and social stability.Therefore, China has prohibited CLB is used for the fowl poultry as fodder additives meat production in 1997, and formulated corresponding residue criterion and detection method, promptly with makings analysis (GC-MS) as Deterministic Methods, high performance liquid chromatography (HPLC) is as the semidefiniteness method.Yet the sensitivity of these methods be subjected to sample purifying, step such as concentrate influence big; Moreover these not available complex instrument in methods most of laboratories of needs, cost an arm and a leg, need the professional to operate, on-the-spot mass detection be can not carry out, and length consuming time, testing cost height detected, can not be extensive use of.Utilizing Immunological Method that clenobuterol hydrochloride residue in the livestock product is detected in recent years is a kind of new technology that has development prospect and application prospect.That this method has is easy and simple to handle, batch detection, highly sensitive and expense is low, but the key of this technology is the monoclonal antibody that needs the anti-Clenbuterol hydrochloride of acquisition high specific, the specificity and the avidity of the monoclonal antibody of the domestic anti-Clenbuterol hydrochloride of having reported are not high at present, it is tired and the highlyest also has only about 100,000, its major cause is that also synthetic clenbuterol complete antigen three-dimensional arrangement is not strong, stimulates animal body can not effectively produce the antibody of high specific and high-affinity.Therefore, the monoclonal antibody that obtain the anti-Clenbuterol hydrochloride of high specific at first needs one to have good immunogenic antigen Clenbuterol hydrochloride.But the Clenbuterol hydrochloride molecular weight only is 313.7, generally do not have immunogenicity for molecular weight less than 1000 daltonian micromolecular compounds, can not produce specific antibody by the direct immunization animal, must connect and compose effectively artificial conjugate with macromolecular carrier is complete antigen synthesis, specificity position with outstanding its molecule stereo structure is an antigenic determinant, could produce at the micromolecular specific antibody of this target by immune animal.
The preparation method of present existing clenbuterol complete antigen generates the Clenbuterol hydrochloride hapten compound by free Clenbuterol hydrochloride and Succinic anhydried (being succinyl oxide) reaction, utilize carbodlimide method then, the Clenbuterol hydrochloride haptens is connected on the bovine serum albumin.The main drawback of this method is that preparation is loaded down with trivial details, needs preparation Clenbuterol hydrochloride haptens earlier; In utilizing the carbodlimide method process, need N, volatile organic solvents such as dinethylformamide, N-hydroxy-succinamide easily cause poisoning, and operator ' s health is caused very big harm; The connecting arm that in this process, can connect a certain-length, in next step immune operation test, easily produce antibody at connecting arm, not only influence generation, also will influence the specificity and the avidity of antibody of clenbuteral hydrochloride at antibody of clenbuteral hydrochloride.
Summary of the invention
The invention provides a kind of clenbuterol complete antigen and anti-Clenbuterol hydrochloride MONOCLONAL ANTIBODIES SPECIFIC FOR method, overcome the complex operation step that prior art exists, hazardous agents easily produces at shortcomings such as the antibodies specific of the antibody of other antigenic determinants and generation and avidity are low.
The present invention is directly connecting the Clenbuterol hydrochloride molecule of some amount by the diazonium method on the bovine serum albumin, prepare the complete antigen CL-BSA that can be used for immune animal, with the specific antibody of its immune Balb/c mouse inbred lines generation at Clenbuterol hydrochloride, and obtain the cell strain of the anti-Clenbuterol hydrochloride monoclonal antibody of stably excreting by cytogamy, this cell strain is cultivated by purifying obtained to have the anti-Clenbuterol hydrochloride monoclonal antibody of high-affinity, high specific at last.The acquisition of this monoclonal antibody will provide the material that using value is arranged very much for immuno analytical methods such as enzyme linked immunologicals, thereby improve the sensitivity of immuno analytical method detection Clenbuterol hydrochlorides such as enzyme linked immunological.
