CN1880447A - Process for preparing high-flux monoclonal antibody - Google Patents
Process for preparing high-flux monoclonal antibody Download PDFInfo
- Publication number
- CN1880447A CN1880447A CN 200510026873 CN200510026873A CN1880447A CN 1880447 A CN1880447 A CN 1880447A CN 200510026873 CN200510026873 CN 200510026873 CN 200510026873 A CN200510026873 A CN 200510026873A CN 1880447 A CN1880447 A CN 1880447A
- Authority
- CN
- China
- Prior art keywords
- chip
- hybridoma
- monoclonal antibody
- protein
- cell
- 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
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a monoclonal antibody preparing method of high-flux, which comprises the following steps: (a) proceeding immune inoculation for non-human mammal animal through multiple immunogen; (b) compounding splenocyte of immune inoculation animal and bone marrow oncocyte to fuse into cross oncocyte; (c) collecting the cross oncocyte; culturing; preparing cross oncocyte suspension; (d) screening cross oncocyte through biological chip to produce cross oncocyte system of immunogen monoclonal antibody.
Description
Technical field
The present invention relates to biological field, relate more specifically to a kind of the associating use protein chip, polypeptide chip and organization chip high-throughput ground preparation monoclonal antibody method.
Background technology
At present in the MONOCLONAL ANTIBODIES SPECIFIC FOR process, traditional method is to use a kind of albumen or antigen immune mouse, determines the hybridoma that produces antibody by the ELISA method, clones and builds strain, further the monoclonal antibody of purifying is identified then, detected its epitope again as needs.This preparation method often can only obtain a strain specific antibodies.
In real work, often obtain the hybridoma of a large amount of generation antibody when fusion rate is high, but can't clone simultaneously, can only screen positive cell according to the OD value that microplate reader is read, clone and build strain, so very likely cause the monoclonal antibody of the different cell strains that obtain to act on same epitope.
Along with the enforcement and the propelling of the Human Genome Project, life science has entered the genome times afterwards comprehensively.In these epoch, the main research object of life science is a functional genomics, comprises research of structure gene group and proteome research etc.Although the genome of existing a plurality of species is checked order now, from the genome to protein, there are transcriptional level, translation skill and three kinds of regulatory mechanisms of translation back level, the information that obtains in the genomics can not be represented protein expression level comprehensively.
Protein is the executive of physiological function, is the direct agent of biological phenomena, will directly illustrate the change mechanism of life under physiology or pathological conditions to the research of protein structure and function.The existence form of protein itself and mechanics as problems such as posttranslational modification, protein-protein interaction and protein conformations, still depend on directly proteinic research are solved.Though special propertys such as proteinic mutability and diversity have caused the protein research technology more complicated and much more difficult than nucleic acid technology far away, these mutabilities and diversity participate in and affect whole vital process just.
Traditional mode that single protein is studied can't satisfy the requirement of genome times afterwards comprehensively.This be because: the generation of (1) biological phenomena is multifactor impact often, must relate to a plurality of protein.(2) a plurality of proteinic participations are woven into network, or parallel generation, or are the cascade cause and effect.(3) proteinic performance is various, dynamic when carrying out physiological function, and it is constant not resemble the genome basic fixed.Therefore to comprehensive and deep understanding be arranged to the complicated activity of life, must on the level of whole, dynamic, network, study protein.Therefore in the mid-90 in last century, produced a new branch of science-proteomics (Proteomics) in the world, it be with all protein in the cell exist and manner is a research object.We can say that carrying out of proteome research be not only the milestone that life science enters the genome times afterwards comprehensively, also is one of core content of genome times afterwards comprehensively life science.
Antibody be research protein properties and function and with the important tool of the relation of disease.Antibody is the ideal tools of analysis of cells protein group, specificity height not only, and can separate very delicately and read proteinic relative abundance in the cell pyrolysis liquid sample, use various antibody, by quantitatively monitoring the variation of protein expression profiles at different cell proteins; Utilize antibody chip or the same tissue of different tissues and different situations is carried out parllel screening, can determine differentially expressed protein with same antibody, or even the albumen of new agnoprotein or disease-related.
Effectively preparation monoclonal anti body method is the hybridoma method at present, promptly comprise step: use a kind of immunogen mouse, carry out fusion growth with the myeloma cell, carry out hybridoma screening and strain is built in cloning, further the monoclonal antibody that obtains is identified then by the ELISA method.Yet the monoclonal antibody that this preparation method obtains can only be discerned specific antigenic determinant, and in the real work, once merges the hybridoma that can obtain several generation antibody, and the factors such as manpower that need consume when screening are very huge.Because workload is huge, makes this high-tech technology of MONOCLONAL ANTIBODIES SPECIFIC FOR become the technology of manpower consumption's type, thereby also just becomes restrictive factor at work, also becomes the factor of speed limit.The important aspect of another one is, specificity to the antigenic determinant identification of the character of monoclonal antibody especially its recognition site, usually need after acquisition antibody, can further verify, greatly reduce the screening efficiency in the monoclonal antibody production process.
