Monoclonal Dev

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Pacific West Western Regional Rsch. Ctr. (Albany, Ca) Foodborne Contaminants Research

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You are here: Research / Research Project: DEVELOPMENT OF Project Team Advanced Search DETECTION TECHNOLOGIES FOR TOXINS AND THEIR Brandon, David VALIDATION IN FOOD Stanker, Larry MATRICES Carter, John - Mark Location: Foodborne Cheng, Luisa Wai Wai Contaminants Research Title: Development and Partial Hernlem, Bradley - Brad Programs and Characterization of High-affinity Projects Rasooly, Reuven Monoclonal Antibodies for Subjects of He, Xiaohua Botulinum Toxin Type A and Investigation their use in Analysis of Milk by Sandwich ELISA Authors Stanker, Larry Publications Merrill, Paul Publications Scotcher, Miles Cheng, Luisa Wai Wai Submitted to: Journal of Immunological Methods Publication Type: Peer Reviewed Journal Publication Acceptance Date: March 4, 2008 Publication Date: April 9, 2008

Related National Programs Food Safety, (animal and plant products) (108)

Citation: Stanker, L.H., Merrill, Related Projects P.A., Scotcher, M.C., Cheng, L.W. 2008. Development and DEVELOPMENT OF Partial Characterization of HighDETECTION affinity Monoclonal Antibodies TECHNOLOGIES FOR for Botulinum Toxin Type A and BACTERIAL their use in Analysis of Milk by NEUROTOXINS AND Sandwich ELISA. Journal of THEIR VALIDATION Immunological Methods, 336:1IN FOOD MATRICES 8. Interpretive Summary: Botulism is a serious, often fatal neuroparalytic disease in humans and animals caused by a protein toxin (botulinum toxin, BoNT) produced by the bacterium Clostridium botulinum. BoNT is considered the most toxic biological toxin

Monoclonal Antibody Development ProSci can help you develop custom monoclonal antibodies and we understand researchers have varying needs for levels of involvement, depending on the target. We have developed monoclonal antibody development packages for different levels of involvement in the project, from researcher screening (you screen positive wells following fusion for clones of interest) to ProSci sending western blot and immunocytochemistry positive final clones. We offer custom monoclonal antibody development packages for both protein and peptide antigens. Our screening options include: • • •

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ProSci sends final clones, guaranteed ELISA positive. (no additional charge) Researcher screening of positive wells, following fusion. (starting at $500) Western Blot screening: ProSci screening of positives during Phase 2 (Fusion) and Phase 3 (Sub-cloning) for Western Blot positive clones. o Protein antigens: guarantee of up to 3 western blot positive clones. ($2200-$2600) o Peptide antigens: cannot guarantee reactivity, but >70% success rate; see Terms & Conditions for more details. ($2200-$2600) ICC screening: ProSci screening of positives during Phase 2 (Fusion) and Phase 3 (Sub-cloning) for immunocytochemistry positive clones o Protein antigens: guarantee of up to 3 immunocytochemistry positive clones. ($2600) o Peptide antigens: cannot guarantee reactivity, but >70% success rate; see Terms & Conditions for more details. ($2600) Western blot & immunocytochemistry screening: ProSci screening of positives during Phase 2 (Fusion) and Phase 3 (Sub-cloning for Western Blot AND immunocytochemistry positive clones. ($4200)

More pricing information can be found at the mouse monoclonal antibody developement page. ProSci’s fusion success rate for custom monoclonal antibody developement is greater than 95%, using techniques developed in-house. All work is performed by skilled technicians on premises in our San Diego, California laboratories. Your custom monoclonal antibody production project, and can be scaled up to almost any magnitude. We can screen against multiple antigens (additional charges apply) to develop the specific clone you need. ProSci has repeatedly proven our abilities to produce hybridomas against difficult targets. We use 5 Balb/c mice as a production strain for hybridoma development. Balb/c is a very common species used throughout the industry for monoclonal antibody development due to their large spleen to body size ratio. This yields a higher number of spleenocytes at fusion.

Protein vs. Peptide Antigens: We are frequently asked about the merits of using recombinant or fusion proteins versus peptides as antigens. To obtain IgG antibodies that work for multiple applications (i.e.: western blot, immunostaining, immunoprecipitation, etc.) a protein antigen works much better for custom monoclonal antibody development. Proteins have a much higher number of linear and conformational epitopes for the mice’s immune systems to respond to. The more diverse immune response generally results in antibodies against many different locations on the protein, each of which has unique properties. Peptide antigens have fewer epitopes and are normally only linear in nature. Using longer peptides (~20-30 residues in length) increases the total number of epitopes available for the mouse immune system to respond to and increases likelihood of success of your custom monoclonal antibody development project. Our peptide-antigen packages include 25 residues, to help increase the project’s success. Shorter peptide sequences (fewer than 20 residues in length) often work only for ELISA. Fusion Partners (protein antigens): If you are submitting a protein antigen for custom monoclonal antibody developement, we highly recommend cleaving large fusion partners such as GST tags. These tags tend to be highly immunogenic, and if they are not removed prior to immunization, a dual screening process must be performed. This ensures positive responses are to the protein, not the fusion partner. To do this we perform dual ELISA screening, screening for high ELISA titers against the protein and low ELISA titers against the fusion partner. This additional service is available at $2400 per additional screening.

Custom Monoclonal Antibody Development Antigen Requirements:

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Purity: Antigens should be as pure as possible. An antibody can be made against impurities just as easily as against the protein itself. Solubility: Antigens need to be soluble for screening. Antigen Types: ProSci Incorporated can work with protein, enzymes, peptides, and other small molecules. At this time we do not accept cells, viruses, or bacteria as antigens for custom monoclonal antibody production. Antigen Quantities: From start to finish ProSci needs ~5 mg of antigen. 1mg is used for immunizations and 4mgs are used for screening. We can start a project with as little as 0.5 mg, and additional quantities can be supplied throughout the project Conjugation of Antigen: Small antigens (such as peptides or antigens with a molecular weight of 10 kD or less) need to be conjugated to a carrier protein to have a large enough size to initiate a strong immune response. ProSci requires 2-3mg of material to conjugate to the carrier protein (or 1mg conjugate if provided by researcher), and an additional 4mgs of antigen for screening

Each of our monoclonal antibody development packages include:

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Immunization of antigen into 5 Balb/c mice

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Screening of test bleeds against immunizing antigen to isolate a mouse with high IgG titer and low IgM titer Fusion of one mouse’s spleen to the SP 2/0 myeloma cell line Screening of positive wells following fusion for IgG specific clones using immunizing antigen Cell line stabilization of up to 3 cell lines Isotype report on each final clone provided, confirming Ig class Cell line banking for 1 year following final clone shipment

Additional options and services include:

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Peptide synthesis (with antigen design assistance) Dual ELISA screening (i.e.: screen against positive hybridomas against GST or other large fusion partner) ($2400) Researcher screening of positive wells following fusion; 7-day screening time (starting at $500) Western blot or immunocytochemistry screening ($2200-$2600) Purchase of additional clones (from $600) Ascites production and purification Cell line isotyping ($50 per cell line) Cell line banking Cell bank generation