Clenbuterol complete antigen and MONOCLONAL ANTIBODIES SPECIFIC FOR method thereof is characterized in that preparation method's step of described clenbuterol complete antigen is as follows:
A) preparation 1mol/L hydrochloric acid soln is inserted precooling in 4 ℃ of refrigerators with it; Take by weighing Clenbuterol hydrochloride (CLB) and place beaker, slowly drip the hydrochloric acid soln of precooling, and be stirred to dissolving with glass stick, the above-mentioned solution that dissolving is finished is put into the ice bath pot then;
B) the ice bath pot is positioned on the magnetic stirring apparatus, opens switch, slowly stir, and in beaker, drip 30% Sodium Nitrite (NaNO gradually
2) solution, drip NaNO
2Pick a little drop in the beaker with glass stick during solution, with the check of starch KI test paper, after treating to continue reaction 45min when it is changed to intense violet color, Dropwise 5 % Ammonium sulfamate termination reaction in beaker again obtains the CLB of azoization;
C) take by weighing bovine serum albumin (BSA) 300mg in another beaker, the phosphate buffered night (PBS) that slowly drips 0.01mol/L, pH=7.2-7.4 is to dissolving fully;
D) draw the CLB solution of azoization in the beaker with drop-burette, slowly splash in another beaker in the dissolved BSA solution, glass stick stirs, and question response liquid color becomes glassy yellow and ends;
E) in above-mentioned mixed solution, drip 1mol/L NaOH solution simultaneously, the pH value is maintained about 9.0, in this beaker, behind the adding rotor, be positioned on the magnetic stirring apparatus;
F) this beaker and magnetic stirring apparatus are put into refrigerator, open the magnetic stirring apparatus switch, lucifuge slowly stirred 24 hours, and during the stirring, every interval 1h detects its pH value, dripped NaOH solution, and the pH value is maintained about 9.0;
G) reaction finishes, and sample is added in the dialysis tubing by the glass stick water conservancy diversion, places 0.01M phosphate buffered saline buffer (PBS) dialysis, 3-4h changes liquid once, dialysis 72h, and dialysis finishes, with the clenbuterol complete antigen sample packing that obtains, place-20 ℃ of refrigerator storages standby.
Clenbuterol hydrochloride (CLB) small molecules is as follows through the azo-compound molecular structure that the azo process obtains:
Coupling rate through Clenbuterol hydrochloride molecule and bovine serum albumin in the clenbuterol complete antigen that produces with bovine serum albumin phenolic hydroxyl group generation chemical reaction is 17, and its molecular structure is as follows:
Described method for preparing monoclonal antibody comprises the steps:
1) select the female pure lines Balb/c mouse of 6-8 week size, body weight 18-22g for use, the immunizing dose fundamental immunity be 50 μ g/ only, booster immunization be 100 μ g/ only; Dilute the clenbuterol complete antigen of purchasing with the PBS damping fluid; Add isopyknic Fu Shi Freund's complete adjuvant during initial immunity, fully emulsified, the abdominal injection method is adopted in emulsion droplet indiffusion in splashing into water; 2-3 carries out booster immunization after week, carry out booster immunization once more every two weeks later on, adopts freund 's incomplete adjuvant during booster immunization; From immunity for the third time, the eye socket blood sampling was carried out in each immunity on the 3rd day, measured and tired and specificity; Treat immune serum tire qualified after, prepare to carry out cytogamy;
2) detect qualified serum by indirect enzyme-linked immunosorbent method (IELISA) after, this mouse is got spleen is broken into one cell suspension and SP2/0 myeloma cell according to 3-10: 1 ratio mixing, the centrifugal supernatant liquor that removes; Using with PBS damping fluid dissolved mass volume ratio is that 50% Macrogol 4000 joins in the cell of mixing and carries out warm 1min, in the fusion process, fusion liquid is slowly shaken up;
3) after fusion finishes, fused cell is added in the HAT substratum, and this cell culture fluid is added in the 96 porocyte culture plates, place 5%CO
2Cultivate in 37 ℃ of incubators; Cultivate with the HT substratum two week backs, and pair cell nutrient solution supernatant carries out IELISA and detect, and finally screens and obtain the high specific hybridoma cell strain, through four subculture in vitro separately and cryopreservation resuscitation, cell well-grown, and stably excreting antibody, it is tired greater than 3 * 10
6
4) get 6-8 week Balb/c mouse, only to its abdominal injection whiteruss, pneumoretroperitoneum inoculations in seven days are in the hybridoma of growth logarithmic phase according to 1ml/, every injection 1 * 10
6Individual, after three days, observe the mouse ascites production every day at interval, treat that its belly becomes big, hypotrichosis, dying when motionless, collect 6mL ascites, ascites is centrifugal, obtain the supernatant of 5mL, add the ammonium sulfate method of 50g/100mL then, obtain precipitating the monoclonal antibody that is anti-Clenbuterol hydrochloride after centrifugal, it with an amount of PBS buffered soln dissolving, is positioned over the medium-term and long-term preservation of-20 degree refrigerators after the packing.