If use the former while immunity of panimmunity for improving flux, once merge, the screening means are subjected to the restriction of common ELISA screening method again.Traditional E LISA screening needs to use a large amount of immune primordial covering flat boards, use a large amount of cell culture fluid supernatants to detect simultaneously, and in real work, immunogen is very micro-often, very precious, and the culture supernatant of each hybridoma also has only the 100-200 microlitre, is difficult to the flux of raising Antibody Preparation from the actual experiment operation.
In sum, still there is not the gratifying multiple monoclonal antibody method for preparing effectively and quickly up to now.Therefore this area press for exploitation effectively, fast, high-throughput ground prepares at the monoclonal antibody method of synantigen and/or different epitopes not.
Summary of the invention
Purpose of the present invention just provide a kind of effectively, fast, high-throughput ground preparation monoclonal antibody method, this method can be by once immunity, prepares in a large number at the antibody of synantigen and/or different epitopes not.
In a first aspect of the present invention, a kind of high-throughout method for preparing monoclonal antibody is provided, may further comprise the steps:
(a) with 3-10000 kind immunogen non-human mammal is carried out immunization;
(b) will get the splenocyte that white above-mentioned immunization is crossed immunogenic non-human mammal, mix, and merge the formation hybridoma with the myeloma cell of non-human mammal of the same race;
(c) collect hybridoma, cultivate, be prepared into the hybridoma suspension;
(d) with the biochip that is selected from down group: protein chip, polypeptide chip, organization chip or its combination, wherein said biochip is coated with the described immunogenic point of sample of 3-10000 kind, described hybridoma is screened, thereby secreted hybridoma cell line respectively at the described immunogenic monoclonal antibody of 3-10000 kind.
In another preference, in step (d), screen with protein chip earlier, screen with polypeptide chip again.
In another preference, the white group down of described immunogen choosing: protein, polypeptide, cell, tissue, cell lysate or extract, lipid, carbohydrate, nucleic acid, pathogenic agent, viral capsid or its mixture.
In another preference, described non-human mammal is rat, mouse.
In another preference, described immunogenic quantity is the 4-5000 kind, more preferably the 5-1000 kind.
In another preference, described method also comprises step:
(e) use the hybridoma that a step obtains, produce monoclonal antibody;
(f) separate the monoclonal antibody that described monoclonal antibody obtains purifying.
In another preference, described step (e) is by being 1-5 * 10 with the cell count with hybridoma
6Cell inoculation is collected mouse ascites in mouse after 5-10 days; Perhaps make its quantity reach 10 by cultivating hybridoma
7Individual-10
10Individual (more preferably 2 * 10
7Individual-1 * 10
9Individual), get cell conditioned medium then.
In another preference, described step (f) is to use the affinity column of Protein G to carry out purifying, obtains pure monoclonal antibody protein.
In another preference, described method also comprises step:
(g) with the monoclonal antibody point sample of purifying on chip substrate, make the monoclonal antibody chip.
In another preference, described biochip is a porous plate, and described porous plate has 4-10000 hole, and a chip substrate is arranged in each hole, and point sample has the described immunogen of 3-10000 kind on the chip substrate.
In a second aspect of the present invention, the monoclonal antibody that obtains with the inventive method screening also is provided, and with the application of described monoclonal antibody.
Description of drawings
Fig. 1 is the synoptic diagram of a single hole in monoclonal antibody screening protein chip or the polypeptide chip.
Fig. 2 has shown the protein chip in example of the present invention.Wherein Fig. 2 A uses different dot matrix respectively with 2B: Fig. 2 A protein chip array adopts 3 * 3 and 4 * 4 two kinds, and Fig. 2 B protein chip array is unified to adopt 4 * 4.
Fig. 3 has shown single hole in the protein chip.Wherein, this hole is 4 * 4 array chips, and four lines is from top to bottom respectively with A, B, C, D name, four row are from left to right respectively with 1,2,3,4 names, the wherein negative contrast of A1 and A4, the positive contrast of D2, D4, other 12 points are represented 6 kinds of antigens (each antigen has two point of samples) respectively.
Embodiment
The inventor is through extensive and deep research, Monoclonal Antibody technology and protein chip, polypeptide chip and the tissue array technology of routine are organically combined, thereby realized first that by once immunity a large amount of preparations are at the antibody of synantigen and/or different epitopes not.Finished the present invention on this basis.