Monoclonal Antibody and Hybridoma Development Program The identification of genetic biomarkers and pathways has revolutionized the process of drug discovery and created a flurry of novel therapeutic targets and methods. In today’s regulatory environment, it is critical to have high quality assay reagents to provide proof of concept, and more importantly, the ability to track the effectiveness of a therapy through clinical trials and beyond. MBS has partnered with top pharmaceutical companies, using 19 years of antibody development experience and proprietary protocols, to provide the best possible antibodies to monitor their therapeutic applications. Price Range For Hybridoma Development $7,300-$16,500 As a response to customer feedback, MBS offers a rapid immunization protocol resulting in a fusion at day 28 and your monoclonal cell line in 16-20 weeks. The protocol has been used with great success for over 4 years. A more traditional protocol, taking approximately 26 weeks for immunizations, is also available by request. Our hybridoma development team can help you to decide which method best fits your project parameters. The success of your project depends heavily on frequent communication with our staff throughout the project. Our goal is to help you get your diagnostic test or therapeutic assay to the market sooner. The Hybridoma Guarantee explains our commitment to quality. Phase 1 Project Initiation

This phase provides you with the opportunity to discuss the overall goals of the project and tailor our protocols to meet your requirements. To begin this stage, please fill out a Hybridoma Development Checklist. This form will supply MBS with all of the non-proprietary information and help us to get started with your development. Hybridoma initiation checklist Phase 2 : Immunization of your Mice or Rats An antibody response is generated in mice or rats to your specific immunogen. Up to 8 animals can be immunized if required. A standard project requires 3-4 mgs of immunogen, however if less is available the rapid hybridoma development can be modified. Immunogens less than 20kD should be conjugated. We recommend conjugating 2mgs to KLH for immunizations and 2mgs to BSA for screening of test bleeds and supernatants. After 28 days, the selected animal goes to fusion. Important Immunogen Factors To Consider Phase 3: Fusion Spleen cells from the selected animal are fused with myeloma cell line to develop a hybridoma that secretes a specific antibody. Once your mice or rats are ready for fusion per ELISA screening results, a fusion is performed. The fusion is plated and screened. The primary screening is a direct ELISA against your antigen to capture all positive fusion products. The positive fusion products are scaled up and screened again. The secondary screening differentiates between IgG and IgM clones, and verifies that the selected positive fusion products are still producing antibody prior to cryopreservation. You will choose the number of fusion wells for scale-up. Not all wells that are scaled up will survive or continue to produce specific antibody. The supernatants of the positives are shipped to you for further testing. At this point, MBS is able to offer small amounts of purified antibody from the fusion products through a process called MultiPure. Learn more about MultiPure Phase 4: Subcloning A monoclonal antibody-producing cell line is isolated from selected parental clone. MBS performs subcloning to render a hybridoma fusion product, or cell line, monoclonal. The selected fusion clones are subcloned by limiting dilution. MBS has three proprietary approaches to subcloning, which are used depending on the stability of the clone. Once colonies are established, the wells are screened by a direct ELISA assay. Clones from each parental are selected for freeze down and storage. The supernatants are shipped to you for further evaluation. This phase takes 3-4 weeks. Phase 5: Antibody Production Sufficient antibody is generated through ascites or in vitro production to allow you to assay in your specific application. For cell lines developed in Balb/c mice, we typically use 10 Balb/c mice to produce Ascites. If your cell line was developed in rats, we will produce a Spinner Flask production or run a nude mice ascites production. The ascites or spinner flask production can be purified by Protein A or Protein G when the antibody is an IgG. The final monoclonal antibody is shipped to you within 7-8 weeks of selecting a subclone.

Contact us today to learn more about our Monoclonal Antibody and Hybridoma Development Program.

Technical Resources Useful Tools & Links Following are scientific tools and website resource links that we hope you will find useful. Please let us know if you have another site of interest or other resource suggestion that we may consider adding to this area. Virtual Library for Biotechnology http://www.cato.com/biotech/index.html Virtual Library, a directory of companies providing products and services in biotechnology and pharmaceutical development, public service of Cato Research Ltd. MedLine Pub-Med Search http://www4.infotrieve.com/newmedline/search.asp Find journal current and past journal articles on-line XE.com Currency Conversion chart http://www.xe.com/ucc/ Converts currencies: Euros, Canadian dollars, Deutsche Marks, Francs, Yen, Guilders, Lires, Swiss Francs and more. The Protein Data Bank http://www.rcsb.org/pdb/index.html Repository for the processing and distribution of 3-D biological macromolecular structure data. Protocol On-line http://www.protocol-online.org/prot/Immunology/ Immunochemistry Protocols Pharma-Lexicon Search Medical abbreviation, Pharmaceutical Companies and Associations. Refer to existing MBS site www.mainebiotechnology.com for more info:

STEPS IN HYBRIDOMA PRODUCTION 1. Immunization of mice ( in vitro or in vivo)

2. Spleen removal and preparation of a single cell suspension 3. Myeloma cell preparation 4. Fusion of spleen cells and myeloma cells 5. Post-fusion cells cultured in hybridoma selection medium (HAT) 6. Collection and dispersion of peritoneal macrophages 7. Addition of fused cells to microtiter plates with macrophages 48 hours post-fusion ( 2-4x 106 cells/ml) 8. Culture of cells: 370C, 5% CO2 [ feed with HAT medium] 9. 7 to-21 days post fusion: observe and numerate hybridoma clones 10. Screen for specific antibody production 11. Expand cultures positive by screening test 12. Reclone by a limiting dilution technique all positive hybridoma clones to assure monoclonality and to select for the fastest growing cell line with the greatest antibody production. Hybridomas should be recloned periodically (after 3-4 months of culture) to prevent overgrowth of your preferred culture by mutants or cells expressing an altered phenotype. 13. Inject 2-10x106 recloned hybridoma cells into BALB/c mice which had received an i.p injection of 0.3 ml of pristane 7 days previously. Collect ascites 7-21 days latter. 14. Guard against loss of hybridoma by storing several amps of each clone in liquid nitrogen.

REAGENTS Media: M-O: RPMI-1640 plus 1% of 45% D-glucose M-10: MO plus 10% heat inactivated FCS Hy-HAT: to make 100 ml

RPMI-1640 - 77 ml NCTC-135 - 10 ml FCS (hyclone) - 10 ml 45% D-glucose (w/v) - 1 ml (0.2 micron filtered) 200 mM L-glutamine - 0.1 ml (0.2 micronfiltered) 0.1 mm Na2SeO3 - 0.1 ml (0.2 micron filtered) HAT concentrate (100x) - 1 ml (0.2 micron filtered) Gentamycin (50 mg/ml) - 0.1 ml Hy-HT: Replace HAT concentrate in Hy-HAT with HT concentrate (100x) HyM-(HT or HAT): Hy-(HT or HAT) - 98.5 ml LPS conc. - 1.0 ml (aseptically) Dextran sulfate conc. - 0.5 ml (0.45 micron filtered) HAT concentrate (100x): Hypoxanthine - 135.00 mg (H)= 1x10-4M Aminopterin - 1.91 mg (A)= 4x10 -7M Thymidine - 38.60 mg (T)= 1.6x10-5 M Add (H) to approximately 60 ml of distilled water plus 1.2 ml of 1 M NaOH. Stir on magnetic stir table for 10-30 min. If not dissolved, add more 1 M NaOH in 0.1 ml volumes at 5 min. intervals until a complete solution is achieved. Next, add (T), stir until dissolved and then add (A). When all ingredients are in solution, bring the volume to 100 ml and filter sterilize through 0.2 micron filter. When stored at 40 C, it is stable for several months.

SIMPLE METHOD: BY FROM GIBCO the HAT and HT concentrate. HT concentrate (100x): Same as HAT without aminopterin.