It is IgG1 that described monoclonal antibody detects the hypotype of determining this antibody by test kit, and its light chain is κ.
The present invention adopts the diazonium method directly bovine serum albumin to be connected to Clenbuterol hydrochloride and is prepared into complete antigen, and this preparation process is easy, and agents useful for same is an inorganic reagent, and is little to the human body potential hazard; Need not connecting arm, directly Clenbuterol hydrochloride small molecules antigen is connected on the bovine serum albumin, farthest preserve its three-dimensional arrangement, avoid producing antibody at other antigenic determinants, and be beneficial to the specific antibody of its immune animal generation at Clenbuterol hydrochloride, and can obtain the anti-Clenbuterol hydrochloride cell strain of monoclonal antibody of stably excreting, and then this cell strain is cultivated the anti-Clenbuterol hydrochloride monoclonal antibody that can obtain to have the high affinity high specific by purifying by cytogamy.The monoclonal antibody that this method prepares will provide the material that using value is arranged very much for immuno analytical methods such as enzyme linked immunologicals, thereby improve the sensitivity and the specificity of enzyme immunoassay technology for detection Clenbuterol hydrochloride.
Description of drawings
Fig. 1 is an electrophorogram.
M: marker, 1: coupling sample, 2: the standard bovine serum albumin.
Embodiment
The preparation method of comparatively detailed clenbuterol complete antigen below is provided.
Basic skills is at first the Clenbuterol hydrochloride small molecules to be placed activation under the acidic conditions, is placed on then and carries out diazotization under the condition of ice bath, carries out coupling with the carrier proteins bovine serum albumin at last under alkaline condition.
Embodiment 1: the synthetic and purifying of complete antigen
Preparation 1mol/L hydrochloric acid soln is inserted precooling in 4 ℃ of refrigerators with it.Take by weighing Clenbuterol hydrochloride (CLB) 80.00mg and place beaker, slowly drip the hydrochloric acid soln of precooling, and be stirred to the CLB dissolving with glass stick, the above-mentioned solution that dissolving is finished is inserted in the ice bath pot then, and adds rotor.The ice bath pot is positioned on the magnetic stirring apparatus, opens switch, slowly stir, and drip 30%NaNO gradually
2Solution.Drip NaNO
2Pick a little reaction solution drop with glass stick during solution,, treat to stop when it is changed to intense violet color adding 30%NaNO with the check of starch KI test paper
2Solution.Reaction solution is proceeded, and treats Dropwise 5 % Ammonium sulfamate 1ml termination reaction behind the 45min.Take by weighing BSA 300mg in beaker, slowly drip 0.01mol/L pH=7.2-7.4 phosphate buffered saline buffer (PBS) to dissolving fully.CLB solution with drop-burette absorption azoization slowly is added dropwise in the dissolved BSA solution, and glass stick stirs, and question response liquid color becomes glassy yellow and ends.Simultaneously, drip 1mol/LNaOH solution, the pH value is maintained about 9.0.After in beaker, adding rotor, be positioned on the magnetic stirring apparatus.Beaker and magnetic stirring apparatus are put into refrigerator, open the magnetic stirring apparatus switch, lucifuge slowly stirred 24 hours.During the stirring, every 1h detects its pH value, drips NaOH solution, and the pH value maintains about 9.0.Reaction finishes, and sample is added in the dialysis tubing by the glass stick water conservancy diversion, places the PBS damping fluid to dialyse.3-4h changes liquid once, dialysis 72h.Dialysis finishes, and obtains Clenbuterol hydrochloride-bovine serum albumin coupling liquid (CLB-BSA), and it is standby to be placed in-20 ℃ of refrigerators storage after packing.
