CN101173923B - Method for detecting immune body affinity - Google Patents
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Abstract
The invention discloses a method for testing the affinity of antibody, which mainly comprises the following steps: firstly, coating the antibody with microspheres; secondly, testing the antibody affinity; thirdly, processing data. The invention has the advantages of simple and fast test, high sensitivity, good repeatability, and stabler and more reliable testing result.
Description
Technical field
The invention belongs to the medicine bioengineering class, relate to a kind of method that detects affinity of antibody specifically.
Technical background
Affinity of antibody is one of important parameter of determining antibody character, simultaneously, also is the important indicator of the secreted monoclonal antibody of hybridoma (McAb) stability.The tightness degree that its expression antibody combines with antigen.The antibody of high-affinity is useful for detection by quantitative and guiding diagnosis and treatment, and that the antibody of low-affinity is used for immune purifying is better.Therefore, select antibody, detect with regard to the first antagonist affinity of needs according to application aims with certain affinity.
Affinity of antibody is meant the firm degree of antibody and antigen combination, and affinity costant Ka commonly used represents.Ka be just/ratio of backward reaction rate constant, unit is dilutability unit (L/mol), expression needs how many 1mol antibody dilutions risen to, just can make Ag-Ab in conjunction with descending 50%; And the inverse of Ka is dissociation constant (Kd), and its unit is concentration unit (mol/L).For sensitivity, precision and the accuracy that improves immunoassay, it is very crucial selecting high antibody of Ka value or antiserum for use.
The radiommunoassay (RIA) of competition generally adopt equilibrium dialysis, enzyme linked immunosorbent assay (ELISA) or to(for) the detection of affinity of antibody at present is in conjunction with test etc.Whole antigen-antibody reaction processes of equilibrium dialysis are finished in the homogeneous liquid-phase system, and the disturbing factor that the dynamic process of reaction is subjected to is less, meet the precondition requirement of the mass action law basically fully.But this method only can be measured the affinity costant between micromolecule antigen and corresponding antibodies, require simultaneously reaction substrate is carried out radioactive label, need again after reaction is finished antigen antibody complex and free antigen antibody are separated, experimentation is loaded down with trivial details, complicated, is difficult for extensively carrying out.Though RIA is highly sensitive, can cause radioactive contamination and harm, and exist radionuclide halflife commonly used short, can't automated analysis etc. many deficiencies.It is also very imperfect to adopt noncompetitive ELISA method to measure the antibody affinity costant in theory.ELSIA operation steps complexity, it is more to influence reaction factor, particularly the bag of solid phase carrier is killed in a disaster and is reached unanimity between each individuality, and therefore in quantitative measurement, every BT(batch testing) must be with normative reference product production standard curve under identical condition of a series of variable concentrations.Simultaneously, the bag of antigen is spent the night for 4 ℃ by need, and the washing step of multistep is arranged, and required time is long.
The liquid-phase chip system is full-fledged technology of biochip field.
The liquid-phase chip system comprises Luminex instrument and different fluorescence-encoded microballoons etc.The Luminex instrument is high detection of an automaticity and analytical instrument.Fluorescence-encoded microballoon has 100 kinds at present, can detect 100 kinds of different types of target molecules of phase coupling with it.These microballoons are suspended in the liquid-phase system, just can a plurality of different detected object in the same sample be detected simultaneously, this detection technique is called xMAP (flexible Multi-Analyte Profiling) technology.
In the manufacture process of microballoon, mixed two kinds of different red fluorescence dyestuffs, according to the ratio difference of these two kinds of dyestuffs, can be divided into 100 kinds to microballoon.In the liquid-phase chip system, in order to distinguish different probes, each microballoon that is used for label probe all has the fluorescence-encoded of a uniqueness.Different microballoon covalent bond at the albumen (antibody or antigen are used for immune detection) of difference thing to be detected as probe molecule, detect in the antibody with biotin labeling, and use high-sensitive fluorescent dyeing.These microballoons and determinand, detection antibody, fluorescent marker just form complete microballoon detection architecture and are used for reading of Luminex system.Excitated red respectively laser of Luminex reading system and green laser are used for the detection of microsphere system, and wherein red laser detects the red classification of microsphere surface intensity of fluorescence, and according to different color in the microballoon and number class, thereby determine the type of reactant; Green laser then detects the fluorescence intensity of fluorescent marker in the sample, detects microballoon kind, quantity by machine and computing machine automatic statistical analysis laser again, thereby judges the concentration of sample to be tested plurality of target tester.
