CN101407548B - Method for preparing micro-cantilever beam modified with antibody - Google Patents
Method for preparing micro-cantilever beam modified with antibody Download PDFInfo
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- CN101407548B CN101407548B CN2008102274496A CN200810227449A CN101407548B CN 101407548 B CN101407548 B CN 101407548B CN 2008102274496 A CN2008102274496 A CN 2008102274496A CN 200810227449 A CN200810227449 A CN 200810227449A CN 101407548 B CN101407548 B CN 101407548B
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Abstract
The invention discloses a method for preparing a micro-cantilever decorated with an antibody, which uses hydrochloride mercaptan imine as a sulfhydrylation agent, and the antibody is sulfhydrylized, and the sulfhydrylation antibody is fixed on a gold membrane of the micro-cantilever to obtain the micro-cantilever decorated with the antibody. The result shows that by introducing the sulfhydrylation agent-hydrochloride mercaptan imine, the method for preparing the micro-cantilever decorated with the antibody has the advantages of short reaction time, simple operation, few loss of antibody activity and the like, compared with the existing method.
Description
Technical field
The present invention relates to the method that a kind of preparation is modified with the micro-cantilever of antibody.
Background technology
Appearance along with AFM (AFM) and MEMS (MEMS); The micro-cantilever sensing technology develops rapidly becomes a kind of new method for sensing; Its principle is: the change that when biochemical reaction takes place on the micro-cantilever surface, can produce surface stress; Thereby cause micro-cantilever to produce flexural deformation, monitor this distortion, just can know that whole biochemical reaction carries out the information of process in conjunction with optics or electrical method.Micro-cantilever immune sensing technology through relying on immunologic opsonin identification to set up is compared with traditional enzyme-linked immunosorbent assay technology (ELISA); This technology need not testing sample is carried out mark when detecting; Eliminated the added influence that possibly produce by mark, and highly sensitive, can realize in real time, the generating process of parallel monitoring whole biochemical reaction; Thereby obtain abundanter bioinformation; Realize the significant advantage that big scale, high-throughput, parallel array are measured easily, be specially adapted to be difficult to the tracer of mark, like the target molecule to be detected of resorcinolphthalein or tracer isotope.In recent years, micro-cantilever immune sensing technology has all obtained using widely at aspects such as cancer detection, DNA hybridization and transcription factors.
The key of micro-cantilever immune sensing technical application is the activity that how effectively is attached to antibody highly sensitive, high specificity on the golden film of micro-cantilever and keeps antibody as far as possible.About antibody being fixed to the method for on the micro-cantilever micro-cantilever being modified; The domestic and foreign literature reported method mainly is to carry out through the sulfhydrylization reagent with bifunctional group; Main method has following two types: first kind method is earlier sulfhydrylization reagent to be attached on the micro-cantilever; Carboxyl or amino on the activation sulfhydrylization reagent then; Make it with antibody on amino or carboxyl combine used sulfhydrylization reagent such as 11-thiol carboxylic acid (11-MUA), 2-aminoothyl mercaptan (AET) or 3-thiohydracrylic acid (MPA).This type way has increased activation step, influences antibody fixation stability property and consistence, and the activity of antibody is reduced, and the result's is repeated relatively poor; In addition, the time that continues with antibody response after the sulfhydrylization reagent activation is also longer, generally needs about 3-5 hour.Second class methods are earlier antibody to be carried out sulfhydrylation; And then be attached on the golden film of micro-cantilever; Sulfhydrylization reagent is with sulfo group HOSu NHS base-6-(3 '; 2-pyridine two sulphur-propionic acid amide)-the two sulfosuccinimide propionic esters (DTSSP) of capronate (Sulfo-LC-SPDP) and 3,3 '-two sulphur are main.The principle of this type way is to form disulfide linkage at antibody and sulfhydrylization reagent reaction back; Add WR 34678 (DTT) again and restore free sulfhydryl group; Its shortcoming is owing between the heavy chain of antibody and the heavy chain and between heavy chain and the light chain many disulfide linkage are arranged, may destroy the structure of antibody when reducing with DTT and then influence the activity of antibody and the limit of detection of micro-cantilever beam sensor.In addition, cup fragrant crown ether (Calixarene) verivate Prolinker
TMDeng being applied for a patent abroad.
Summary of the invention
A kind of new purposes that the purpose of this invention is to provide the hydrochloric acid mercaptan imine.
To be it be modified with the application in the micro-cantilever of antibody as sulfhydrylization reagent in preparation to the new purposes of hydrochloric acid mercaptan imine provided by the present invention.