Ultraviolet light absorption method is identified clenbuterol complete antigen
The coupling liquid of respectively Clenbuterol hydrochloride, bovine serum albumin bletilla being purified through dialysis under the 200-500nm wavelength carries out uv-absorbing scanning, measures and obtains Clenbuterol hydrochloride and bovine serum albumin maximum absorption wavelength.According to the stackable principle of absorbancy, bovine serum albumin in this research (BSA), conjugate (CLB-BSA), at BSA, the optical density of the maximum absorption wave strong point of CLB is as the absorption value of calculating the coupling rate, to reduce the error of experiment.By using the scanning of UV1700 ultraviolet spectrophotometer, BSA and CLB maximum absorption wavelength are respectively 280nm and 296nm.
With BSA is index, and above CLB-BSA coupling solution allocation is become the CLB-BSA solution of corresponding BSA concentration, and disposes the standard BSA solution of same concentrations, detect at 280nm and 296nm place light absorption value, and it is poor to calculate extinction.Stackable according to light absorption value, under same BSA concentration, the increase of absorption value comes from CL on crosslinked to its contribution, typical curve contrast with this contribution margin and preparation, determine the concentration of CLB in sample, again by formula: coupling rate=CLB molecule number/BSA molecule number draws the coupling rate.Ratio through calculation result Clenbuterol hydrochloride small molecules and bovine serum albumin is: 17: 1.
Electrophoretic method is identified clenbuterol complete antigen
Adopt the Xylene Brilliant Cyanine G method to detect the concentration that obtains sample in the CLB-BSA coupling solution, and it is diluted to 10 μ g/ml. resolving gel concentration concentration 12%, concentrate gum concentration 5%, with Xylene Brilliant Cyanine G 6250 dyeing, obtain as shown in Figure 1 through the evaluation of gel imaging instrument, as seen in Figure 1, the molecular weight of the coupling sample 1 obviously molecular weight than standard bovine serum albumin is big, the coupling success is described, obtains clenbuterol complete antigen.
The sero-fast evaluation of Clenbuterol hydrochloride
After the clenbuterol complete antigen for preparing carried out five immunity to the Balb/c mouse inbred lines, by the indirect enzyme-linked immunosorbent method measure with phosphate buffered saline buffer respectively and in the mice serum after containing 1000 times of the phosphate buffered saline buffer dilutions of 5%BSA anti-antibody of clenbuteral hydrochloride tire, the results are shown in following table.
As can be seen from the table, 1,2,3,6, the serum of No. 7 mouse is behind the antibody that neutralizes the anti-BSA that may exist, and its indirect enzyme-linked immunosorbent method (IELISA) detected result at Clenbuterol hydrochloride shows positive (OD
450nm＞1.0 is promptly positive), show that immune effect is obvious, obtained high serum of tiring.Above result proves absolutely that also this research of employing synthetic complete antigen immune mouse can produce the antibody at Clenbuterol hydrochloride.
Indirect enzyme-linked immunosorbent method measurement result table
| Sample | Neutral experiment * | Non-neutral experiment material | ||
| 1 2 3 4 5 6 | CLB-BSA*** 1.967 1.630 1.666 0.003 -0.005 0.879 | BSA*** 0.032 0.074 0.045 -0.010 0.006 0.004 | CLB-BSA*** 1.466 1.146 1.069 0.006 0.013 1.196 | BSA*** 1.254 1.163 1.166 -0.010 O.029 1.001 |
| 7 | 1.545 | 0.201 | 1.282 | 1.273 |
* sample uses the PBS damping fluid that contains 5%BSA to dilute
The * sample uses the PBS damping fluid to dilute
* * CLB-BSA and BSA are coated on the enzyme plate respectively
Embodiment 2: animal immune prepares anti-Clenbuterol hydrochloride monoclonal antibody
1) selects the female pure lines Balb/c mouse of 6-8 week size, body weight 18-22g for use.The immunizing dose fundamental immunity is 50 μ g/, and booster immunization is 100 μ g/.Dilute quantitative artificial antigen mixture respectively with the PBS damping fluid, add isopyknic Fu Shi Freund's complete adjuvant during initial immunity, fully emulsified, emulsion droplet indiffusion in splashing into water.Adopt the abdominal injection method.2-3 carries out booster immunization after week, carry out booster immunization once more every two weeks later on, adopts freund 's incomplete adjuvant during booster immunization.From immunity for the third time, the eye socket blood sampling was carried out in each immunity on the 3rd day, measured and tired and specificity.Treat immune serum tire qualified after, prepare to carry out cytogamy.