Dextran sulfate conc. (200x): (not needed if macrophages are used) Dissolve 40 mg of dextran sulfate (17% S) MW=500,000 (Sigma), in 10.0 ml of distilled water. Filter through 0.2 micron filter and store at 40 C. Stable for several weeks. LDS concentrate (100x): (not needed if macrophages are used) Aseptically add 20 ml of distilled water to 100 mg vial of E. coli lipopolysaccharide, W, 055:B5 (Difco) and aliquot 1 ml per tube. Store frozen at -20ð0 C. Stable for several months. Na2SeO3 stock solution (1000 x): Dissolve 1.73 mg of sodium selenite in 100 ml of distilled water. Filter sterilize through a 0.2 micron filter. Caution: Sodium selenite is a possible carcinogen. Fusion reagent: Polyethylene glycol-4000 (Baker) 50% : PEG-4000 - 10 g (melted) DMSO - 1 ml 0.15 M HEPES pH 7.5 - 9 ml Filter sterilize with 0.45 micron filter. Store at room temperature. Stable for several months. Hemolytic reagent: Tris base - 0.2 g NH4Cl - 0.83 g Adjust pH to 7.2 with HCl. Dissolve in 100 ml d. water. Filter sterilize with 0.2 micron filter. Store at 40 C. Stable for several months. Ethanol 70%

INSTRUMENTS FOR SPLENECTOMY OF ONE MICE Sterile: three scissors and two forceps. One 150 ml beaker containing 80-100 ml of 70% ethanol.

Two 5 ml syringes fitted with a 26 g x 1/2 inch needle and each containing 5 ml of M10 medium. Two 60 mm petri dishes, one with 3 ml and the other containing 10 ml of M-10 medium. One tissue sieve (100 squares/inch) and pestle in a 100 mm petri dish. The sieve screen and pestle face should be wetted with M-10 and the petri dish contain 15 ml of this medium.

PREPARATION FOR CELL FUSION Instruments should be conveniently organized in a biological hood with a "stack" of three or four paper towels placed near the front of the hood upon which the alcohol cleansed mouse will be placed for the splenectomy. For each mouse's spleen used in the fusion, 5 ml of Tris-NH4Cl and 15 ml of M-10 medium should be cooled in an ice bath. The immunized mouse is sacrificed by cervical dislocation and is completely submerged in the 70% ethanol for approximately one minute. Excess alcohol is drained from the mouse and it is placed with its head to the technicians left on its back, upon the paper towels in the biological hood. The gloved technician uses the first pair of scissors to make a lateral incision between the hind legs. These appendages are pinned down with the right hand and the skin to the left of the incision grasped with the left hand. By moving the left hand towards the head and parallel to the body, the skin is peeled back completely exposing the abdomen. Using the second scissors and forceps, the visceral organs are exposed by carefully opening the abdominal musculature. The spleen is a dark red flattened and oblong shaped organ on the left side of the body situated above the kidney and adjacent to the stomach. Using the last scissors and forceps, remove the spleen and carefully trim off as much connective tissue and fat as possible. First rinse the spleen in the 60 mm petri dish containing 10 ml of M-10 medium then place in the second 60 mm petri dish containing 3 ml of M-10. With a medium-filled syringe in each hand, poke 30-50 holes in the spleen. Now perfuse the spleen using the whole 10 ml of M-10. Do not draw cells back into the syringe as they will be damaged by the small 26 ga. needle. Using sterile forceps, transfer the spleen to the tissue sieve. Pipet the cells obtained perfusion into a 15 ml capped test tube. Carefully and gently push the red spleen tissue through the sieve screen with the pestle. Connective and fatty tissues will be left on top of the screen. Pipet the medium from around the sieve and rinse cells from the screen. Remove sieve for cleaning. Carefully pipet cells from petri dish avoiding as many cell clumps as possible and put in a second test tube. Centrifuge spleen cells at 1000 rpm (PR-6000 IEC centrifuge) for 5 minutes. Combine cell pellets and resuspend cells in 5 ml of ice cold hemolytic reagent. Incubate at room temperature for 10 minutes and add 5 ml of ice cold M-10 medium. Immediately centrifuge at 1,000 rpm for 5 minutes . The cell pellet should be erythrocyte-free and contain approximately 100 x 106 cells. If the cell pellet has any red coloration indicating the presence of unlysed erythrocytes, the cells should be

suspended in additional 10 ml of ice cold M-10 medium and recentrifuged. When the spleen cell pellet is free of red blood cells, combine with 100 x 106 NS-1 BALB/c myeloma cells by resuspending the cells in 15 ml of M-0 medium at room temperature. Centrifuge at 1,000 rpm for 5 minutes, aspirate of the supernatant and resuspend cell pellet for a second wash in M-0 medium. Centrifuge as above and aspirate off all the supernatant to give a "dry" cell pellet. CELL FUSION Set a timer for 6 minutes. Quickly resuspend the cells in 0.5 ml of 50% PEG-4000 and start the timer. After 2 minutes, add 0.5 ml of M-0 medium and carefully mix. After each additional minute, add another 0.5 ml of M-0 until the end of 6 minute period. At this time, finish filling the test tube with M-0. Carefully invert to mix thus diluting the PEG. Centrifuge at 750 rpm for 3 minutes and aspirate off the supernatant. Carefully add 10 ml of HAT medium at a rate sufficient to dislodge the pellet without dispersing the cells. Allow the fused cells to "rest" for 10 minutes then draw the cell pellet into a pipet and expel with just enough force to disperse the cells. Pipet the cells into a 75cm² tissue culture flask in a total of 50 to 60 ml of HAT medium. Incubate in a 5% CO2 incubator at 370 C for 2 days, then distribute the cells evenly into eight 96-well microtiter plates. The plates have had either 3,000-6,000 peritoneal macrophages added per well in 60-100 µl of HAT medium or the HAT medium should be supplemented with 50 µg/ml of LPs and 20 µg/ml of dextran sulfate. If the mouse's spleen cells respond well to these mitogens, the hybridomas will probably grow faster and produce more clones than with peritoneal macrophages. A drawback to using mitogen stimulation is that some hybridomas may develop a nutritional requirement for LPs or dextran sulfate. Cultures should be feed with one drop per well ( approx. 60 µl ) of fresh medium every 3-5 days. When a well is full, one-half of the volume of the well should be removed by aspiration using a sterile pasteur pipet and a drop of fresh medium added.

SCREENING THE CLONES A suitable assay to detect mouse antibodies to the antigen(s) of interest should be operational before cell fusion is attempted. Only a week or so is available from the time the cultures are producing sufficient amounts of antibodies to be assayed until the clones must be transferred to larger flasks or recloned to ascertain and maintain their monoclonality. Tests must be sensitive and require a small amount of hybridoma conditioned medium ( 100 µl or less )

RECLONING

When a microtiter well tests positive for the antibody of interest it is important to reclone as soon as possible to avoid the potential loss of the positive clone due to overgrowth by non-secreting cells. Recloning can be accomplished by either growing the hybridoma in soft agar or by a limiting dilution technique. I have personally better success with the latter method. Limiting dilution recloning first requires an accurate cell count of the viable hybridoma cells suspended in the medium of the microtiter well in which the cells are growing. Next the cells are diluted so that when one drop of cell suspension is placed in each well of a 96-well microtiter plate, the average number of hybridoma cells will be 0.5, 1 and 3 cells per well. At least one plate ( 96 wells ) should be prepared for each dilution. If there is growth in 5 wells or less, the odds are greater than 95 % that the clones are monoclonal. If less than 80 % of the clones tested are positive, the hybridoma should be recloned a second time. When the dilutions listed above give to little or to much growth an appropriate adjustment to either a higher or lower dilution is required. The cloning efficiency of hybridomas can be greatly enhanced by the addition of 3,000-6,000 peritoneal macrophages per well or by including LPs and dextran sulfate in the cloning medium. The fastest growing clones producing the most antibody are selected for clonal expansion. Several ampules of each clone chosen for expansion should be prepared for storage in liquid nitrogen using a standard cell freezing technique. Antibody can be purified and concentrated from the hybridoma conditioned growth medium or high titer ascitic fluid can be prepared.