The advantage of liquid-phase chip platform:
(1) high flux, the parallel detection of many indexs: liquid-phase chip can be analyzed simultaneously to different molecular in the same sample, can detect reaching 100 kinds of different indexs in 35-60 minute; Compare with the detection one by one of classic method, this is a qualitative leap.
(2) susceptibility height, good reproducibility: traditional enzyme linked immunological absorption (ELISA) technology and plane chip technology are that the light signal to elements collection detects and obtains the result, and only are implemented to half-quantitative detection; Liquid-phase chip technology then can realize detection by quantitative, and single microballoon fluorescence signal is detected, and calculates 100 microballoon fluorescent values, gets its intermediate value (Median), thus the degree of accuracy height, good reproducibility.Sensitivity and sensing range that the early stage double antibody sandwich method that uses liquid phase protein chip carries out verified this type of technology of Study of cytokines are more high than ELISA.
(3) simple to operate, save time sample and cost: liquid-phase chip is reflected in the liquid phase of suspension and carries out, and liquid phase environment can keep the native conformation and the activity of albumen better, also more helps the reaction of probe and detected material, so the reaction time is short.Usually need not clean promptly after the reaction and can directly detect, overcome plane solid phase chip probe points pattern take place easily point sample dislocation, consuming time, parallel equivalent application of sample difficulty, feed head repeatedly application of sample shortcomings such as pollution, poor repeatability between sample easily take place, detection efficiency is much higher than the plane solid phase chip.
Summary of the invention
The technical issues that need to address of the present invention provide a kind of reliable and stable, detect the method for multiple affinity of antibody the time simple to operate.
The technical scheme that solves the problems of the technologies described above is: a kind of method that detects affinity of antibody may further comprise the steps:
(1) antigen coated microballoon:
-with the microballoon activation of 50 μ L, the microballoon after the activation is centrifugal;
-inhale and to abandon supernatant, microballoon is resuspended in the solution of MES damping fluid of 200-300 μ L 50mM of pH=5.0 vortex vibration 10-30s, sonic oscillation 10-30s; Microballoon is centrifugal; This step repeats once;
-inhale and abandon supernatant, microballoon is resuspended in the MES damping fluid (pH5.0) of 100 μ L 50mM the about 10-30s of vortex vibration, the about 10-30s of sonic oscillation;
-in the microballoon that suspends, add 0.8-1.2 μ g antigen, with MES damping fluid (pH5.0) solution of 50mM cumulative volume is mended the L to 450-550 μ; With vortex oscillator mixing, room temperature lucifuge vibration 1.5-3hr;
Microballoon behind the-coupled antigen is centrifugal;
-inhale and to abandon supernatant, with microballoon be resuspended in 500 μ L PBS-TBN (be to contain 0.1%BSA among the PBS, 0.02%Tween 1,0.05%NaN, pH7.4) in the solution, the about 20s of vortex vibration, ultrasonic about 20s; Room temperature lucifuge vibration 30min;
-microballoon behind the coupled antigen is centrifugal, inhale and abandon supernatant, microballoon is resuspended in the 0.8-1.2mL PBS-TBN solution, vortex vibration 10-30s is centrifugal with microballoon behind ultrasonic about 10-30s; Repeat this step once;
-inhale and abandon supernatant, microballoon is resuspended in the 250-1000 μ L PBS-TBN solution;
-Bao is kept in Dark Place in 2-8 ℃ by counting the back by good microballoon;
(2) detection of affinity of antibody:
-take out 96 micropore filter plates, determine and mark position; Antibody to be detected is carried out gradient dilution, for example be diluted to concentration and be respectively: 100,10,1,0.1,0.01,10
-3μ g/ml; Antibody with variable concentrations; Corresponding respectively joining in the micropore of filter plate, the amount in every hole is 23-28 μ l;
-antigen coated microballoon vortex vibration is diluted to 35-45/μ l after the sonicated;
-in micropore, adding antigen coated microballoon 25 μ l respectively, 25-35min is hatched in 37 ℃ of vibrations;
-suction filtration is washed plate three times.
-every hole adds the antiantibody (PE-Ab) of the 2 μ g/ml phycoerythrin marks of 45-55 μ l, and 25-35min is hatched in vibration about 37 ℃;
-suction filtration is washed plate three times;
-read fluorescent value;
(3) data processing.