2) detect qualified serum by indirect enzyme-linked immunosorbent method (IELISA) after, this mouse is got spleen is broken into one cell suspension and SP2/O myeloma cell according to 3-10: 1 ratio mixing, the centrifugal supernatant liquor that removes.Use is that 50% Macrogol 4000 joins in the cell of mixing and carries out warm 1min with PBS buffering dissolved mass volume ratio at night, in the fusion process, slowly shakes up merging liquid.
3) after fusion finishes, fused cell is added in the HAT substratum, and this cell culture fluid is added in the 96 porocyte culture plates, place 5%CO
2Cultivate in 37 ℃ of incubators.Cultivate with the HT substratum two week backs, and pair cell nutrient solution supernatant carries out IELISA and detect, and finally screens and obtain the high specific hybridoma cell strain, through four subculture in vitro separately and cryopreservation resuscitation, cell well-grown, and stably excreting antibody, it is tired greater than 3 * 10
6The high specific hybridoma cell strain is kept at～standby in 70 ℃ of refrigerators.
4) get 6-8 week Balb/c, with whiteruss injection 1ml/ only, pneumoretroperitoneum inoculations in seven days are in the hybridoma of growth logarithmic phase, every injection 1 * 10
6Individual, after three days, observe the mouse ascites production every day at interval, treat that its belly becomes big, hypotrichosis, dying when motionless, collect 6mL ascites, ascites is centrifugal, obtain the supernatant of 5mL, add the ammonium sulfate method of 50g/100mL then, obtain precipitating the monoclonal antibody that is anti-Clenbuterol hydrochloride after centrifugal, it with an amount of PBS buffered soln dissolving, is positioned over the medium-term and long-term preservation of-20 degree refrigerators after the packing.
Claims (4)
1. clenbuterol complete antigen and MONOCLONAL ANTIBODIES SPECIFIC FOR method thereof is characterized in that preparation method's step of described clenbuterol complete antigen is as follows:
A) preparation 1mol/L hydrochloric acid soln is inserted precooling in 4 ℃ of refrigerators with it; Take by weighing Clenbuterol hydrochloride (CLB) and place beaker, slowly drip the hydrochloric acid soln of precooling, and be stirred to dissolving with glass stick, the above-mentioned solution that dissolving is finished is put into the ice bath pot then;
B) the ice bath pot is positioned on the magnetic stirring apparatus, opens switch, slowly stir, and in beaker, drip 30% Sodium Nitrite (NaNO gradually
2) solution, drip NaNO
2Pick a little drop in the beaker with glass stick during solution, with the check of starch KI test paper, after treating to continue reaction 45min when it is changed to intense violet color, Dropwise 5 % Ammonium sulfamate termination reaction in beaker again obtains the CLB of azoization;
C) take by weighing bovine serum albumin (BSA) 300mg in another beaker, the phosphate buffered night (PBS) that slowly drips 0.01mol/L, pH=7.2-7.4 is to dissolving fully;
D) draw the CLB solution of azoization in the beaker with drop-burette, slowly splash in another beaker in the dissolved BSA solution, glass stick stirs, and question response liquid color becomes glassy yellow and ends;
E) in above-mentioned mixed solution, drip 1mol/L NaOH solution simultaneously, the pH value is maintained about 9.0, in this beaker, behind the adding rotor, be positioned on the magnetic stirring apparatus;
F) this beaker and magnetic stirring apparatus are put into refrigerator, open the magnetic stirring apparatus switch, lucifuge slowly stirred 24 hours, and during the stirring, every interval 1h detects its pH value, dripped NaOH solution, and the pH value is maintained about 9.0;
G) reaction finishes, and sample is added in the dialysis tubing by the glass stick water conservancy diversion, places 0.01M phosphate buffered saline buffer (PBS) dialysis, 3-4h changes liquid once, dialysis 72h, and dialysis finishes, with the clenbuterol complete antigen sample packing that obtains, place-20 ℃ of refrigerator storages standby.