ASCITIC FLUID PRODUCTION The NS-1 myeloma cell line is BALB/c origin so if the spleen cells are from any other mouse strain then ascites should be produced in the F1 cross between BALB/c and the other mouse strain. In all of the fusions I have prepared the spleen cells were from BALB/c mice and therefore this mouse strain was used to produce ascitic fluid. The mice are first primed with an intraperitoneal injection of 0.3 to 0.5 ml of pristane. Pristane is a C14 branched oily hydrocarbon which induces, in primed mice, an oilgranuloma. This environment is optimal for acceptance and growth of hybridomas and allows the antibody titer in ascitic fluid to reach levels 100 to 10,000 times greater than can be achieved in tissue culture medium. Seven days after the mice are primed, one to ten million hybridoma cells are washed with sterile PBS and injected into each mouse. Seven to twenty one days latter the ascites is collected by inserting a 1 1/2" x 19 GA needle into the swollen peritoneum in the inguinal area and parallel to the spine. The mouse is positioned with the needle hub over a test tube and the ascitic fluid is collected by gravity flow. 1-8 ml of fluid can be obtained from each mouse.

Now the ascitic fluids allowed to clot and the cells and fibrin are removed by centrifugation. Heat inactivate at 560 C for 20-25 minutes. Next, centrifuge at high speed to clarify and aliquot in 1 ml to vials and store in -700 C freezer. PREPARATION OF PERITONEAL MACROPHAGES The mouse is prepared as for splenectomy except the abdominal musculature is left intact. A 5 cc. glass syringe is fitted with a 1 1/2" x 18 GA needle and filled with 5 ml of refrigerator cold (4-80 C) 0.34 M sucrose ( dissolved in distilled water and filter sterilized through a 0.2 micron filter). A medial and enteral part of the abdominal musculature is held away from visceral organs with a sterile forceps to form a space for 3 ml of the sucrose solution to be injected. The needle is withdrawn and the abdomen massaged for about 15 seconds to insure maximum suspension of resident macrophages. The abdominal musculature is again held with the forceps and the needle reinserted. The remaining 2 ml of sucrose in the syringe is forcefully injected into the abdominal cavity thus mixing with the cell suspension. This cell mix is then withdrawn into the syringe, care being taken not to puncture any organs with the needle while removing as much of the cell suspension as possible. The cell suspension is transferred into a sterile Teflon tube, enumerated, then capped and centrifuged at 1,200 rpm in a IEC PR-6000 centrifuge for 7 minutes. A tube of Teflon is used as macrophages will tenaciously adhere to glass and most plastic, but will not adhere to Teflon. The cells form a lose pellet so care must be exercised when aspirating the supernatant not to disturb the pellet. 0.5 to 0.7 ml of sucrose solution can remain with the cells with no adverse effect on the hybridoma culture. 80-90 % of the resident peritoneal cells should be macrophages, while the majority of remaining cells are lymphocytes. 2-4 million cells should be recovered per mouse. The cells are now ready to be diluted in Hy-HAT medium and used as hybridoma feeder cells. The following dilutions are recommended: 1. 96 well microtiter plate: 3,000-6,000 macroph./well 2. 24 well plate: 50,000 macroph./Well 3. 25 cm² tissue culture flask: 7x105 to 1.4x106 macroph. Author Information Primary Author Jody D. Berry ( [email protected] ) Affiliation University of Manitoba , Canada Co-Author(s) Franscesca Ranada Protocol Information Protocol ID / PID PUB335 Category Cell Biology Molecular Biology Title The production of monoclonal antibody by hybridoma fusion: Immortalization of sensitized B lymphocytes from immune mice. Overview We outline a simple and contemporary protocol for the development of monoclonal antibodies using hybridoma fusion in immune mice (1). While the basic style of this fusion is similar to others (2,3), this protocol has several subtle but significant modifications. These include the use of: spleen perfusion rather than crushing to separate the spleen cells (which reduces the amount of contaminating fibroblast and lipoidal material) ; commercial Hybridoma CLoning Factors (rather

than feeder layers); commercially prepared semi-solid HAT containing agarose, rather than limiting dilution. Elements of this protocol span several research institutions and many years of experience (4,5,6). Material Sterile 10, 25, and 50 ml serological pipettes, Pipet-Aid, 15 and 50 ml centrifuge tubes (Falcon sterile), Tissue culture flasks (25 cm2, 75 cm2 and 125 cm2), indelible waterproof marker. Sterile 1 ml pipette tips for Gilson p1000 pipetteman. 370C waterbath and thermometer. Humidified 370C, 5% CO2 tissue culture cabinet. Class II Biological Safety Cabinet. Inverted Microscope. Benchtop centrifuge containing a 4 swinging bucket rotor, at room temperature. Stopwatch or timer. Multichannel pipettor and appropriate sterile tips, sterile disposable petri dishes. Sterile 96-well flat-bottomed cell culture plates. Reagents: 1-3 x 10 8-9 immune spleen cells 1-6 x10 7-8 myeloma cells in log phase of growth Complete Media No Sera (CMNS) for washing of the myeloma and spleen cells. Hybridoma medium CM - HAT {Cell Mab (BD), 10% FBS (or HS) ; 5% Origen HCF (hybridoma cloning factor) containing 4mM L-glutamine and antibiotics} to be used for plating hybridomas after the fusion. Hybridoma medium CM - HT (NO AMINOPTERIN) {Cell Mab (BD), 10% FBS 5% Origen HCF containing 4mM L-glutamine and antibiotics} to be used for fusion maintenance stored in the refrigerator at 4-60C. feeding fusions on days 4, 8, and 12, and subsequent passages. Thawed inactivated and pre-filtered commercial Fetal Bovine serum (FBS) or Horse Serum (HS) stored in the refrigerator at 40C. Must be pretested for myeloma growth from single cells. L-glutamine, 200mM, 100X solution stored at -200C freezer. The L-gln is thawed and warmed until completely in solution. The L-gln is dispensed into media to supplement growth. L-gln is added to 2 mM for myelomas, and 4 mM for hybridoma media. Penicillin, Streptomycin, Amphotericin (antibacterial-antifungal) is stored at -200C until needed and it is thawed and added to Cell Mab Media to 1%. Cell Mab Media, Quantum Yield from BD is stored in the refrigerator at 40C in the dark. Myeloma growth media is Cell Mab Media with added L-gln to 2 mM and antibiotic/antimycotic solution to 1% and is called CMNS. Do not add antibiotics to media when growing myelomas for stock, only for pre-fusion growth. Indicate presence or absence of each reagent on the bottle label. Do not adjust the pH as it contains HEPES biological buffer already. 1 bottle of PEG 1500 in Hepes (Roche) (use fresh bottle that has never been opened) 8-Azaguanine is stored as the dried powder supplied by SIGMA at -700C until