Biochip technology is based on the interactional large-scale parallel analytical procedure of biomacromolecule (nucleic acid, protein etc.), has become one of current life science field technology with fastest developing speed.
Protein chip is to detect interactional biochip between the protein, it is based on antigen and antibodies specific bonded principle, utilize microarray technology and multiple proteins to be combined on the solid-phase matrix, thereby traditional biological analysis means can be finished in minimum scope fast, reach the purpose that once experiment is analyzed a plurality of biological samples simultaneously or detected multiple disease.On the protein biochip technology theory, can in single test, provide a large amount of biological informations.
Polypeptide chip promptly is that the peptide section with different lengths is fixed on the solid support, by antibody antigen specificity bonded principle corresponding sample is detected, screens, identifies.
Organization chip is that the tissue sample with for example dozens of even thousands of different patients is integrated into a micro-array tissue.
Referring to Fig. 1.Wherein shown and be used for monoclonal antibody screening protein chip of the present invention or single hole of polypeptide chip.Wherein, described single hole is the single hole in the porous plate (as 96 orifice plates or 384 orifice plates), and every hole is provided with positive control area, negative control area and screening zone respectively.Positive control point of sample and negative control point of sample are arranged respectively in positive control and negative control area.In protein chip, corresponding screening is arranged with antigen or albumen at the regional mid point of screening; In polypeptide chip, at the epitope of the screening usefulness that has of the regional mid point of screening.Entire chip is integrated by a large amount of identical single holes, perhaps direct pattern with 96 orifice plates, 384 orifice plates, or be integrated on other moulds.
In the present invention, by optimizing the antigen immune condition in the Monoclonal Antibody, with biochip technology, be that organization chip, protein chip and polypeptide chip are united as a kind of screening platform application in MONOCLONAL ANTIBODIES SPECIFIC FOR, can reach a large amount of preparations of once immunity at the antibody of synantigen and/or different epitopes not.
In a preference, high-throughput method for preparing monoclonal antibody of the present invention may further comprise the steps:
(1) uses 3-10000 kind (preferably 4-1000 kind) immunogen (as protein, polypeptide) immune mouse simultaneously.For example, use cell, tissue and cell lysate, natural or recombinant expressed albumen, synthetic polypeptide or its mixture immunity non-human mammal (for example mouse, rat) of purification.Immunization ways is the routine immunization of this area, but booster immunization in case of necessity;
(2) spleen of getting mouse is made cell suspension, and (as 5: 1-2: 1) mix, prepare hybridoma under the fusogen effect, hybridoma is merged in collection, cultivates for some time (as 7-8 days), is prepared into suspension with myeloma cell's ratio routinely;
(3) use the biochip at complete antigen of difference and/or epitope of prepared beforehand (comprising protein chip, polypeptide chip and/or organization chip) as required, the hybridoma of being cultivated is carried out the screening of antibody validity, the hybridoma cell line that screening obtains having the stably excreting monoclonal antibody;
(4) with the cell count 1-5 * 10 with the hybridoma cell line that obtains
6Injection cell is to the Balb/C mouse, gets mouse ascites after waiting for about a week, perhaps hybridoma is cultured to quantity in Tissue Culture Flask and reaches 10
7More than, get cell conditioned medium;
(5) use the affinity column of ProteinG that mouse ascites or cell conditioned medium are carried out purifying, obtain pure monoclonal antibody protein;
(6) use a series of Monoclonal Antibody biochips that obtain, carry out the detection and the proteome research of tumour.
On the other hand, the also disclosed a kind of preparation of the present invention is used for the preparation method of the inventive method biochip, and it may further comprise the steps:
(1) for protein chip,, different cell lysates, the natural or recombinant expressed albumen of purifying are integrated into a microarray according to the monoclonal antibody of desire screening;
(2) for polypeptide chip, according to the monoclonal antibody of desire screening, use the possible epitope of information biology means analysis, synthetic different lengths polypeptide is integrated into a microarray;
(3) for organization chip, on purpose or at random select dozens of even thousands of different patients' tissue sample to be integrated into a micro-array tissue;
(4) chip joint is the different zones that above three kinds of biochips is structured in simultaneously a chip;
As for the pre-treatment of solid phase biological chip carrier or liquid phase biochip carrier and the sealing and the post-processed of biochip, can adopt the routine techniques in biochip field.