2. clenbuterol complete antigen according to claim 1 and MONOCLONAL ANTIBODIES SPECIFIC FOR method thereof is characterized in that:
Clenbuterol hydrochloride (CLB) small molecules is as follows through the azo-compound molecular structure that the azo process obtains:
Coupling rate through Clenbuterol hydrochloride molecule and bovine serum albumin in the clenbuterol complete antigen that produces with bovine serum albumin phenolic hydroxyl group generation chemical reaction is 17, and its molecular structure is as follows:
3. clenbuterol complete antigen and MONOCLONAL ANTIBODIES SPECIFIC FOR method thereof is characterized in that method for preparing monoclonal antibody comprises the steps:
1) select the female pure lines Balb/c mouse of 6-8 week size, body weight 18-22g for use, the immunizing dose fundamental immunity be 50 μ g/ only, booster immunization be 100 μ g/ only; Dilute the clenbuterol complete antigen of purchasing with the PBS damping fluid; Add isopyknic Fu Shi Freund's complete adjuvant during initial immunity, fully emulsified, the abdominal injection method is adopted in emulsion droplet indiffusion in splashing into water; 2-3 carries out booster immunization after week, carry out booster immunization once more every two weeks later on, adopts freund 's incomplete adjuvant during booster immunization; From immunity for the third time, the eye socket blood sampling was carried out in each immunity on the 3rd day, measured and tired and specificity; Treat immune serum tire qualified after, prepare to carry out cytogamy;
2) detect qualified serum by indirect enzyme-linked immunosorbent method (IELISA) after, this mouse is got spleen is broken into one cell suspension and SP2/0 myeloma cell according to 3-10: 1 ratio mixing, the centrifugal supernatant liquor that removes; Using with PBS damping fluid dissolved mass volume ratio is that 50% Macrogol 4000 joins in the cell of mixing and carries out warm 1min, in the fusion process, fusion liquid is slowly shaken up;
3) after fusion finishes, fused cell is added in the HAT substratum, and this cell culture fluid is added in the 96 porocyte culture plates, place 5%CO
2Cultivate in 37 ℃ of incubators; Cultivate with the HT substratum two week backs, and pair cell nutrient solution supernatant carries out IELISA and detect, and finally screens and obtain the high specific hybridoma cell strain, through four subculture in vitro separately and cryopreservation resuscitation, cell well-grown, and stably excreting antibody, it is tired greater than 3 * 10
6
4) get 6-8 week Balb/c mouse, only to its abdominal injection whiteruss, pneumoretroperitoneum inoculations in seven days are in the hybridoma of growth logarithmic phase according to 1ml/, every injection 1 * 10
6Individual, after three days, observe the mouse ascites production every day at interval, treat that its belly becomes big, hypotrichosis, dying when motionless, collect 6mL ascites, ascites is centrifugal, obtain the supernatant of 5mL, add the ammonium sulfate method of 50g/100mL then, obtain precipitating the monoclonal antibody that is anti-Clenbuterol hydrochloride after centrifugal, it with an amount of PBS buffered soln dissolving, is positioned over the medium-term and long-term preservation of-20 degree refrigerators after the packing.