Another object of the present invention provides the method that a kind of preparation is modified with the micro-cantilever of antibody.
Preparation provided by the present invention is modified with the method for the micro-cantilever of antibody; Be to be sulfhydrylization reagent with the hydrochloric acid mercaptan imine, antagonist carries out sulfhydrylation, obtains sulfhydrylation antibody; Again said sulfhydrylation antibody is fixed on the micro-cantilever that has golden film, obtains being modified with the micro-cantilever of antibody
In the aforesaid method, the mol ratio of said antibody and said hydrochloric acid mercaptan imine is 1: (1-6), be specially 1:3.
In the aforesaid method, said antibody can be zoogenous polyclonal antibody such as mouse, rabbit or monoclonal antibody etc., like the anti-clenbuterol monoclonal antibody.
Available existing sulfhydrylation method utilizes said sulfhydrylization reagent-hydrochloric acid mercaptan imine antagonist to carry out sulfhydrylation.During concrete preparation, the reaction conditions of said antibody and said hydrochloric acid mercaptan imine does, reacts 0.5-2.0h under the room temperature, specifically can be under the room temperature and reacts 1h.
Reaction conditions on the said golden film that sulfhydrylation antibody is fixed to micro-cantilever is that 30.0-37.5 ℃ of reaction 0.5-2.0h specifically can be 37 ℃ of reaction 1h.
Utilize the micro-cantilever that is modified with antibody of above-mentioned arbitrary method preparation also to belong to protection scope of the present invention.
In view of micro-cantilever immune sensing technical antagonism body fixed importance, the present invention as sulfhydrylization reagent and antibodies, is fixed to the hydrochloric acid mercaptan imine on the micro-cantilever with the antibody behind the sulfhydrylation then.The result shows, through the introducing of sulfhydrylization reagent-hydrochloric acid mercaptan imine, of the present invention antibody the method on the micro-cantilever of being fixed to is compared with existing method has short, simple to operate, advantage such as the antibody activity loss is few of reaction times.
Embodiment
Below in conjunction with specific embodiment the method that antibody is fixed on the micro-cantilever of the present invention is further specified, but do not limit the present invention.
Embodiment 1, preparation are modified with the micro-cantilever of antibody
1, utilize the hydrochloric acid mercaptan imine with the antibody sulfhydrylation
(1) accurately taking by weighing anti-clenbuterol monoclonal antibody (available from U.S. USBiological company) 10.0mg is dissolved in and obtains the antibody-solutions that concentration is 10g/L among the 1.0mL PBS;
(2) taking by weighing 2.0mg hydrochloric acid mercaptan imine (available from U.S. Sigma company) is dissolved in and obtains the sulfhydrylization reagent that concentration is 2.0g/L (at present joining existing usefulness) in the 1.0mL zero(ppm) water;
(3) sulfhydrylization reagent with the antibody-solutions of 1.0mL above-mentioned steps (1) and 45.8 μ l above-mentioned steps (2) mixes, and makes that the mol ratio of anti-clenbuterol monoclonal antibody and hydrochloric acid mercaptan imine is 1:3 in the mixing solutions, and gentle agitation is reacted 1h under the room temperature condition;
(4) with the PBS damping fluid of 20mM (contain 0.15M NaCl and 1.0mM EDTA, pH7.2) to the reaction solution dialysis 48h of above-mentioned steps (3), during every 2h change a dialyzate, obtain the sulfhydrylation antibody-solutions.
2, the mole binding ratio of antibody and mercapto groups in Ellman ' the s method mensuration sulfhydrylation antibody
(1) take by weighing 100mg5,5-dithio two (2-nitrobenzoic acid) (DTNB) is dissolved in the 25mL0.1M PBS damping fluid (pH8.0);
(2) take by weighing 3.5mg halfcystine (Cys) and be dissolved in the 10mL0.1M PBS damping fluid (pH8.0), obtain the halfcystine that concentration is 2.0mM (Cys) solution; Halfcystine (Cys) solution that with above-mentioned concentration is 2.0mM is diluted to halfcystine (Cys) solution that concentration is respectively 1.0mM, 0.5mM, 0.25mM, 0.125mM and 0.0625mM successively with 0.1M PBS damping fluid (pH8.0);
(3) the sulfhydrylation antibody-solutions that halfcystine (Cys) solution of getting the different concns that above-mentioned steps (2) obtains and above-mentioned steps 1 obtain is 250 μ L respectively; Add respectively in the DTNB solution of 50 μ L above-mentioned steps (1) preparation; Abundant mixing; Room temperature condition reacted 15 minutes down, and the light absorption value of solution is measured at the 412nm place;
(4) volumetric molar concentration with halfcystine (Cys) solution is an X-coordinate; With the light absorption value is ordinate zou; The drawing standard curve; Calculate the volumetric molar concentration of sulfydryl in the sulfhydrylation antibody that above-mentioned steps 1 obtains, be converted into the sulfydryl number of every mole of antibody molecule, promptly obtain mole binding ratio of antibody and mercapto groups in the sulfhydrylation antibody.