In a preference, the mistake of utilizing protein chip, polypeptide chip and/or organization chip to screen can may further comprise the steps:
(1) screening of protein chip
A. the surface that different hybridoma suspension (each hole on corresponding 96 porocyte culture plates or the 384 porocyte culture plates) is added protein chip, protein chip has been fixed different known antigens marks, repeat region on the chip adds different cell suspensions, hatches 30-60 minute in 37 ℃ of wet boxes;
B. use phosphate buffered saline buffer washing chip 3-5 time;
C. add anti-mouse specific antibody on protein chip, antibody was hatched 30-60 minute in 37 ℃ of wet boxes with one or more marks in fluorescence dye or colour developing or the chemoluminescence substrate for enzymatic activity;
D. use phosphate buffered saline buffer washing chip 3-5 time;
E. for the anti-mouse specific antibody that uses fluorochrome label, protein chip directly detects with chip scanner, uses the protein chip of colour developing or chemoluminescence substrate for enzymatic activity mark, after the reaction of adding colour developing liquid, detects with corresponding scanner;
F. repeated experiments, the positive colony that obtains at synantigen not, are set up the hybridoma storehouse respectively.
(2) screening of polypeptide chip
A. use set up previously at the antigenic hybridoma of difference storehouse, different hybridoma suspension (each hole on corresponding 96 porocyte culture plates or the 384 porocyte culture plates) is added the antigenic surface of polypeptide chip corresponding polypeptide, the different zones of polypeptide chip has been fixed different polypeptide antigens, repeat region on the chip adds different cell suspensions, hatches 30-60 minute in 37 ℃ of wet boxes;
B. use phosphate buffered saline buffer washing chip 3-5 time;
C. add anti-mouse specific antibody on polypeptide chip, antibody was hatched 30-60 minute in 37 ℃ of wet boxes with one or more marks in fluorescence dye or colour developing or the chemoluminescence substrate for enzymatic activity;
D. use phosphate buffered saline buffer washing chip 3-5 time;
E. use the polypeptide chip of detection of fluorescent dyes directly to detect, use the polypeptide chip of colour developing or chemoluminescence substrate for enzymatic activity mark, after the reaction of adding colour developing liquid, detect with corresponding scanner with chip scanner;
F. repeated experiments, the positive colony that obtains is set up corresponding hybridoma storehouse respectively at different epitopes.
Certainly, also can directly screen the hybridoma suspension with polypeptide chip.
(3) screening of organization chip
A. add the surface of organization chip with what obtain previously at the hybridoma suspension of synantigen and different epitopes (each hole on corresponding 96 porocyte culture plates or the 384 porocyte culture plates) not, hatched 30-60 minute in 37 ℃ of wet boxes;
B. use phosphate buffered saline buffer washing chip 3-5 time;
C. add anti-mouse specific antibody on organization chip, antibody was hatched 30-60 minute in 37 ℃ of wet boxes with one or more marks in fluorescence dye or colour developing or the chemoluminescence substrate for enzymatic activity;
D. use phosphate buffered saline buffer washing chip 3-5 time;
E. use the organization chip of detection of fluorescent dyes directly to detect, use the organization chip of colour developing or chemoluminescence substrate for enzymatic activity mark, after the reaction of adding colour developing liquid, detect with corresponding scanner with chip scanner;
F. repeated experiments, positive findings illustrate that this antigen expresses in the tissue that organization chip comprises, set up corresponding hybridoma storehouse respectively.
Certainly, also can directly screen the hybridoma suspension with organization chip.
For the hybridoma that filters out, available ordinary method manufacture order clonal antibody also carries out purifying.For example in a preference, monoclonal antibody protein production and purge process may further comprise the steps:
(1) with the cell count 1-5 * 10 with the hybridoma cell line that obtains
6Injection cell is to the Balb/C mouse, gets mouse ascites after waiting for about a week, perhaps hybridoma is cultured to quantity in Tissue Culture Flask and reaches 10
7More than, get cell conditioned medium;
(2) use the affinity column of ProteinG that mouse ascites or cell conditioned medium are carried out purifying, obtain pure monoclonal antibody protein.
Prepare monoclonal antibody with the inventive method, both can be applicable to proteome research, also can be applicable to the exploitation of clinical medicine and diagnostic reagent.
Major advantage of the present invention is:
First, immunization can be used 3-10000 kind (preferably 4-1000 kind) immunogen (comprise natural antigen or use natural tissues or its lysate etc.), thereby by realizing once (or minority) immunity, obtain in a large number at the antibody of synantigen and/or different epitopes not, overall cost can descend at double.
The second, simple to operate, do not need the repetition immune mouse, do not need to repeat to merge, saved plenty of time and cost.