4. clenbuterol complete antigen according to claim 3 and MONOCLONAL ANTIBODIES SPECIFIC FOR method thereof is characterized in that:
It is IgG1 that monoclonal antibody detects the hypotype of determining this antibody by test kit, and its light chain is κ.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100477148A CN101182356A (en) | 2007-11-01 | 2007-11-01 | Clenbuterol complete antigen and method for preparing monoclonal antibody thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100477148A CN101182356A (en) | 2007-11-01 | 2007-11-01 | Clenbuterol complete antigen and method for preparing monoclonal antibody thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101182356A true CN101182356A (en) | 2008-05-21 |
Family
ID=39447795
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007100477148A Pending CN101182356A (en) | 2007-11-01 | 2007-11-01 | Clenbuterol complete antigen and method for preparing monoclonal antibody thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101182356A (en) |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101813696A (en) * | 2010-04-09 | 2010-08-25 | 镇江出入境检验检疫局检验检疫综合技术中心 | Immunology quick detection method of diminazene in animal-derived food |
| CN101955541A (en) * | 2010-05-06 | 2011-01-26 | 北京维德维康生物技术有限公司 | Clenbuterol detection immunoassay kit and special antibody thereof |
| CN102168072A (en) * | 2010-12-20 | 2011-08-31 | 江苏出入境检验检疫局动植物与食品检测中心 | A monoclonal antibody of anti-clenobuterol hydrochloride and a kit for detecting clenobuterol hydrochloride |
| CN101942415B (en) * | 2010-01-21 | 2012-02-01 | 泰州康正生物技术有限公司 | Hybridoma cell strain and anti-hydrochloric acid clenbuterol monoclonal antibody prepared from hybridoma cell strain |
| CN102464719A (en) * | 2010-11-11 | 2012-05-23 | 中国科学院上海生命科学研究院 | Murine anti-clenbuterol monoclonal antibody and its application |
| CN102659949A (en) * | 2012-04-24 | 2012-09-12 | 杭州隆基生物技术有限公司 | Anti-clenbuterol antibody and preparation method and application thereof |
| CN103012593A (en) * | 2011-09-21 | 2013-04-03 | 北京勤邦生物技术有限公司 | Preparation and applications of clenbuterol monoclonal antibody |
| CN103159852A (en) * | 2013-03-28 | 2013-06-19 | 江南大学 | Method for synthesizing specific artificial antigen of clenbuterol |
| CN104678093A (en) * | 2015-03-20 | 2015-06-03 | 上海理工大学 | Preparation method of immobilized anti-clenbuterol hydrochloride monoclonal antibody |
| CN103012593B (en) * | 2011-09-21 | 2016-12-14 | 北京勤邦生物技术有限公司 | The preparation of clenbuterol monoclonal antibody and application thereof |
| CN109628408A (en) * | 2018-12-24 | 2019-04-16 | 北京望尔生物技术有限公司 | A kind of hybridoma cell strain that secreting anti-clenbuterol monoclonal antibody and its application |
| CN113960305A (en) * | 2021-10-15 | 2022-01-21 | 北京勤邦生物技术有限公司 | Immunomagnetic bead for clenbuterol enrichment and purification and preparation method and application thereof |
-
2007
- 2007-11-01 CN CNA2007100477148A patent/CN101182356A/en active Pending
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942415B (en) * | 2010-01-21 | 2012-02-01 | 泰州康正生物技术有限公司 | Hybridoma cell strain and anti-hydrochloric acid clenbuterol monoclonal antibody prepared from hybridoma cell strain |
| CN101813696A (en) * | 2010-04-09 | 2010-08-25 | 镇江出入境检验检疫局检验检疫综合技术中心 | Immunology quick detection method of diminazene in animal-derived food |
| CN101955541A (en) * | 2010-05-06 | 2011-01-26 | 北京维德维康生物技术有限公司 | Clenbuterol detection immunoassay kit and special antibody thereof |
| CN101955541B (en) * | 2010-05-06 | 2012-10-03 | 北京维德维康生物技术有限公司 | Clenbuterol detection immunoassay kit and special antibody thereof |
| CN102464719A (en) * | 2010-11-11 | 2012-05-23 | 中国科学院上海生命科学研究院 | Murine anti-clenbuterol monoclonal antibody and its application |
| CN102464719B (en) * | 2010-11-11 | 2014-06-04 | 中国科学院上海生命科学研究院 | Murine anti-clenbuterol monoclonal antibody and its application |
| CN102168072A (en) * | 2010-12-20 | 2011-08-31 | 江苏出入境检验检疫局动植物与食品检测中心 | A monoclonal antibody of anti-clenobuterol hydrochloride and a kit for detecting clenobuterol hydrochloride |
| CN102168072B (en) * | 2010-12-20 | 2013-03-06 | 江苏出入境检验检疫局动植物与食品检测中心 | A monoclonal antibody of anti-clenobuterol hydrochloride and a kit for detecting clenobuterol hydrochloride |