Three repetitions are established in experiment, and the result shows that the mole binding ratio of anti-clenbuterol monoclonal antibody and mercapto groups is 1:3 in the sulfhydrylation antibody that above-mentioned steps 1 obtains.
3, active damaed cordition behind the direct competitive ELISA method mensuration antibody sulfhydrylation
(1) encapsulate: to add the concentration after the 100 μ L dilution respectively be the anti-clenbuterol monoclonal antibody of 2.5 μ g/mL and the sulfhydrylation anti-clenbuterol monoclonal antibody of above-mentioned steps 1 acquisition, 37 ℃ of incubation 3h in every hole in the enzyme plate;
(2) wash plate: outwell the antibody-solutions of above-mentioned steps (1), enzyme plate is dried, on automatic washer, wash plate 4 times, dry with the washings of 200 μ L on filter paper;
(3) sealing: it is the sealing of 5% BSA solution (dissolving with PBS) that every hole adds 100 μ L quality percentage compositions, 37 ℃ of incubation 30min;
(4) wash plate: outwell confining liquid, enzyme plate is dried, on automatic washer, wash plate 4 times, dry with 200 μ L washingss on filter paper;
(5) competition: every hole adds the clenbuterol solution that 50 μ L concentration are 50ng/mL (available from U.S. Sigma company) successively and concentration is the clenbuterol horseradish peroxidase connection thing (available from U.S. Sigma company) of 1.0mg/L, 37 ℃ of incubations competition 30min;
(6) wash plate: outwell reaction solution, enzyme plate is dried, on automatic washer, wash plate 4 times, dry with 200 μ L washingss on filter paper;
(7) colour developing: every hole adds 100 μ L substrate solutions (tmb substrate uses liquid and substrate buffer solution to press the volume ratio mixing of 1:9, adds the 4.0mg urea peroxide in the above-mentioned mixing of every 10mL also), color development at room temperature;
(8) stop: behind the 15min, every hole adds 50 μ L stop buffer termination reactions, and the 450nm wavelength reads the OD value respectively.
Analyzing with Origin7.0 software, is X-coordinate with the concentration of clenbuterol solution, with B/B
0(B and B
0Representative suppresses the colour developing value that Kong Yufei suppresses the hole respectively) be ordinate zou, draw four parametric equation curves, 0 hole colour developing value (B of the direct ELISA typical curve that relatively the anti-clenbuterol monoclonal antibody is participated in before and after the sulfhydrylation
0) and 503nhibiting concentration (IC
50Value), judge the active influence of sulfhydrylation antagonist.
Three repetitions are established in experiment, and the result shows, the A of the typical curve of anti-clenbuterol monoclonal antibody correspondence before and after the sulfhydrylation
0Value is respectively 1.458 and 1.397, IC
50Value is respectively 3.13 μ g/L and 3.65 μ g/L.The active basic of anti-clenbuterol monoclonal antibody do not lose after sulfhydrylation was described.
4, sulfhydrylation antibody and micro-cantilever gold film combine and direct competitive ELISA method is estimated the situation that combines of sulfhydrylation antibody and the golden film of micro-cantilever
The micro-cantilever (available from American I BM company) that clean and air dried is coated with golden film is put into the enzyme plate aperture; Adding 100 μ L concentration is the sulfhydrylation anti-clenbuterol monoclonal antibody of above-mentioned steps 1 acquisition of 2.5 μ g/mL; 37 ℃ of incubation 1h promptly obtain being fixed with the micro-cantilever of antibody.