needed. Reconstitute 1 vial / 500 ml of media and add entire contents to 500 ml media (eg. 2 vials/ litre). Myeloma Media is CM which has 10% FBS (or HS) and 8-Aza (1 X) stored in the refrigerator at 40c. Clonal cell medium D (Stemcell, Vancouver) contains HAT and methyl cellulose for semi-solid direct cloning from the fusion. This comes in 90 ml bottles with a CoA and must be "melted at 37Oc in a waterbath in the morning of the day of the fusion. Loosen the cap and leave in CO2 incubator to sufficiently gas the medium D and bring the pH down. Hybridoma supplements HT [hypoxanthine, thymidine] are to be used in medium for the section of hybridomas and maintenace of hybridomas through the cloning stages respectively. Origen HCF can be obtained directly from Igen and is a cell supernatant produced from a macrophage-like cell-line. It can be thawed and aliqouted to 15 ml tubes at 5 ml per tube and stored frozen at -200C. Positive Hybridomas are fed HCF through the first subcloning and are gradually weaned. It is not necessary to continue to supplement unless you have a particularly difficult hybridoma clone. This and other additives have been shown to be more effective in promoting new hybridoma growth than conventional feeder layers. Procedure At least one week prior to expected fusion date thaw a fresh vial of myeloma cells. It is advisable to keep several flasks at different densities so that you can choose the best one on the day of the fusion. We generally try to use a flask that is actively dividing and at a cell density of 3-6x105 cells/ml. Do not let them overgrow or they will enter a decline phase. Two to five days before the scheduled fusion give a final injection of ~5ug of antigen in PBS i.p. or intravenously in tail vein of the mouse (with high titer already determined). 1. Spin down myelomas and wash with 30 ml serum free media (CMNS has glutamine). Use tabletop centrifuge at 850 rpm for 12 minutes. Perform viable cell count with trypan blue exclusion principle, and wash cells with 30 ml of RPMICMNS. Spin down as above, resuspend in CMNS and disperse. Leave at 37°C until spleens are retrieved. Test aminopterin sensitivity. Keep 1 million myeloma cells for control plate and transfer into a 15ml conical. To do so, add 15 ml of HAT media to the million myeloma cells and plate out 2 drops/well on a 96 well plate. 2. Remove spleen from mouse in the biohazard facility. Euthanise the mice and submerge it in 70% ETOH. Let the mouse air dry on its right side on a paper towel. Remove spleen using sterile instruments and carefully put into labeled 10 ml of RPMI-CM with antibiotics and 20% FCS for transport back to the lab. Dispose of mouse and leave facility.

3. Place spleen into sterile petri dishes. Add 10 ml of RP-I-CMNS and perfuse the cells out of the spleen. Poke the spleen 8-10 times with an 18 ga needle (hold with sterile forceps). Use a 21 ga on a 3 ml syringe to draw up some RPMI. Inject the RPMI slowly into the spleen about 50-100 times until nearly all the cells are washed out. Discard the spleens into the biohazards bag. 4. Collect and transfer the spleen cells to a new 50 ml conical tube. Rinse out the dish 2X with 10 ml of RPMI-CMNS and pool with the first 10 ml (the use of perfusion removes the production of large debris seen with grinding, and obviates the need to let the debris settle). Spin down at 900 rpm for 12 minutes. Discard the supernatant to bleach container. Wash the cells with another 30 ml RPMI-CMNS. Remove a small sample and count the viable cell/ml and spin again as above. Combine the cells at a ratio of 5:1 (spleen cells: myeloma cells) and never 1X10 myeloma cells. 5. Wash both the myeloma and spleen cells 2 more times with 30 ml of RPMI-CMNS. Spin at 800 rpm for 12 minutes. 6. Remove supernatant and resuspend cells in 5 ml of RPMI-CMNS and pool together. Fill volume to 30 ml and spin down as before. 7. Aspirate all fluid into bleach vessel. Break up pellet by gently tapping on the flow hood surface. Add 1 ml of BMB REG1500 (prewarmed to 37°C) dropwise with 1 cc needle over 1 minute. Swirl and tap the conical gently while adding the PEG to resuspend the cells. 8. Add 1 ml of RPMI-CMNS to the PEG cells gently over 1 minute while swirling (to dilute the PEG). 9. Add 8 ml RPMI-CMNS over 2 minutes to slowly dilute out the PEG. 10. Incubate the cells in the 37°C waterbath for 10 minutes. Centrifuge the cells at 700 rpm for 10 minutes (the membranes are still very weak). 11. Aspirate all fluid, and add 5 ml of RPMI-CM-10% FCS WITHOUT RESUSPENDING THE CELLS! The cells will disperse adequately by simply adding the media at this point. 12. Incubate @ 37°C another 10 minutes. (12.5 Meanwhile put aside 1 ml of Clonacell medium D for myeloma testing. ) 13. Gently dilute cells in 5 ml of Complete media and transfer into 95 ml of Clonacell Medium D (HAT) media (with 5 ml of HCF) and plate out 10 ml per small petri plate. 14. Dilute about 1000 P3X63 Ag8.653 myeloma cells into 1 ml of mediu D and transfer into a single well of a 24 well plate. This is the myeloma/HAT control. P 15. Place plates in incubator two plates inside of a large petri plate, with an additional petri plate full of dH20 without a lid for humidity. Leave for 10-18 days under 5% CO2 overlay at 37 degrees.

15. Pick clones from semisolid agarose into 96 well plates containing 150-200 ul of CM- HT. Screen sups 4 days later in ELISA. Move positive clones up to 24 well plates. 16. Heavy growth will require changing of the media at day 8 (+/- 150 ml). Should see macroscopic colonies at this time. At this time can decrease the HCF to 0.5% (gradually- 2%, then 1%, then 0.5%) in the cloning plates. 17. Isotype via supernatants and grow up for ascites/ large flask production and further freeze down. Troubleshooting I strongly reccommend to use Southern Biotech Goat anti-Mouse Ig (H+L)HRP Chains for screening supernatants. We have screened and developed Mabs to various antigens and have surprisingly found out that using some secondary reagents from several other major companies can result in weak or no reactivity.

Reference 1) Kohler G, and C. Milstein Continuous cultures of fused cells secreting antibody of predefined specificity.1975. Nature 256: 495-497. 2) Lane, R.D. A short duration polyethylene glycol fusion technique for increasing production of monoclonal antibody-secreting hybridomas. 1985. J. Immunol. Meth. 81:223-228. 3) Harlow, E. and D. Lane. Antibodies: A laboratory manual.Cold Spring Harbour Laboratory Press. 1988. 4) Kubitz, D. The Scripps Research Institute. La Jolla. Personal Communication. 5) Zhong, G., Berry, J.D., and Choukri, S. (1996) Mapping epitopes of Chlamydia trachomatis neutralizing monoclonal antibodies using phage random peptide libraries. J. Indust. Microbiol. Biotech. 19, 71-76. 6) Berry, J.D. , Licea, A., Popkov, M., Cortez, X., Fuller, R., Elia, M., Kerwin, L., and C.F. Barbas III. (2003) Rapid monoclonal antibody generation via dendritic cell targeting in vivo. Hybridoma and Hybridomics 22 (1), 23-31.

Monoclonal Antibody Production Mouse Immunization

Order 6 six week old Balb/C mice and let the ARC know they are coming. Have your antigen ready for when they arrive. Once they get there earmark the mice and perform a pre-bleed on them to be used as an ELISA control for monitoring the titer and screening of the hybridomas:

Bleeding Mice 1. 2. 3. 4.

5. 6. 7. 8.

Place the mouse in a mouse restrainer. Sterilize the tail with 70% ethanol. With a razor blade, nip off the last 2 mm of the tip of the tail. Using a milking motion, pull blood down and let drip off the end of the tail until you have collected ~200 µL. (You may have to pre-bleed twice, with a week or so between bleeds). Take the collected blood and place at 37 °C for 30 min. to remove complement. Place blood at 4 °C overnight to clot. Centrifuge samples at 10,000g 10 min. Pipet off the serum supernatant. Store at -20 °C. This is your prebleed control.