| CN103012593A (en) * | 2011-09-21 | 2013-04-03 | 北京勤邦生物技术有限公司 | Preparation and applications of clenbuterol monoclonal antibody |
| CN103012593B (en) * | 2011-09-21 | 2016-12-14 | 北京勤邦生物技术有限公司 | The preparation of clenbuterol monoclonal antibody and application thereof |
| CN102659949A (en) * | 2012-04-24 | 2012-09-12 | 杭州隆基生物技术有限公司 | Anti-clenbuterol antibody and preparation method and application thereof |
| CN103159852A (en) * | 2013-03-28 | 2013-06-19 | 江南大学 | Method for synthesizing specific artificial antigen of clenbuterol |
| CN104678093A (en) * | 2015-03-20 | 2015-06-03 | 上海理工大学 | Preparation method of immobilized anti-clenbuterol hydrochloride monoclonal antibody |
| CN109628408A (en) * | 2018-12-24 | 2019-04-16 | 北京望尔生物技术有限公司 | A kind of hybridoma cell strain that secreting anti-clenbuterol monoclonal antibody and its application |
| CN113960305A (en) * | 2021-10-15 | 2022-01-21 | 北京勤邦生物技术有限公司 | Immunomagnetic bead for clenbuterol enrichment and purification and preparation method and application thereof |
| CN113960305B (en) * | 2021-10-15 | 2023-10-17 | 北京勤邦科技股份有限公司 | Immunomagnetic bead for clenbuterol Luo Fuji purification and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101182356A (en) | Clenbuterol complete antigen and method for preparing monoclonal antibody thereof | |
| CN101013129B (en) | Enzyme linked immunosorbent reagent casing for detecting furacilin metabolite and uses thereof | |
| CN103820394B (en) | Monoclonal antibodies against morphine, produce the cell strain of this antibody, morphine detection kit and preparation method thereof | |
| CN110498766B (en) | Fluazinam hapten, artificial antigen and antibody, and preparation method and application thereof | |
| CN103146652B (en) | Anti-olaquindox monoclonal antibody and its hybridoma cell line, preparation methods of antibody and cell line, and kit for detecting olaquindox in forage | |
| CN101863981A (en) | Preparation method of anti-bisphenol A monoclonal antibody | |
| CN101962358A (en) | Ciprofloxacin hapten, artificial antigen and antibody and preparation method and application thereof | |
| CN105044365B (en) | The preparation method of the time resolved fluoro-immunoassay test paper of detection enrofloxacin residual | |
| CN103787946A (en) | Isokwas tenuazonowy artificial antigen and antibody as well as preparation method and application thereof | |
| CN111943882B (en) | Pythium hapten, artificial antigen and antibody as well as preparation method and application thereof | |
| CN101021535B (en) | Enzyme-linked immunalogical kit for detecting gentamicin medicine and method | |
| CN101358967B (en) | Method for detecting chlorpromazine and special ELISA kit thereof | |
| CN100489530C (en) | Method for assaying sulfaquinoxaline and special enzyme-linked immune reagent kit | |
| CN100533148C (en) | Method for detecting tylosin and special enzyme-linked immune reagent kit thereof | |
| CN106831498A (en) | Furacilin metabolite SEM derivatizations haptens, the preparation method and applications of artificial antigen | |
| CN102020713A (en) | Immune colloidal gold test strip used for detecting aureomycin residue and preparation method thereof | |
| CN102168072B (en) | A monoclonal antibody of anti-clenobuterol hydrochloride and a kit for detecting clenobuterol hydrochloride | |
| CN100489532C (en) | Method for detecting salinomycin and spcial enzyme-linked immune reagent kit thereof | |
| CN103018451A (en) | Enzyme-linked immunoassay kit for chloramphenicol detection, and applications thereof | |
| CN1790025A (en) | Test paper for lateral flow immunochromatographic semi-quantitative detection of ractopamine | |
| CN101393218A (en) | Preparation of monoclonal antibody and uses thereof | |
| CN107118159A (en) | Cistofuran metabolite AHD derivatizations haptens, the preparation method and applications of artificial antigen | |
| Zhou et al. | Development of a novel antibody probe useful for domoic acid detection | |
| CN2653511Y (en) | Kelunteluo fast semi quantitative detection test paper | |
| CN100338467C (en) | Enzyme-linked immunological kit for detecting tetracycline drug |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20080521 |