Direct competitive ELISA method is estimated the situation that combines of sulfhydrylation antibody and the golden film of micro-cantilever, and detailed process is following:
(1) clean: with the careful micro-cantilever of antibody that taken out two said fixing of tweezers, with the washings flushing for several times, nitrogen dries up;
(2) sealing: the micro-cantilever after nitrogen dried up is put into enzyme plate respectively, adds 100 μ L quality percentage compositions and be 5% BSA solution (dissolving with PBS) sealing, 37 ℃ of incubation 30min;
(3) clean: carefully take out micro-cantilever with tweezers, with the washings flushing for several times, nitrogen dries up;
(4) compete: the micro-cantilever after nitrogen is dried up is put into two aperture (marked of enzyme plate respectively; Suppress Kong Yufei and suppress the hole); It is the clenbuterol solution (available from U.S. Sigma company) of 100 μ g/L through the concentration of sample diluting liquid (containing the PBS that final concentration is 0.1mol/LpH7.5, the tween 20 of 0.1% volumn concentration and the gelatin of 0.1% quality percentage composition) dilution that the inhibition hole adds 50 μ L, and non-inhibition hole adds 50 μ L sample diluting liquids as contrast; And then adding the clenbuterol horseradish peroxidase connection thing 50 μ L (available from U.S. USBiological company) that concentration is 1.0mg/L respectively, 37 ℃ of incubations are competed 30min;
(5) clean: with the careful micro-cantilever that takes out in the above-mentioned inhibition Kong Yufei inhibition hole of tweezers, with the washings flushing for several times, nitrogen dries up;
(6) develop the color: two micro-cantilevers after nitrogen is dried up are put into two apertures of enzyme plate respectively; Every hole adds 100 μ L chromophoric solutions, and (tmb substrate uses liquid and substrate buffer solution to press the volume ratio mixing of 1:9; Add the 4.0mg urea peroxide in the above-mentioned mixed solution of every 10mL), color development at room temperature;
(7) stop: behind the 15min, every hole adds 50 μ L stop buffer termination reactions, takes out micro-cantilever, and the 450nm wavelength reads the OD value respectively.
Three repetitions are established in experiment, and the result shows that the absorbance under the 450nm wavelength in inhibition hole and non-inhibition hole is respectively 0.147 and 0.598, explains that the anti-clenbuterol monoclonal anti physical efficiency behind the sulfhydrylation effectively is attached on the golden film of micro-cantilever.
Embodiment 2, preparation are modified with the micro-cantilever of antibody
1, utilize the hydrochloric acid mercaptan imine with the antibody sulfhydrylation
(1) accurately taking by weighing anti-clenbuterol monoclonal antibody (available from U.S. USBiological company) 10.0mg is dissolved in and obtains the antibody-solutions that concentration is 10g/L among the 1.0mL PBS;
(2) taking by weighing 2.0mg hydrochloric acid mercaptan imine (available from U.S. Sigma company) is dissolved in and obtains the sulfhydrylization reagent that concentration is 2.0g/L (at present joining existing usefulness) in the 1.0mL zero(ppm) water;
(3) sulfhydrylization reagent with the antibody-solutions of 1.0mL above-mentioned steps (1) and 91.6 μ L above-mentioned steps (2) mixes, and makes that the mol ratio of anti-clenbuterol monoclonal antibody and hydrochloric acid mercaptan imine is 1:6 in the mixing solutions, and gentle agitation is reacted 2h under the room temperature condition;
(4) with the PBS damping fluid of 20mM (contain 0.15M NaCl and 1.0mM EDTA, pH7.2) to the reaction solution dialysis 48h of above-mentioned steps (3), during every 2h change a dialyzate, obtain the sulfhydrylation antibody-solutions.
2, the mole binding ratio of antibody and mercapto groups in Ellman ' the s method mensuration sulfhydrylation antibody
(1) take by weighing 100mg5,5-dithio two (2-nitrobenzoic acid) (DTNB) is dissolved in the 25mL0.1M PBS damping fluid (pH8.0);
(2) take by weighing 3.5mg halfcystine (Cys) and be dissolved in the 10mL0.1M PBS damping fluid (pH8.0), obtain the halfcystine that concentration is 2.0mM (Cys) solution; Halfcystine (Cys) solution that with above-mentioned concentration is 2.0mM is diluted to halfcystine (Cys) solution that concentration is respectively 1.0mM, 0.5mM, 0.25mM, 0.125mM and 0.0625mM successively with 0.1M PBS damping fluid (pH8.0);
(3) the sulfhydrylation antibody-solutions that halfcystine (Cys) solution of getting the different concns that above-mentioned steps (2) obtains and above-mentioned steps 1 obtain is 250 μ L respectively; Add respectively in the DTNB solution of 50 μ L above-mentioned steps (1) preparation; Abundant mixing; Room temperature condition reacted 15 minutes down, and the light absorption value of solution is measured at the 412nm place;
(4) volumetric molar concentration with halfcystine (Cys) solution is an X-coordinate; With the light absorption value is ordinate zou; The drawing standard curve; Calculate the volumetric molar concentration of sulfydryl in the sulfhydrylation antibody that above-mentioned steps 1 obtains, be converted into the sulfydryl number of every mole of antibody molecule, promptly obtain mole binding ratio of antibody and mercapto groups in the sulfhydrylation antibody.