Hybridoma Fusion For one fusion you will need: 1. 2. 3. 4. 5. 6.

8 500 mL bottles of DMEM media 3 500 mL bottles of fetal calf serum (FCS) 2 50 mL bottles of 100X penicillin-streptomycin 10 mL of 100X HAT selection solution 10 mL of 100X HT solution 10 sterile flat-bottomed 96-well plates

Assaying for Positive Clones Run an indirect ELISA using the antigen you want the MAb directed against. If you are raising the MAbs against a small hapten that you coupled to a carrier protein for immunization of the mice, then use the hapten coupled to a different carrier protein for the screen.

Expanding Positive Clones

1. The day prior to any expansion, obtain feeder cells. For this first

2.

3. 4. 5.

6.

7.

expansion from the 200 µL cultures in 96-well plates to 500 µL cultures in 24-well plates, add 200 µL of feeder cells/well in DMEM/20% FCS + HT to the 24-well plates the day before expansion. Let the feeder cells grow overnight in the CO2 incubator. For the initial expansion, resuspend the positive testing hybridoma cells and place all but the last 10 µL (200 µL) in the well of the 24well plate with the feeder cells. Then bring to 500 µL with DMEM/20% FCS +HT. Add 200 µL of DMEM/20% FCS +HT to the little bit of cells left in the 96-well plate. This serves as a backup for your positive clones. Place all plates in the CO2 incubator. When the cells are beginning to acidify the media (gets orangeyellow), change the media and double the culture volume by pipetting off 400 µL, and replace with 900 µL fresh DMEM/20% FCS +HT. Harvest more feeder cells for your next expansion. Put 0.5 mL f feeder cells in DMEM/20% FCS (note the removal of the HT) in a T25 flask (one for each positive clone) for later use When the cells are ready for expansion out of the 24-well plate, resuspend them and pipet to the T25 flasks with feeder cells. Again leave a little bit in the 24-well plate and refill with DMEM/20 FCS as a backup. Volume to the T25 culture 5 mL with DMEM/20% FCS (add 3.5 mL) Place all cultures in the incubator.

Subcloning Positive Clones Immunological Techniques Making Monoclonal Antibodies Introduction Monoclonal antibodies are great! Why? Specificity! Each one recognizes only one site of the antigen structure. Why else? Immortality! Mass cultures can be generated from a single clone. Mice are small! They require less antigen than a rabbit to get an antibody reaction. The antigen travels through the circulation system. Blood is channeled through the spleen where B lymphocyte cells recognize the foreign bodies and produce antibodies. Spleen cells, however, cannot survive alone in tissue culture media. Myeloma cells can! Why not fuse them? We can select for fused cells by using a HAT (hypoxanthine, aminopterin, thymidine) supplemented media. The mutant myeloma cell line SP2/0 cannot survive in HAT media because it lacks HGPRT. The aminopterin in the HAT supplement blocks DNA synthesis. The enzyme HGPRT can overcome

this block. Spleen cells do have HGPRT. Fuse the spleen and SP2/0 cells and you'll find survivors in HAT tissue culture media.

Notes: Remember, Sterile Technique! Wash yo' hands. If you pour (you really should pipet) DON'T SPILL! Contamination occurs because of spillage outside of bottles and tubes. Ethanol and flame when you're feeling paranoid. For your own safety, wear gloves when dissecting. Use disPo products to avoid problems with detergent residue on washed glassware.

Supplies: BALB/c female mice for immunization one mouse / feeder layer disPo pipets - 10 ml and 25 ml tissue culture flasks (5 / feeder layer) 24 well tissue culture plates (about 12 per fusion) 3 autoclaved sets of forceps and scissors 1 autoclaved screen for spleen cell dispersion 5cc syringe (plunger used to disperse cells on screen) 1cc syringe 18g & 25g (1/2 inch) needles 100 mm tissue culture plates 15 & 50 ml sterile conical tubes *see attached for details on ordering

Immunization Cutting out protein bands from a polyacrylamide gel. Coomassie stain for an hour & destain (10% acetic acid, 45% methanol) as quickly as possible, cutting the bands as soon as they are discernible to limit the time that the gel is in acid. Rinse with PBSa & cut out the bands. Protein can be stored with a small amount of PBSa in the -20 freezer. Make first injection of protein at 1:1 ratio with Freund's Complete or TiterMax.

1. Homogenize protein with a small amount of PBSa so protein doesn't get too thick. Keep protein cold while homogenizing. 2. If using Freund's, mix up dead bacteria in the bottle. Draw Freund's into a syringe and force it out into a small beaker containing your protein sample. Mix the Freund's and your protein until it takes on a white foamy consistency. You may have to change needles and syringes because the Freund's reacts with the plastic. e.g. To inject 6 mice with emlc and rmlc each, I ran one 12% gel, cut out the bands, homogenized the protein and combined each protein sample with 450ul of Freund's. This gave me a little over 600ul of each sample to inject. 3. To inject. Refer to the animal care manual for further instructions. notes: • Inject 100ul per mouse. • Your sample must be concentrated but not so thick that you cannot get it through a 1/2 inch, 25g needle. • Sterility is important for the health of the mouse! Two weeks later, make second injection of protein with Freund's Incomplete Adjuvant or TiterMax. Two weeks later, do a test bleed. If negative, do another boost with Freund's Incomplete Adjuvant or TiterMax. Five days before the fusion, do a final boost of protein without Freund's.

Test Bleeds Mark mice's tails. Apply grease to tail (an area of 3/4 inch, from base toward tip). Use extremely sharp blades to make slice (EM blades). Be careful not to cut through the tail but make cut large enough to draw about 2.5 heprinized capillary tubes. Apply pressure to tail wound to stop the bleeding. Spin down the blood at max speed for 5 minutes in the microfuge. Draw off and store sera at -20o until you can test on a blot at 1:100 or at 1:50.

Preparation for Fusion Day WED/THURS Thaw out a plate of SP2/0, split one to 3. (must split during weekend). Need 10-12 100mm plates in log phase for fusion. Thaw tube quickly in 37o bath. Put into falcoln tube with 10ml of warm media. Let sit for 5 min. Spin down at low speed. Resuspend in 10 ml and spin. Resuspend in 30ml and put into 3 plates.

THURS Run gel for final boost.

FRI Do final boost of protein without Freund's or Titermax.

MON Prepare Feeder Layer

TUES Do Fusion!

FEEDER LAYER DAY Collect animal board- ethanoled and then sterilized under UV light 2 x 10 ml petri dishes autoclaved forceps, scissors, & screen 5 ml syringe, 10 ml syringe, 18g needle beaker with 95% ethanol disPo 10 ml pipets Warm the following in the 37o water bath: • 50 ml aliquots of DMEM- without additives • 500 ml bottle of DMEM two which the following has been added: 5 ml Sodium pyruvate 5 ml L-glutamine HAT(with syringe, pump 5ml DMEM into HAT bottle, add to DMEM) 5 ml anti-biotic/anti-mitotic (Don't warm past room temp) 50 ml Hyclone Fetal Calf Serum Put into 5 x 50 ml conical tubes (to later be transferred to 5 flasks for feeder layer). • Set up the sterile dissecting utensils.

• Get the normal un-immunized mouse. • Place screen in plate with 10 ml of DMEM-.