The 3rd, when using the antigen immune mouse, can use a small amount of native protein or tissue, can use recombinant protein or synthetic polypeptide in the time of chip manufacturing, but the antibody that screening obtains is the antibody of anti-natural antigen epi-position, has saved a large amount of antigens and fund;
The 4th, Application of Biochips has realized high-throughout screening, greatly the preparation of enhancing antibody and research process;
The 5th, a large amount of monoclonal antibodies that provide provide effective research tool for proteome research, for the exploitation of clinical medicine, diagnostic reagent provides raw material.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
The high-throughput preparation of tumor markers monoclonal antibody
One, mouse immune and hybridoma preparation
(1) chooses known tumor markers CEA, CA19-9, CA242, CA50, CA724 stone kind tumor markers, as the antigen of preparation monoclonal antibody;
(2) to the antigen of mouse peritoneal injection total amount 200 micrograms, every kind 40 microgram, antigen mixes with Fu Shi Freund's complete adjuvant equal-volume, booster immunization after two weeks, the antigen total dose is 200 micrograms, antigen mixes with the freund adjuvant equal-volume, after two weeks, inject 20 microgram hybrid antigens to mouse peritoneal, after the week, to mouse tail vein injection 20 microgram hybrid antigens;
(3) spleen of getting mouse after the last immunity on the 3rd day is made cell suspension, mixes according to a certain percentage with the myeloma cell, prepares hybridoma under the fusogen effect, collects to merge hybridoma, cultivates 7-8 days, is prepared into suspension;
Two, the preparation of protein chip
Use tumor markers CEA, CA19-9, CA242, five kinds of tumor markerses of CA50, CA724 to prepare protein chip.
(1) the film chip is adopted in the preparation of protein chip, use point sample instrument by certain array in proper order with above-mentioned five kinds of antigen dot matrix to be integrated on the film in 96 orifice plates, the positive and negative control are set simultaneously;
(2) use 37 ℃ of sealings of 5% milk powder (containing 0.1% tween) a hour, rearmounted 4 degree of washing are preserved stand-by.
The result:
The protein chip that makes is shown in Fig. 2 A and 2B.Wherein Fig. 2 A uses different dot matrix respectively with 2B: Fig. 2 A protein chip array adopts 3 * 3 and 4 * 4 two kinds, and Fig. 2 B protein chip array is unified to adopt 4 * 4.
Three, the protein chip of hybridoma screening
A. because the protein chip for preparing previously is 96 well plate format, different hybridoma suspension (each hole on the corresponding 96 porocyte culture plates) 50 microlitres in the hybridoma storehouse that obtains are previously added in the corresponding hole of protein chip, hatched 30-60 minute in 37 ℃ of wet boxes;
B. use phosphate buffered saline buffer washing chip 3-5 time;
C. add anti-mouse specific antibody on protein chip, antibody was hatched 30-60 minute in 37 ℃ of wet boxes with horseradish peroxidase-labeled;
D. use phosphate buffered saline buffer washing chip 3-5 time;
E. use the horseradish peroxidase substrate to develop the color, visual inspection perhaps scans with corresponding scanner.
Show the male hybridoma for protein chip, use limiting dilution assay to carry out the cloning experiment, the cell strain that obtains continues to use protein chip to screen, and screens respectively at CEA, CA19-9, three kinds of antigenic corresponding antibodies cell strains of CA242.
The result:
The result of a chip monoclonal antibody as shown in Figure 3.This hole is 4 * 4 array chips, four lines is from top to bottom respectively with A, B, C, D name, four row are from left to right respectively with 1,2,3,4 names, the wherein negative contrast of A1 and A4, the positive contrast of D2, D4, other 12 points are represented 6 kinds of antigens respectively, comprise five kinds of tumor markerses of CEA, CA19-9, CA242, CA50, CA724 and recombinant expression protein P8 that immunity is used, and every kind of antigen two repeats.Test sample is the training liquid of hybridoma cell strain.
The monoblock chip operation is good, wherein for the positive Hybridoma Cell Culture liquid of antigens c EA result, carries out cloning by limiting dilution assay, finally obtains secreting cell strain 3 strains at the antibody of antigens c EA.In addition, secrete the hybridoma of anti-CEA antibody, further screen and identify, find 2 strains at same epi-position, and another strain is at different epi-positions with the polypeptide chip of epitope polypeptide for 3 strains.
For the positive Hybridoma Cell Culture liquid of antigens c A19-9 result, carry out cloning by limiting dilution assay, finally obtain secreting cell strain 2 strains at the antibody of antigens c EA.
For the positive Hybridoma Cell Culture liquid of antigens c A242 result, carry out cloning by limiting dilution assay, finally obtain secreting cell strain 2 strains at the antibody of antigens c EA.
For the positive Hybridoma Cell Culture liquid of antigens c A50 result, carry out cloning by limiting dilution assay, finally obtain secreting cell strain 1 strain at the antibody of antigens c EA.
For the positive Hybridoma Cell Culture liquid of antigens c A724 result, carry out cloning by limiting dilution assay, finally obtain secreting cell strain 2 strains at the antibody of antigens c EA.