The computing method of antibody affinity costant K and the rice of enzymatic reaction-Man constant are similar.Under the constant situation of antigen concentration, the antibody (M) of concentration known is carried out gradient dilution.When if antibody concentration is high and the combination rate of enzyme-labelled antigen be 100% (claim Bmax, the MFI value is the highest), then combination rate is that the reciprocal value of 50% pairing antibody concentration is affinity.According to this principle, the inventor has designed following data processing method:
-be mol/L with the antibody concentration unit of being converted to;
-be to take the logarithm at the end with 10, get opposite number again;
-make horizontal ordinate with the above-mentioned data of calculating, make ordinate with corresponding MFI value, make smooth curve figure;
-simulate curvilinear equation;
-antibody high concentration platform MFI value deducts low concentration platform MFI value, divided by two, and the above-mentioned curvilinear equation of substitution, the corresponding horizontal ordinate numerical value of calculating promptly can be used as a typical value of this antibody and antigen affinity.Also can continue to draw the affinity costant (L/mol) of antibody by the reverse computing of above-mentioned steps.
Preferably, described microballoon activation may further comprise the steps:
-usefulness vortex oscillator or ultrasound wave suspension microballoon, approximately 20s;
-to get 50 μ L microballoons centrifugal;
-remove supernatant, microballoon is resuspended in the distilled water of 100 μ L, with vortex oscillator or the about 20s of ultrasound wave suspension microballoon, centrifugal;
-inhale and abandon supernatant, add the phosphate buffer (pH6.2) of 80 μ L, the about 20s of vortex vibration, the about 20s of ultrasound wave suspension microballoon;
-add 10 μ L 50mg/mL Sulfo-NHS (N-Hydroxysulfosuccinimide sodium salt, N-hydroxy thiosuccinimide), use vortex oscillator mixing lightly;
-add 10 μ L 50mg/mL EDC (1-Ethyl-3-(3-dimethyllaminopropyl) carbodiimidehydrochloride, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride), use vortex oscillator mixing lightly;
-room temperature lucifuge leaves standstill 20min, every 10min vortex oscillator mixing lightly.
Preferably, described centrifugal speed 〉=8000g, the time is 1-2min.
The present invention utilizes antigen, PE labelled antibody to set up a kind of brand-new affinity of antibody detection method on the liquid-phase chip platform, has the following advantages:
1) be applied to the raw material quality inspection and the antibody screening of liquid-phase chip detection technique platform, detection method of the present invention does not need cyclic washing, simple and fast;
2) susceptibility height, good reproducibility, testing result is more reliable and more stable;
3) can carry out affinity to multiple antibody simultaneously and detect, improve detection efficiency greatly.
Description of drawings
Fig. 1 is embodiment 1 a testing result synoptic diagram;
Fig. 2 is embodiment 2 testing result synoptic diagram;
Fig. 3 is embodiment 3 testing result synoptic diagram.
Embodiment
The present invention is described further below in conjunction with embodiment and accompanying drawing.
Among the embodiment, the prescription of described various solution is as follows:
1.50mM MES damping fluid (pH5.0) prescription (250ml):
| Reagent | The source | Final concentration | The consumption of every 250ml |
| MES (2[N-Morpholino] ethanesulfonic acid) | Sigma?M-2933 | 0.05M | 2.44g |
| 5M?NaOH | Fisher?SS256-500 | --- | 5 |
2.PBS prescription:
| Reagent | Catalog number (Cat.No.) | Final concentration | The consumption of every 1L |
| PBS | Sigma-3813 | 138mM?NaCl 2.7mM?KCl | 1 bag |
3.PBS-TBN prescription (be to contain 0.1%BSA among the PBS, 0.02%Tween-20,0.05%Na3N, pH7.4)
| Reagent | Catalog number (Cat.No.) | Final concentration | The consumption of every 1L |
| PBS?pH7.4 | Sigma?P-3813 | 138mM?NaCl 2.7mM?KCl | 1 bag |
| BSA | Sigma?A-9647 | 0.1％ | 1g |
| Tween-20 | Sigma?P-9416 | 0.02％ | 0.2ml |
| Sodium?Azide | Sigma?S-8032 | 0.05％Azide | 500mg |
Embodiment 1
With three kinds of different antibodies of β-HCG (β-subunit human chorionic gonadotropin, hCG-) (clone number is respectively M94139.7,220-5011, affinity 220-5013) detects and is example.