Three repetitions are established in experiment, and the result shows that the mole binding ratio of anti-clenbuterol monoclonal antibody and mercapto groups is 1:6 in the sulfhydrylation antibody that above-mentioned steps 1 obtains.
3, active damaed cordition behind the direct competitive ELISA method mensuration antibody sulfhydrylation
(1) encapsulate: to add the concentration after the 100 μ L dilution respectively be the anti-clenbuterol monoclonal antibody of 2.5 μ g/mL and the sulfhydrylation anti-clenbuterol monoclonal antibody of above-mentioned steps 1 acquisition, 37 ℃ of incubation 3h in every hole in the enzyme plate;
(2) wash plate: outwell the antibody-solutions of above-mentioned steps (1), enzyme plate is dried, on automatic washer, wash plate 4 times, dry with the washings of 200 μ L on filter paper;
(3) sealing: it is the sealing of 5% BSA solution (dissolving with PBS) that every hole adds 100 μ L quality percentage compositions, 37 ℃ of incubation 30min;
(4) wash plate: outwell confining liquid, enzyme plate is dried, on automatic washer, wash plate 4 times, dry with 200 μ L washingss on filter paper;
(5) competition: every hole adds the clenbuterol solution that 50 μ L concentration are 50ng/mL (available from U.S. Sigma company) successively and concentration is the clenbuterol horseradish peroxidase connection thing (available from U.S. Sigma company) of 1.0mg/L, 37 ℃ of incubations competition 30min;
(6) wash plate: outwell reaction solution, enzyme plate is dried, on automatic washer, wash plate 4 times, dry with 200 μ L washingss on filter paper;
(7) colour developing: every hole adds 100 μ L substrate solutions (tmb substrate uses liquid and substrate buffer solution to press the volume ratio mixing of 1:9, adds the 4.0mg urea peroxide in the above-mentioned mixing of every 10mL also), color development at room temperature;
(8) stop: behind the 15min, every hole adds 50 μ L stop buffer termination reactions, and the 450nm wavelength reads the OD value respectively.
Analyzing with Origin7.0 software, is X-coordinate with the concentration of clenbuterol solution, with B/B
0(B and B
0Representative suppresses the colour developing value that Kong Yufei suppresses the hole respectively) be ordinate zou, draw four parametric equation curves, 0 hole colour developing value (B of the direct ELISA typical curve that relatively the anti-clenbuterol monoclonal antibody is participated in before and after the sulfhydrylation
0) and 503nhibiting concentration (IC
50Value), judge the active influence of sulfhydrylation antagonist.
Three repetitions are established in experiment, and the result shows, the A of the typical curve of anti-clenbuterol monoclonal antibody correspondence before and after the sulfhydrylation
0Value is respectively 1.447 and 1.382, IC5
0Value is respectively 3.15 μ g/L and 3.71 μ g/L.The active basic of anti-clenbuterol monoclonal antibody do not lose after sulfhydrylation was described.
4, sulfhydrylation antibody and micro-cantilever gold film combine and direct competitive ELISA method is estimated the situation that combines of sulfhydrylation antibody and the golden film of micro-cantilever
The micro-cantilever (available from American I BM company) that clean and air dried is coated with golden film is put into the enzyme plate aperture; Adding 100 μ L concentration is the sulfhydrylation anti-clenbuterol monoclonal antibody of above-mentioned steps 1 acquisition of 2.5 μ g/mL; 37.5 ℃ incubation 2h promptly obtains being fixed with the micro-cantilever of antibody.