Dissection WORK QUICKLY! (The slower you are, the more difficult it is to work with the animal. Also, you don't want the media to cool.) • Sacrifice the mouse. Ethanol until saturated. Pin the animal in place. • Use one set of utensils to cut through the skin, pin back the flap. ETHANOL! • Use second set of utensils to cut through the peritoneum. • Use third set to pull out the spleen, cutting away the connective tissue. Changing utensils is very important to avoid contamination.

Wash Cells • Put spleen in the dish with 10 ml of DMEM-. Quickly and carefully disperse spleen cells with plunger of a 5 ml syringe against the screen. • Leaving connective tissue on the grid, pipet up and down and put cells into a 15ml conical tube. Let large chunks settle to the bottom of the tube for 2-3 min. • Transfer supernatant to another tube bringing the volume up to 15 ml with more DMEM- to wash. Spin down for 10 min at 170g on the table top centrifuge. • Carefully, suck off supernatant with sterile pasteur pipet. Wash spleen cells again with 10ml DMEM-. • Suck off super. Resusp. in 5ml DMEM-, incubate for 10 min. in 37o bath. Pour 5x 50ml conicals of DMEM+ into tc flasks. Put 1ml of spleen cells in each flask. • Incubate o/n in 6% CO2 37o chamber. Lay flasks on sides with caps loosened.

FUSION DAY I. Collect immunized spleen cells. II. Collect SP2/0 cells. III. Combine with feeder layer.

Collect • All of the same supplies used for the feeder layer on previous day. • Transfer 50% PEG into a conical tube.

• Warm the PEG, wash media, and the remaining DMEM+ in 37o water bath. Filter sterilize leftover DMEM+ media from yesterday followed by feeder layer. The feeder layer is more likely to clog the filter. (Make sure you have media in the container before turning on the vacuum otherwise you could break the filter membrane).

Prepare the Immunized Spleen Cells Follow protocol used to dissect the mouse for the feeder layer. It is important to minimize the time that the spleen cells are either not in the animal or not in the incubator. For this reason and depending on how quickly you are able to dissect the mouse, you may want to collect the SP2/0 cells first.

Collect the SP2/0 Cells Triterate cells from 10 plates and put into 2 x 50 ml conical tubes. Spin down the cells. Wash cells 2x. Pellet for 10 min. at 170g. pellet spleen cells

WHILE

triterating the SP2/0 cells

resusp spleen cells, wash, pellet

WHILE

pellet SP2/0 cells

resusp spleen cells, wash, pellet

WHILE

resusp one tube in 5ml, transfer to other tube, wash, pellet

resusp spleen cells in 10 ml, count,

WHILE

resusp SP2/0, wash, pellet let sit for 10 min in 37o resusp in 20-30 ml, count

1 x 108 spleen cells

ADD TO

5 x 107 SP2/0 cell

• Pellet cells together at higher speed (between 170 - 200g). • Suck off media • Add 1.5 ml PEG over 1 min. under agitation-- THIS IS THE FUSION!!! Stir gently with tip of pipet to mix in PEG • Wait 90 seconds, stirring gently. • Over the next min, add 1ml of warmed DMEM- while stirring. • Repeat previous step. • Add 8 ml DMEM- over 5 min. • Wait 10 min. Then, disrupt cells by pipetting gently 5 times.

• Dilute into sterile filterized DMEM+ with Feeder Layer. • Aliquot 2ml/ well in 24 well plates (~ 10-12 plates) • Incubate in 6% CO2, 37o chamber for one week. • Feed and incubate for ~one week.

Screening one week after first media feeding: Test wells where media is turning yellow. Draw off 500ul & test on blot strips, incubate overnight. (Don't forget positive controls including mouse antibody - use mouse polyclonal sera and a marker.) Grow up positive wells in larger plates. Retest the well and its corresponding plate. Both should be positive. Continue to split positive plates and freeze down (in 10% DMSO) as back ups. Triterate and dilute cells from the positive well into a 96 well plate at a dilution of 1 cell to ~3 wells. We're going for the single clone! Transfer cells in the 96 well plates to 24 well plates and Retest. Pass through dilution again until all wells are testing positive and you are convinced that you have a monoclonal cell line. Eventually, try and wean the cells from 10% FCS to 5% FCS, and from HAT to HT to unsupplemented media.

Affinity Purification of IgG Using G-Beads (Pharmacia) Buffers: 0.1M NaPO4, pH 7.0 monobasic - 5.52g in 200 mL for 0.2M monobasic dibasic - 8.52g in 300 mL for 0.2M dibasic start with the dibasic solution and titer with the monobasic until pH 7.0 is reached dilute 1:1 with water for use

1.0M acetic acid - 0.1M glycine buffer, pH 3.0 30.12 mL acetic acid 3.75 g glycine

bring to 500 mL with water

Protocol 1. Equilibrate column with several volumes of pH 7.0 buffer. 2. Put sample over column - 10 mL rabbit serum for IgG isolation or entire volume of papain digestion - take gel samples. (Flow rate at 1mL/4 min) 3. Save pass through - in papain digest the pass through contains the Fab - take gel samples. 4. Wash to baseline with pH 7.0 buffer (1-2 hours). 5. Elute with pH 3.0 buffer - takes 15-20 minutes to see something. 6. Collect 0.5 mL fractions and neutralize solution with 1.0M Tris to pH 7.4 (use indicator strips)- take gel samples. Spec fractions to find peak. 7. Wash with pH 3.0 buffer for 15 minutes and store in 20% ethanol. 8. Dialyze IgG or Fab against 4L PBS overnight at 4° C then at room temperature with 2 changes over 1-2 days. 9. Determine concentration using spec at absorbance of 280nm with A280 of 1.35 for IgG or 1.5 for Fab. 10. Store at -80° C in aliquots.

Affinity Purification of Antiserum Using DPT Paper 1. Run SDS-PAGE curtain. (Use as much protein as possible without having so much that you risk running bands together and contaminating the band of interest). 2. Place unstained gel in 0.5 M KCl for 10-15 seconds -- until bands begin to appear. The bands will appear clear, while the rest of the gel will turn white as the SDS precipitates. Remove the gel onto a glass plate and cut out band of interest as rapidly as possible. If you wait too long, the bands will turn white as well. 3. Place gel strip(s) into 50 mM NaPO4, pH 6.5 with 0.1% SDS and rinse 3 times, with gentle shaking, for 30 min. per rinse. 4. At third gel wash, begin activation of the APT paper. Do these steps in the cold room on an ice bath. First, place an appropriately-sized piece (slightly larger than gel) of APT paper in cold 1.2N HCl (100 ml) to which has been added 3 ml of sodium nitrite (NaNO2). The NaNO2 is made in water at 10 mg/ml immediately before use. Incubate paper on ice with occassional shaking for 15-30 min. The paper should turn bright yellow. Activation is followed by two or more rapid ice water washes and two rapid washes with 50 mM NaPO4 pH 6.5, taking 5 minutes for the whole process. (When I originally did this, I used a protocol which called for 5 X 5 minute washes with ice cold water and a subsequent 10 min wash in cold 50 mM NaPO4, pH 6.5. However, Bio-Rad emphasizes the necessity of short washes. Both seemed to work for me.) 5. Immediately after activation, set up the transfer in the cold using 50 mM NaPO4, pH 6.5 as transfer buffer. Transfer for 4 hours at 0.6 amps (Hoefer TE-52). The paper