The step of described affinity of antibody detection method is as follows:
Antigen coated microballoon:
-usefulness vortex oscillator or ultrasound wave suspension microballoon (U.S. Luminex company), approximately 20s;
-get 50 μ L microballoons in the centrifuge tube of 1.5ml, 〉=8, the centrifugal 1-2min of the speed of 000g;
-remove supernatant, microballoon is resuspended in the distilled water of 100 μ L, with vortex oscillator or the about 20s of ultrasound wave suspension microballoon, the centrifugal 1-2min of the speed of 〉=8000g;
-inhale and abandon supernatant, add the phosphate buffer (pH6.2) of 80 μ L, the about 20s of vortex vibration, the about 20s of ultrasound wave suspension microballoon;
-add 10 μ L 50mg/mL Sulfo-NHS, use vortex oscillator mixing lightly;
-add 10 μ L 50mg/mL EDC, use vortex oscillator mixing lightly;
-room temperature lucifuge leaves standstill 20min, every 10min vortex oscillator mixing lightly;
Microballoon after the-activation, 〉=8000g, centrifugal 1-2min;
-inhale and to abandon supernatant, microballoon is resuspended in the solution of MES (pH5.0) of 250 μ L 50mM the about 20s of vortex vibration, ultrasonic about 20s, microballoon 〉=8000g, centrifugal 1-2min; Repeat this step once;
-inhale and to abandon supernatant, microballoon is resuspended in the solution of MES (pH5.0) of 100 μ L 50mM the about 20s of vortex vibration, ultrasonic about 20s;
-in the microballoon that suspends, add 1 μ g β-HCG antigen, with MES (pH5.0) solution of 50mM cumulative volume is mended to 500 μ L;
-usefulness vortex oscillator mixing, room temperature lucifuge vibration 2hr;
Microballoon behind the-coupled antigen is with the speed of 〉=8000g, centrifugal 1-2min;
-inhale and abandon supernatant, microballoon is resuspended in the 500 μ L PBS-TBN solution the about 20s of vortex vibration, ultrasonic about 20s;
-room temperature lucifuge vibration 30min;
-microballoon 〉=8000g, centrifugal 1-2min;
-inhale and to abandon supernatant, microballoon is resuspended in the 1mL PBS-TBN solution, the about 20s of vortex vibration, ultrasonic about 20s, microballoon is in 〉=8000g, centrifugal 1-2min; Repeat this step once;
-inhale and abandon supernatant, microballoon is resuspended in the 250-1000 μ L PBS-TBN solution;
-Bao is counted by the luminex instrument by good microballoon;
-Bao is placed 2-8 ℃ to keep in Dark Place by good microballoon.
The detection of affinity of antibody:
-take out 96 micropore filter plates, determine and mark position;
-(clone number is respectively M94139.7, and 220-5011 220-5013) carries out gradient dilution respectively, is diluted to concentration and is respectively: 20,2,0.2,0.02,0.002,0.0002 μ g/ml joins respectively in the filter plate micropore by mark position with β-HCG antibody;
-antigen coated microballoon vortex vibration (vortex) 30s, sonicated 1min is diluted to 40/μ l with antigen coated microballoon.
Add antigen coated microballoon 25 μ l (i.e. 1,000/hole) and antibody 25 μ l in-the filter plate micropore respectively, 30min is hatched in 37 ℃ of concussions.
-suction filtration is washed plate three times.
-adding the 2 μ g/ml PE-Ab (antiantibody of phycoerythrin mark) of corresponding 50 μ l respectively, 30min is hatched in 37 ℃ of lucifuge vibrations.
-suction filtration is washed plate three times.
-last machine-readable fluorescent value.
-data processing:
-be mol/L with the antibody concentration unit of being converted to;
-be to take the logarithm at the end with 10, get opposite number again;
-make horizontal ordinate with the above-mentioned data of calculating, make ordinate with corresponding MFI value, make smooth curve figure;
-simulate curvilinear equation;
-antibody high concentration platform MFI value deducts low concentration platform MFI value, divided by two, and the above-mentioned curvilinear equation of substitution, the corresponding horizontal ordinate numerical value of calculating promptly can be used as a typical value of this antibody and antigen affinity.Also can continue to draw the affinity costant (L/mol) of antibody by the reverse computing of above-mentioned steps.