Direct competitive ELISA method is estimated the situation that combines of sulfhydrylation antibody and the golden film of micro-cantilever, and detailed process is following:
(1) clean: with the careful micro-cantilever of antibody that taken out two said fixing of tweezers, with the washings flushing for several times, nitrogen dries up;
(2) sealing: the micro-cantilever after nitrogen dried up is put into enzyme plate respectively, adds 100 μ L quality percentage compositions and be 5% BSA solution (dissolving with PBS) sealing, 37 ℃ of incubation 30min;
(3) clean: carefully take out micro-cantilever with tweezers, with the washings flushing for several times, nitrogen dries up;
(4) compete: the micro-cantilever after nitrogen is dried up is put into two aperture (marked of enzyme plate respectively; Suppress Kong Yufei and suppress the hole); It is the clenbuterol solution (available from U.S. Sigma company) of 100 μ g/L through the concentration of sample diluting liquid (containing the PBS that final concentration is 0.1mol/LpH7.5, the tween 20 of 0.1% volumn concentration and the gelatin of 0.1% quality percentage composition) dilution that the inhibition hole adds 50 μ L, and non-inhibition hole adds 50 μ L sample diluting liquids as contrast; And then adding the clenbuterol horseradish peroxidase connection thing 50 μ L (available from U.S. USBiological company) that concentration is 1.0mg/L respectively, 37 ℃ of incubations are competed 30min;
(5) clean: with the careful micro-cantilever that takes out in the above-mentioned inhibition Kong Yufei inhibition hole of tweezers, with the washings flushing for several times, nitrogen dries up;
(6) develop the color: two micro-cantilevers after nitrogen is dried up are put into two apertures of enzyme plate respectively; Every hole adds 100 μ L substrate solutions, and (tmb substrate uses liquid and substrate buffer solution to press the volume ratio mixing of 1:9; Add the 4.0mg urea peroxide in the above-mentioned mixing of every 10mL also), color development at room temperature;
(7) stop: behind the 15min, every hole adds 50 μ L stop buffer termination reactions, takes out micro-cantilever, and the 450nm wavelength reads the OD value respectively.
Three repetitions are established in experiment, and the result shows that the absorbance under the 450nm wavelength in inhibition hole and non-inhibition hole is respectively 0.150 and 0.587, explains that the anti-clenbuterol monoclonal anti physical efficiency behind the sulfhydrylation effectively is attached on the golden film of micro-cantilever.
Embodiment 3, preparation are modified with the micro-cantilever of antibody
1, utilize the hydrochloric acid mercaptan imine with the antibody sulfhydrylation
(1) accurately taking by weighing anti-clenbuterol monoclonal antibody (available from U.S. USBiological company) 10.0mg is dissolved in and obtains the antibody-solutions that concentration is 10g/L among the 1.0mL PBS;
(2) taking by weighing 2.0mg hydrochloric acid mercaptan imine (available from U.S. Sigma company) is dissolved in and obtains the sulfhydrylization reagent that concentration is 2.0g/L (at present joining existing usefulness) in the 1.0mL zero(ppm) water;
(3) sulfhydrylization reagent with the antibody-solutions of 1.0mL above-mentioned steps (1) and 15.3 μ L above-mentioned steps (2) mixes, and makes that the mol ratio of anti-clenbuterol monoclonal antibody and hydrochloric acid mercaptan imine is 1:1 in the mixing solutions, and gentle agitation is reacted 0.5h under the room temperature condition;
(4) with the PBS damping fluid of 20mM (contain 0.15M NaCl and 1.0mM EDTA, pH7.2) to the reaction solution dialysis 48h of above-mentioned steps (3), during every 2h change a dialyzate, obtain the sulfhydrylation antibody-solutions.
2, the mole binding ratio of antibody and mercapto groups in Ellman ' the s method mensuration sulfhydrylation antibody
(1) take by weighing 100mg5,5-dithio two (2-nitrobenzoic acid) (DTNB) is dissolved in the 25mL0.1M PBS damping fluid (pH8.0);
(2) take by weighing 3.5mg halfcystine (Cys) and be dissolved in the 10mL0.1M PBS damping fluid (pH8.0), obtain the halfcystine that concentration is 2.0mM (Cys) solution; Halfcystine (Cys) solution that with above-mentioned concentration is 2.0mM is diluted to halfcystine (Cys) solution that concentration is respectively 1.0mM, 0.5mM, 0.25mM, 0.125mM and 0.0625mM successively with 0.1M PBS damping fluid (pH8.0);
(3) the sulfhydrylation antibody-solutions that halfcystine (Cys) solution of getting the different concns that above-mentioned steps (2) obtains and above-mentioned steps 1 obtain is 250 μ L respectively; Add respectively in the DTNB solution of 50 μ L above-mentioned steps (1) preparation; Abundant mixing; Room temperature condition reacted 15 minutes down, and the light absorption value of solution is measured at the 412nm place;
(4) volumetric molar concentration with halfcystine (Cys) solution is an X-coordinate; With the light absorption value is ordinate zou; The drawing standard curve; Calculate the volumetric molar concentration of sulfydryl in the sulfhydrylation antibody that above-mentioned steps 1 obtains, be converted into the sulfydryl number of every mole of antibody molecule, promptly obtain mole binding ratio of antibody and mercapto groups in the sulfhydrylation antibody.