Embodiment 2
Can discern the high-throughput preparation and the purifying of the monoclonal antibody of cancer of the stomach tumor markers
One, mouse immune and hybridoma preparation
(1) get people's stomach organization, crusher chopping back is centrifugal with organizing at a high speed, obtains total protein, as the antigen of preparation monoclonal antibody;
(2) antigen is injected mouse peritoneal and carry out immunity, immunization ways is a routine immunization, but booster immunization in case of necessity;
(3) spleen of getting mouse is made cell suspension, mixes according to a certain percentage with the myeloma cell, prepares hybridoma under the fusogen effect, collects to merge hybridoma, cultivates 7-8 days, is prepared into suspension;
Two, the preparation of biochip
(1) use is from different patients' stomach organization sample and corresponding negative control, preparation organization chip;
(2) use known tumor markers antigen, the preparation protein chip, the preparation of protein chip can be adopted solid phase chip and two kinds of forms of liquid phase suspending chip;
(3) according to the antigenic aminoacid sequence of known tumor markers, analyze its possible epitope, the synthetic corresponding polypeptide of design, the preparation polypeptide chip, the preparation of polypeptide chip is divided into solid phase chip and two kinds of forms of liquid phase suspending chip;
(4) preparation process of above biochip mainly comprises the preparation of sample, the activation of chip matrix and pre-treatment, processes such as fixing, the sealing of chip and post-processed are identical with embodiment 1 basically.
Three, the screening of monoclonal antibody
1. the screening of organization chip
(1) with the surface of different hybridoma suspension (each hole on corresponding 96 porocyte culture plates or the 384 porocyte culture plates) adding organization chip, the repeat region on the chip adds different cell suspensions, hatches 30-60 minute in 37 ℃ of wet boxes;
(2) use phosphate buffered saline buffer washing chip 3-5 time;
(3) add anti-mouse specific antibody on organization chip, antibody was hatched 30-60 minute in 37 ℃ of wet boxes with one or more marks in fluorescence dye or colour developing or the chemoluminescence substrate for enzymatic activity;
(4) use phosphate buffered saline buffer washing chip 3-5 time;
(5) use the organization chip of detection of fluorescent dyes directly to detect, use the organization chip of colour developing or chemoluminescence substrate for enzymatic activity mark, after the reaction of adding colour developing liquid, detect with corresponding scanner with chip scanner;
(6) repeated experiments, the positive colony that obtains is set up the hybridoma storehouse.
2. the screening of protein chip
A. the surface that the different hybridoma suspension (each hole on corresponding 96 porocyte culture plates or the 384 porocyte culture plates) in the hybridoma storehouse that obtains is previously added protein chip, protein chip has been fixed different known antigens marks, repeat region on the chip adds different cell suspensions, hatches 30-60 minute in 37 ℃ of wet boxes;
B. use phosphate buffered saline buffer washing chip 3-5 time;
C. add anti-mouse specific antibody on protein chip, antibody was hatched 30-60 minute in 37 ℃ of wet boxes with one or more marks in fluorescence dye or colour developing or the chemoluminescence substrate for enzymatic activity;
D. use phosphate buffered saline buffer washing chip 3-5 time;
E. for the anti-mouse specific antibody that uses fluorochrome label, protein chip directly detects with chip scanner, uses the protein chip of colour developing or chemoluminescence substrate for enzymatic activity mark, after the reaction of adding colour developing liquid, detects with corresponding scanner;
F. repeated experiments, the positive colony that obtains at synantigen not, are set up hybridoma storehouse I respectively, and the clone that chip results is negative sets up hybridoma storehouse II, further to study.
3. the screening of polypeptide chip
A. use set up previously at the antigenic hybridoma of difference storehouse I, different hybridoma suspension (each hole on corresponding 96 porocyte culture plates or the 384 porocyte culture plates) is added the antigenic surface of polypeptide chip corresponding polypeptide, the different zones of polypeptide chip has been fixed different polypeptide antigens, repeat region on the chip adds different cell suspensions, hatches 30-60 minute in 37 ℃ of wet boxes;
B. use phosphate buffered saline buffer washing chip 3-5 time;
C. add anti-mouse specific antibody on polypeptide chip, antibody was hatched 30-60 minute in 37 ℃ of wet boxes with one or more marks in fluorescence dye or colour developing or the chemoluminescence substrate for enzymatic activity;
D. use phosphate buffered saline buffer washing chip 3-5 time;
E. use the polypeptide chip of detection of fluorescent dyes directly to detect, use the polypeptide chip of colour developing or chemoluminescence substrate for enzymatic activity mark, after the reaction of adding colour developing liquid, detect with corresponding scanner with chip scanner;
F. repeated experiments, the positive colony that obtains is set up corresponding hybridoma storehouse III respectively at different epitopes.