Experimental result: with β-HCG is example: the MFI value is a fluorescent value in the table, and M94139.7,220-5011,220-5013 are respectively three kinds of different antibodies of β-HCG.
Table 1
Negative logarithm M94139.7 (MFI value) 220-5011 (MFI value) 220-5013 (MFI value) of μ g/ml mol/L concentration
20 0.000125 3.903089987 27734.5 4327.5 27234
2 0.0000125 4.903089987 27391 395.5 24628.5
0.2 0.00000125 5.903089987 25055.5 29.5 5296
0.02 0.000000125 6.903089987 2714.5 14.5 225
0.002 1.25E-08 7.903089987 119.5 7 22
0.0002 1.25E-09 8.903089987 20 12 10
The result is calculated as follows:
The calculating of M94139.7 antibody affinity costant:
Y=(27734.5-20)/2=13857 substitution formula y=-22341x+ 156936
Solve x=6.404312699
Affinity costant K=1/ (10
-6.404312699)=2.50E+06
In like manner, can calculate the affinity costant of 220-5011 and two kinds of antibody of 220-5013, be respectively 2.85E+04,2.97E+05
Embodiment 2
Three kinds of antibody of PSA (Prostate Specific Antigen, prostate specific antigen) (clone number is respectively M701042, PS5, and BM215) affinity detects.
Method therefor is identical with the step of embodiment 1.The result is as follows,
Table 2
Negative logarithm M701042 (MFI value) PS5 (MFI value) BM215 (MFI value) of μ g/ml mol/L concentration
100 6.25E-07 6.20412 6323.5 5369 10054.5
10 6.25E-08 7.20412 6479 4481.5 7657
1 6.25E-09 8.20412 2031.5 1910.5 3447.5
0.1 6.25E-10 9.20412 640 183.5 418
0.01 6.25E-11 10.20412 336.5 97 125
0.001 6.25E-12 11.20412 438.5 52.5 57
The result is calculated as follows:
The calculating of M701042 antibody affinity costant:
Y=(6323.5-438.5)/2=2942.5, substitution formula y=-4447.5x+38174 solves x=7.92164
Affinity costant K=1/ (10
-7.92164)=8.35E+07
In like manner, can calculate the affinity costant of PS5 and two kinds of antibody of BM215, be respectively 9.70E+07,7.66E+07.
Embodiment 3
Three kinds of antibody of TNF-α (Tumor necrosis factor-alpha, tumor necrosis factor) (clone number be respectively TNF-α F6, D2,28401.111) affinity detects.
Method therefor is identical with the step of embodiment 1.The result is as follows,
Table 3
Negative logarithm TNF-α F6 (MFI value) D2 (MFI value) 28401.111 (MFI value) of μ g/ml mol/L concentration
100 0.000625 3.204119983 10233.5 20352 16397
10 6.25E-05 4.204119983 5137.5 15397.5 12433
1 6.25E-06 5.204119983 270.5 6122 5077
0.1 6.25E-07 6.204119983 0 779 722.5
0.01 6.25E-08 7.204119983 0 70.5 73
0.001 6.25E-09 8.204119983 2 15.5 16
0.0001 6.25E-10 9.204119983 16
The result is calculated as follows:
The calculating of TNF-α F6 antibody affinity costant:
Y=(10233.5-2)/2=5116.75, substitution formula y=-4981.5x+26157 solves x=4.2237
Affinity costant K=1/ (10
-4.2237)=1.67E+04
In like manner, can calculate the affinity costant of D2 and 28401.111 two kinds of antibody, be respectively 5.86E+04,6.97E+04.