Three repetitions are established in experiment, and the result shows that the mole binding ratio of anti-clenbuterol monoclonal antibody and mercapto groups is 1:1 in the sulfhydrylation antibody that above-mentioned steps 1 obtains.
3, active damaed cordition behind the direct competitive ELISA method mensuration antibody sulfhydrylation
(1) encapsulate: to add the concentration after the 100 μ L dilution respectively be the anti-clenbuterol monoclonal antibody of 2.5 μ g/mL and the sulfhydrylation anti-clenbuterol monoclonal antibody of above-mentioned steps 1 acquisition, 37 ℃ of incubation 3h in every hole in the enzyme plate;
(2) wash plate: outwell the antibody-solutions of above-mentioned steps (1), enzyme plate is dried, on automatic washer, wash plate 4 times, dry with the washings of 200 μ L on filter paper;
(3) sealing: it is the sealing of 5% BSA solution (dissolving with PBS) that every hole adds 100 μ L quality percentage compositions, 37 ℃ of incubation 30min;
(4) wash plate: outwell confining liquid, enzyme plate is dried, on automatic washer, wash plate 4 times, dry with 200 μ L washingss on filter paper;
(5) competition: every hole adds the clenbuterol solution that 50 μ L concentration are 50ng/mL (available from U.S. Sigma company) successively and concentration is the clenbuterol horseradish peroxidase connection thing (available from U.S. Sigma company) of 1.0mg/L, 37 ℃ of incubations competition 30min;
(6) wash plate: outwell reaction solution, enzyme plate is dried, on automatic washer, wash plate 4 times, dry with 200 μ L washingss on filter paper;
(7) colour developing: every hole adds 100 μ L substrate solutions (tmb substrate uses liquid and substrate buffer solution to press the volume ratio mixing of 1:9, adds the 4.0mg urea peroxide in the above-mentioned mixing of every 10mL also), color development at room temperature;
(8) stop: behind the 15min, every hole adds 50 μ L stop buffer termination reactions, and the 450nm wavelength reads the OD value respectively.
Analyzing with Origin7.0 software, is X-coordinate with the concentration of clenbuterol solution, with B/B0 (B and B
0Representative suppresses the colour developing value that Kong Yufei suppresses the hole respectively) be ordinate zou, draw four parametric equation curves, 0 hole colour developing value (B of the direct ELISA typical curve that relatively the anti-clenbuterol monoclonal antibody is participated in before and after the sulfhydrylation
0) and 503nhibiting concentration (IC
50Value), judge the active influence of sulfhydrylation antagonist.
Three repetitions are established in experiment, and the result shows, the A of the typical curve of anti-clenbuterol monoclonal antibody correspondence before and after the sulfhydrylation
0Value is respectively 1.435 and 1.378, IC
50Value is respectively 3.18 μ g/L and 3.58 μ g/L.The active basic of anti-clenbuterol monoclonal antibody do not lose after sulfhydrylation was described.
4, sulfhydrylation antibody and micro-cantilever gold film combine and direct competitive ELISA method is estimated the situation that combines of sulfhydrylation antibody and the golden film of micro-cantilever
The micro-cantilever (available from American I BM company) that clean and air dried is coated with golden film is put into the enzyme plate aperture; Adding 100 μ L concentration is the sulfhydrylation anti-clenbuterol monoclonal antibody of above-mentioned steps 1 acquisition of 2.5 μ g/mL; 30 ℃ of incubation 0.5h promptly obtain being fixed with the micro-cantilever of antibody.