Four, the purifying of monoclonal antibody protein
(1) hybridoma cell line with the hybridoma storehouse II, the III that obtain is 1-5 * 10 with the cell count
6Injection cell is to the Balb/C mouse, gets mouse ascites after waiting for about a week, perhaps hybridoma is cultured to quantity in Tissue Culture Flask and reaches 10
7More than, get cell conditioned medium;
(2) use the affinity column of ProteinG that mouse ascites or cell conditioned medium are carried out purifying, obtain pure monoclonal antibody protein.
(3) purifying is a monoclonal antibody at known antigens known antigens epi-position from the antibody of hybridoma storehouse III;
(4) antibody spot sample of purifying from hybridoma storehouse II is planar substrate such as glass or pvdf membrane or is coupled on the polystyrene microbeads in material, make the monoclonal antibody chip, be used for proteome research.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. a method for preparing monoclonal antibody is characterized in that, may further comprise the steps:
(a) with 3-10000 kind immunogen non-human mammal is carried out immunization;
(b) will take from the splenocyte that above-mentioned immunization is crossed immunogenic non-human mammal, mix, and merge the formation hybridoma with the myeloma cell of non-human mammal of the same race;
(c) collect hybridoma, cultivate, be prepared into the hybridoma suspension;
(d) with the biochip that is selected from down group: protein chip, polypeptide chip, organization chip or its combination, wherein said biochip is coated with the described immunogenic point of sample of 3-10000 kind, described hybridoma is screened, thereby secreted hybridoma cell line respectively at the described immunogenic monoclonal antibody of 3-10000 kind.
2. the method for claim 1 is characterized in that, in step (d), screens with protein chip earlier, screens with polypeptide chip again.
3. the method for claim 1 is characterized in that, described immunogen is selected from down group: protein, polypeptide, cell, tissue, cell lysate or extract, lipid, carbohydrate, nucleic acid, pathogenic agent, viral capsid or its mixture.
4. the method for claim 1 is characterized in that, described non-human mammal is rat, mouse.
5. the method for claim 1 is characterized in that, described immunogenic quantity is the 4-5000 kind.
6. the method for claim 1 is characterized in that, described method also comprises step:
(e) use the hybridoma that a step obtains, produce monoclonal antibody;
(f) separate the monoclonal antibody that described monoclonal antibody obtains purifying.
7. method as claimed in claim 6 is characterized in that, described step (e) is by being 1-5 * 10 with the cell count with hybridoma
6Cell inoculation is collected mouse ascites in mouse after 5-10 days; Perhaps make its quantity reach 10 by cultivating hybridoma
7Individual-10
10Individual, get cell conditioned medium then.
8. method as claimed in claim 6 is characterized in that, described step (f) is to use the affinity column of Protein G to carry out purifying, obtains pure monoclonal antibody protein.
9. method as claimed in claim 6 is characterized in that, also comprises step:
(g) with the monoclonal antibody point sample of purifying on chip substrate, make the monoclonal antibody chip.
10. the method for claim 1 is characterized in that, described biochip is a porous plate, and described porous plate has 4-10000 hole, and a chip substrate is arranged in each hole, and point sample has the described immunogen of 3-10000 kind on the chip substrate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510026873 CN1880447A (en) | 2005-06-17 | 2005-06-17 | Process for preparing high-flux monoclonal antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510026873 CN1880447A (en) | 2005-06-17 | 2005-06-17 | Process for preparing high-flux monoclonal antibody |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1880447A true CN1880447A (en) | 2006-12-20 |
Family
ID=37518800
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200510026873 Pending CN1880447A (en) | 2005-06-17 | 2005-06-17 | Process for preparing high-flux monoclonal antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1880447A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103255128A (en) * | 2013-04-27 | 2013-08-21 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Methods for preparing monoclonal antibody and hybridoma cell strain thereof by multiple antigens in immune high-flux manner |
| CN103808922A (en) * | 2013-04-27 | 2014-05-21 | 无锡国盛生物工程有限公司 | Method for screening micro antibodies in hybridoma cell supernatant stage |
| CN104062431A (en) * | 2014-07-01 | 2014-09-24 | 上海理工大学 | Method for screening monoclonal antibody hybridoma by utilization of visual antigen chip |