Claims (4)
1. a method that detects affinity of antibody is characterized in that, mainly may further comprise the steps:
(1) antigen coated microballoon:
-with the microballoon activation of 50 μ L, the microballoon after the activation is centrifugal;
-inhale and to abandon supernatant, microballoon is resuspended in the solution of MES damping fluid of 200-300 μ L 50mM of pH=5.0 vortex vibration 10-30s, sonic oscillation 10-30s; Microballoon is centrifugal; This step repeats once;
-inhale and to abandon supernatant, microballoon is resuspended in the MES damping fluid of 100 μ L 50mM of pH=5.0 the about 10-30s of vortex vibration, the about 10-30s of sonicated;
-in the microballoon that suspends, add 0.8-1.2 μ g antigen, with the MES buffer soln of the 50mM of pH=5.0 cumulative volume is mended the L to 450-550 μ; With vortex oscillator mixing, room temperature lucifuge vibration 1.5-3hr;
-with coupling the microballoon behind the antigen centrifugal;
-inhale and abandon supernatant, microballoon is resuspended in the 500 μ L PBS-TBN solution the about 20s of vortex vibration, ultrasonic about 20s; Room temperature lucifuge vibration 30min;
-with coupling the microballoon of antigen centrifugal, inhale and abandon supernatant, microballoon is resuspended in the 0.8-1.2mL PBS-TBN solution, vortex vibration 10-30s, behind ultrasonic about 10-30s that microballoon is centrifugal; Repeat this step once;
-inhale and abandon supernatant, microballoon is resuspended in the 250-1000 μ L PBS-TBN solution;
Above-mentioned PBS-TBN solution is for containing 0.1%BSA, 0.02%Tween-20,0.05%Na
3The PBS solution of N;
-Bao is kept in Dark Place in 2-8 ℃ by counting the back by good microballoon;
(2) detection of affinity of antibody:
-take out the micropore filter plate, determine and mark position; Antibody to be detected is carried out gradient dilution; With the antibody of variable concentrations, corresponding respectively joining in the micropore of filter plate, the amount in every hole is 23-28 μ L;
-antigen coated microballoon vortex vibration is diluted to 35-45/μ L after the sonicated;
-in micropore, adding antigen coated microballoon 25 μ L respectively, 25-35min is hatched in concussion about 37 ℃;
-suction filtration is washed plate three to five times;
-every hole adds the antiantibody of the 2 μ g/mL phycoerythrin marks of 45-55 μ L respectively, and 25-40min is hatched in 37 ℃ of left and right sides lucifuge vibrations;
-suction filtration is washed plate three to five times;
-read fluorescent value;
(3) data processing.
2. the method for detection affinity of antibody according to claim 1 is characterized in that: described microballoon activation may further comprise the steps:
-usefulness vortex oscillator or ultrasound wave suspension microballoon, approximately 20s;
-to get 50 μ L microballoons centrifugal;
-remove supernatant, microballoon is resuspended in the distilled water of 100 μ L, with vortex oscillator or the about 20s of ultrasound wave suspension microballoon, centrifugal;
-inhale and abandon supernatant, add the phosphate buffer of the 80 μ L of pH=6.2, the about 20s of vortex vibration, the about 20s of ultrasound wave suspension microballoon;
-add 10 μ L 50mg/mL Sulfo-NHS, use vortex oscillator mixing lightly;
-add 10 μ L 50mg/mL EDC, use vortex oscillator mixing lightly;
-room temperature lucifuge leaves standstill 20min, every 10min vortex oscillator mixing lightly.
3. the method for detection affinity of antibody according to claim 1 and 2 is characterized in that, described centrifugal speed 〉=8000g, and the time is 1-2min.
4. the method for detection affinity of antibody according to claim 1 and 2 is characterized in that, described data processing may further comprise the steps:
-be mol/L with the antibody concentration unit of being converted to
-be to take the logarithm at the end with 10, get opposite number again
-make horizontal ordinate with the above-mentioned data of calculating, make ordinate with corresponding MFI value, make smooth curve figure
-simulate curvilinear equation
-antibody high concentration platform MFI value deducts low concentration platform MFI value, divided by two, and the above-mentioned curvilinear equation of substitution, the corresponding horizontal ordinate numerical value of calculating promptly can be used as a typical value of this antibody and antigen affinity; Continuation draws the affinity costant of antibody by the reverse computing of above-mentioned steps.
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| CN1825118A (en) * | 2006-04-05 | 2006-08-30 | 北京盈九思科技发展有限公司 | Method for detecting medicine residue |
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| CN1825118A (en) * | 2006-04-05 | 2006-08-30 | 北京盈九思科技发展有限公司 | Method for detecting medicine residue |
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| 孙凯等.液相芯片技术研究应用进展.《中华实验外科杂志》.2005,第22卷(第5期),639-640. * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11892448B2 (en) | 2012-08-08 | 2024-02-06 | Hoffmann-La Roche Inc. | Immunoassay-based determination of in-solution binding kinetics |
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