Direct competitive ELISA method is estimated the situation that combines of sulfhydrylation antibody and the golden film of micro-cantilever, and detailed process is following:
(1) clean: with the careful micro-cantilever of antibody that taken out two said fixing of tweezers, with the washings flushing for several times, nitrogen dries up;
(2) sealing: the micro-cantilever after nitrogen dried up is put into enzyme plate respectively, adds 100 μ L quality percentage compositions and be 5% BSA solution (dissolving with PBS) sealing, 37 ℃ of incubation 30min;
(3) clean: carefully take out micro-cantilever with tweezers, with the washings flushing for several times, nitrogen dries up;
(4) compete: the micro-cantilever after nitrogen is dried up is put into two aperture (marked of enzyme plate respectively; Suppress Kong Yufei and suppress the hole); It is the clenbuterol solution (available from U.S. Sigma company) of 100 μ g/L through the concentration of sample diluting liquid (containing the PBS that final concentration is 0.1mol/LpH7.5, the tween 20 of 0.1% volumn concentration and the gelatin of 0.1% quality percentage composition) dilution that the inhibition hole adds 50 μ L, and non-inhibition hole adds 50 μ L sample diluting liquids as contrast; And then adding the clenbuterol horseradish peroxidase connection thing 50 μ L (available from U.S. USBiological company) that concentration is 1.0mg/L respectively, 37 ℃ of incubations are competed 30min;
(5) clean: with the careful micro-cantilever that takes out in the above-mentioned inhibition Kong Yufei inhibition hole of tweezers, with the washings flushing for several times, nitrogen dries up;
(6) develop the color: two micro-cantilevers after nitrogen is dried up are put into two apertures of enzyme plate respectively; Every hole adds 100 μ L substrate solutions, and (tmb substrate uses liquid and substrate buffer solution to press the volume ratio mixing of 1:9; Add the 4.0mg urea peroxide in the above-mentioned mixing of every 10mL also), color development at room temperature;
(7) stop: behind the 15min, every hole adds 50 μ L stop buffer termination reactions, takes out micro-cantilever, and the 450nm wavelength reads the OD value respectively.
Three repetitions are established in experiment, and the result shows that the absorbance under the 450nm wavelength in inhibition hole and non-inhibition hole is respectively 0.155 and 0.583, explains that the anti-clenbuterol monoclonal anti physical efficiency behind the sulfhydrylation effectively is attached on the golden film of micro-cantilever.
Claims (9)
1. the hydrochloric acid mercaptan imine is modified with the application in the micro-cantilever of antibody as sulfhydrylization reagent in preparation; Said antibody is the anti-clenbuterol monoclonal antibody.
2. method for preparing the micro-cantilever that is modified with antibody; Be to be sulfhydrylization reagent with the hydrochloric acid mercaptan imine, antagonist carries out sulfhydrylation, obtains sulfhydrylation antibody; Again said sulfhydrylation antibody is fixed on the micro-cantilever that has golden film, obtains being modified with the micro-cantilever of antibody; Said antibody is the anti-clenbuterol monoclonal antibody.
3. method according to claim 2 is characterized in that: in the said sulfhydrylation, the mol ratio of said antibody and said hydrochloric acid mercaptan imine is 1: (1-6).
4. method according to claim 3 is characterized in that: in the said sulfhydrylation, the mol ratio of said antibody and said hydrochloric acid mercaptan imine is 1: 3.
5. according to arbitrary described method among the claim 2-4, it is characterized in that: in the said sulfhydrylation, the reaction conditions of said antibody and said hydrochloric acid mercaptan imine is to react 0.5-2h under the room temperature.
6. method according to claim 5 is characterized in that: in the said sulfhydrylation, the reaction conditions of said antibody and said hydrochloric acid mercaptan imine is to react 1h under the room temperature.
7. according to arbitrary described method among the claim 2-4, it is characterized in that: the reaction conditions on the said golden film that sulfhydrylation antibody is fixed to micro-cantilever is 30-37.5 ℃ of reaction 0.5-2h.
8. method according to claim 7 is characterized in that: the reaction conditions on the said golden film that sulfhydrylation antibody is fixed to micro-cantilever is 37 ℃ of reaction 1h.
9. the micro-cantilever that is modified with antibody that arbitrary described method prepares among the claim 2-8.
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| CN101694494B (en) * | 2009-10-20 | 2012-10-10 | 中国农业大学 | Method for manufacturing micro-cantilever modified with antibody |
| CN101712451B (en) * | 2009-11-18 | 2012-09-05 | 中国科学院上海微系统与信息技术研究所 | One-step method for making carboxylic acid functional monofilm on surface of micro cantilever beam |
| CN102951600B (en) * | 2011-08-19 | 2016-02-03 | 中国科学技术大学 | Micro-beam preparation method that antibody fragment is modified and micro-beam immune sensing detection system of modifying based on antibody fragment |
| CN102951598A (en) * | 2011-08-19 | 2013-03-06 | 中国科学技术大学 | Preparation method of microcantilever modified by antibody fragments, and microcantilever immune sensing detection system based on antibody fragment modification |
| CN109557150A (en) * | 2019-01-14 | 2019-04-02 | 大连大学 | Digital microcurrent-controlled chip and pathogen immunologic detection method based on it |
| CN113514632B (en) * | 2021-04-20 | 2024-06-25 | 中国科学技术大学 | Micro-cantilever beam immunosensory method based on nano antibody |
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