| CN108107205A (en) * | 2016-11-25 | 2018-06-01 | 中国科学院苏州纳米技术与纳米仿生研究所 | The method and system of high-flux fast screening positive hybridoma cell |
| CN108663522A (en) * | 2018-05-30 | 2018-10-16 | 河北翰林生物科技有限公司 | Carcinoma of urinary bladder detection kit |
| CN108753733A (en) * | 2018-04-11 | 2018-11-06 | 南京健安医疗科技有限公司 | Hybridoma cell strain and its anti-glycosyl monoclonal antibody and preparation method and preparation of generation |
| CN110484974A (en) * | 2019-07-19 | 2019-11-22 | 中国农业科学院郑州果树研究所 | A kind of preparation method of grape antibody library |
-
2005
- 2005-06-17 CN CN 200510026873 patent/CN1880447A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103255128A (en) * | 2013-04-27 | 2013-08-21 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Methods for preparing monoclonal antibody and hybridoma cell strain thereof by multiple antigens in immune high-flux manner |
| CN103808922A (en) * | 2013-04-27 | 2014-05-21 | 无锡国盛生物工程有限公司 | Method for screening micro antibodies in hybridoma cell supernatant stage |
| CN103808922B (en) * | 2013-04-27 | 2015-09-09 | 无锡国盛生物工程有限公司 | A kind of screening technique of hybridoma supernatant stage trace antibody |
| CN104062431A (en) * | 2014-07-01 | 2014-09-24 | 上海理工大学 | Method for screening monoclonal antibody hybridoma by utilization of visual antigen chip |
| CN108107205A (en) * | 2016-11-25 | 2018-06-01 | 中国科学院苏州纳米技术与纳米仿生研究所 | The method and system of high-flux fast screening positive hybridoma cell |
| CN108107205B (en) * | 2016-11-25 | 2023-10-27 | 南京凌芯生物科技有限公司 | Method and system for high-throughput rapid screening of positive hybridoma cells |
| CN108753733A (en) * | 2018-04-11 | 2018-11-06 | 南京健安医疗科技有限公司 | Hybridoma cell strain and its anti-glycosyl monoclonal antibody and preparation method and preparation of generation |
| CN108663522A (en) * | 2018-05-30 | 2018-10-16 | 河北翰林生物科技有限公司 | Carcinoma of urinary bladder detection kit |
| CN108663522B (en) * | 2018-05-30 | 2021-05-11 | 河北翰林生物科技有限公司 | Bladder cancer detection kit |
| CN110484974A (en) * | 2019-07-19 | 2019-11-22 | 中国农业科学院郑州果树研究所 | A kind of preparation method of grape antibody library |
| WO2021012704A1 (en) * | 2019-07-19 | 2021-01-28 | 中国农业科学院郑州果树研究所 | Method of constructing grape antibody library |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1880447A (en) | Process for preparing high-flux monoclonal antibody | |
| CN1610831A (en) | Method for the diagnosis of cancers by measuring the changes of glycosylation of proteins related to tumorigenesis and metastasis and kit for diagnosis of cancers using the same | |
| CN1761682A (en) | Monoclonal antibody and hybridoma producing the same | |
| CN1313622C (en) | High-flux cell biological chip testing technology and reagent case | |
| CN100494399C (en) | Genotype typing method based on DNA chip and use thereof | |
| CN101056992A (en) | Applications of isolated nucleic acid fragments comprising cpg islands | |
| CN108368549A (en) | The method for determining Cell clonality | |
| CN1974601A (en) | New-type Fc fusion protein and its production process | |
| CN1896102A (en) | Anti-hbs monoclonal antibodies | |
| CN1668766A (en) | Apparatus for polynucleotide detection and quantitation | |
| CN1274085A (en) | Protein chip, its preparing process and its application in screening monoclonal antibody | |
| CN1247797C (en) | Protein chip and its preparing method and use | |
| CN100347296C (en) | Method for manufacturing antigen-specific antibody-producing hybridomas employing a single antigen-specific b lymphocyte and method for manufacturing monoclonal antibody | |
| CN1659185A (en) | Method for producing monoclonal antibodies | |
| CN1732387A (en) | Protein chip for analyzing interaction between protein and substrate peptide | |
| CN1696698A (en) | Affinity chromatography-enzyme immunoassay method for PAT protein, and dedicated kit | |
| CN1455813A (en) | Human myeloma cell line | |
| Di Cristina et al. | An antigen microarray immunoassay for multiplex screening of mouse monoclonal antibodies | |
| CN1660907A (en) | Monoclonal antibody of anti lymphocyst virus of and preparation method | |
| CN1795266A (en) | Method for producing retinal neurocyte from neural stem cell derived from iris tissue and retinal, neurocyte produced by the process | |
| CN1858244A (en) | A polynucleotide probe having enhanced binding specificity, a microarray having the probe immobilized thereon and a method for designing the probe | |
| CN1803846A (en) | Hybrid protein of p53 protein epitope(SQAMDDLMLS) and filobactivirus gene 8 protein and application thereof | |
| CN1191275C (en) | Antifucoidan polysaccharide antibody | |
| CN1214118C (en) | DNA antibody and uses thereof | |
| CN1900315A (en) | Method for screening high quality spirulina princeps strain for large scale production |
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 |