should have turned an orangey (peach) color. Ususally where the gel was, the color is lighter. 6. Block the DPT paper in the following buffer for 2 or more hours: 0.25% gelatin (0.5 g) dissolve first by heating 20 ml ethanolamine 20 ml 1 M Tris-HCl pH 9.0 _______________________ to 200 ml with dH2O 7. Rinse extensively in "Buffer 1" following the blocking step. Do at least 3 20-30 minute washes. Buffer 1: 50 mM Tris-HCl, pH 7.5 5 mM EDTA 150 mM NaCl 8. To check transfer efficiency, cut a strip off one side of DPT paper to which protein has been transferred and incubate with your (appropriately diluted) antibody overnight. (Dilute the antibody in Buffer 1 containing 0.25% gelatin, or 1% BSA [Sigma Fraction V]. I use BSA.) Carry through with normal protocol for immunobloting, i.e. rinse (3 changes of buffer 1 or PBS) 30 min, incubate 2-4 hours in peroxidase conjugated secondary antibody, rinse, develop in 0.05% 4 chloro-1napthol (made up as a 0.3% solution in methanol), 0.01% H2O2 in PBS. The reaction seen on the DPT paper is usually not as distinct as that seen on nitrocellulose. 9. If you are waiting to check efficiency of transfer before using the DPT paper, just store paper in Buffer 1 at 4o C until use. 10. To use antigen-bound DPT paper: a. cut paper into small squares (on a glass plate) with a clean razor blade. b. place squares in 10 ml syringe with large gauge needle c. incubate with 5 ml of an appropriate dilution of antiserum (I use a dilution 2 times more concentrated than that used for immunoblotting). Dilution is made in Buffer 1 with BSA or gelatin. Incubate overnight with vigorous shaking. I do this at R.T. without any problems d. the next morning, squirt out diluted antiserum (from which epitope specific antibodies have presumably been removed) and draw about 10 ml of Buffer 1 into the syringe. Shake rapidly and repeat for a total of 3 rinses (30 minutes each) e. to elute specific antibodies, use 4 ml of 5 M NaI made up fresh in ice H2O. Draw up into syringe and place on ice; agitate by rotation of the syringe on ice for 8 minutes

f. force the NaI solution (now containing eluted antibodies) into a tube (50 ml Falcon type) containing 600 ll of Buffer 1/1% BSA. Force Buffer 1 through DPT paper repeatedly, pooling all of these washes in the same 50 ml tube until a total of 40 to 50 mls is reached. (You may have to dilute even further if the intended use of the antibody is for immunofluorescence. Concentrations of salt that are too high interfere with fluorescence visualization). g. To concentrate antibody: Alternative #1. Concentrate using a Millipore immersible CX-10 filter. Prepare the filter by rinsing with dH2O and then filtering through about 5 ml of 1% BSA/Buffer 1 to block sites (prevents nonspecific sticking to filter). Concentrate down to 2-4 mls, depending on your antibody, to give a final working dilution for immunoblotting. If desired, concentrate further for immunofluorescence, or for dilution with a blocking buffer (e.g. 5% carnation instant milk) of your choice (for immunoblotting). Alternative #2. (faster) a. prepare two amicon centricon filters (30,000 mw cutoff) by adding 300 µl of buffer #1 1% BSA. b. place one half the 5M NaI/antibody solution in the upper reservoir of each of two centricon filters. Using a pasteur pipette, stir to mix the two solutions. c. spin at 5000 x g for 30 minutes. (Use parafilm to make the filters to fit into rotor adaptors. d. after the spin, discard the liquid which has passed through the filter (i.e. the liquid in the lower reservoir). e. bring liquid remaining in the upper reservoir (100-500 µl) to a volume of 2 mls with buffer 1. f. spin again at 5000 x g for 30 minutes. h. discard liquid which has passed through the filter. i. add 250 µl of buffer 1 to each upper reservoir and cap with storage reservoir. j. invert tubes and spin in table to centrifuge for 2-3 minutes to transfer the entire sample into the storage reservoir.

NB. filters can be reused for repeated purification of the same antibody. Store at room temperature with 1 ml of buffer 1 in the upper reservoir. 11. Store DPT squares in the syringe in Buffer 1 at 4oC. I've never used azide but have used squares repeatedly (up to 10-12 times) over a period of 6 months without any problems.

Immunoprecipitation 1. Harvest 5 x 106 - 4 x 107 cells/sample depending on how abundant your protein is. Use 5 x 106 cells/sample to IP myosin, 4 x 107 cells/sample to IP dynein, for example. Wash cells a couple of times in DB, and resuspend in 500 µ l DB/sample. Place on ice. 2. Add an equal volume 2X IP buffer (made fresh) and mix on ice:

2X buffer for dynein IP: 100 mM Pipes, pH 7.5 2% NP40 10 mM EDTA 50 mM NaPPi 200 mM NaF 5 mM DTT 2 mM PMSF 100 µ g/ml leupeptin 100 µ g/ml pepstatin 2 mM ATP

2X buffer for myosin IP: 40 mM Tris, pH 7.5 0.2% NP40 2 mM DTT 10 mM EDTA 2 mM PMSF 200 µ M TPCK 200 µ M TLCK 20 mM NaHSO3

50 mM NaPPi 200 mM NaF 2 mM ATP 200 mM KPO4 3. Add 100 µ g/ml RNAse A. 4. Centrifuge lysate 18K, 30 min, 2 degrees, in SS34 rotor 5. Collect cleared lysate. Use 1 ml lysate per IP sample. Add primary antibody: For NW127 dynein HC Ab, add 50 ul serum/sample For 142, 143, 144 dynein IC Abs, add 50 ul serum/sample For 9E10 anti-myctag Ab, add 200 ul supernatant/sample For 396 myosin Ab, preincubate 25-30 µ l GammaBind or protein A-sepharose beads with 1 ml of 396 supernatant for 4 hours to overnight. Wash beads as per steps 10-11 below and resuspend beads directly in 1 ml cleared lysate. Skip steps 6-7 and go directly to step 8. 6. Rock in cold room for 2 hours to overnight. 7. Add 25-30 ul protein A-sepharose slurry for rabbit and rat primary antibodies; add 25-30 ul GammaBind slurry for mouse primary antibodies. 8. Rock in cold room 1-4 hours. 9. Spin down beads at 5K for 2 minutes in microfuge. 10. Wash beads twice with 1 ml each time of 1X IP buffer with 1 mg/ml BSA (to reduce background, adjust pH of IP buffer to 7.0 for washes). Spin down as above. 11. Wash beads twice with 1 ml each time of 1X IP buffer, pH 7.0 without BSA. Spin down as above. 12. If desired, do a final wash in 1X IP buffer with 0.5M NaCl. This will further reduce your background. HOWEVER, it will strip the dynein IC off the HC when doing IPs with NW127 Ab! 13. Remove all traces of wash buffer, and resuspend beads in 20-30 µ l 2X SDS sample buffer. Boil 5 minutes. Spin down beads, load super onto an SDS gel. IgG heavy chain runs at 50 kd; IgG light chain runs at 22 kd. notes: If you have a lot of background, it may help to preincubate the cleared lysate with beads (without antibody!) for a couple of hours to adsorb out any non-specific binding. Spin down the beads, recover the lysate, and add primary antibody. Proceed with step 5. 1/95, Trivinos

Specific Details for Myosin Ips

1. Cells were harvested and washed in Ca2+-free MES starvation buffer (20 mM MES, 2 mM MgSO4). 2. Ice-cold 2 x lysis buffer was then added to the cells suspension. (40 mM Tris-Cl pH7.5, 0.2% NP40, 2mM DTT , 10 mM EDTA, 2 mM PMSF, 2 mM TAME, 200 µ M TPCK, 200 µ M TLCK, 20 mM NaHSO3, 100 µ g/ml RNAse A, 50 mM Sodium pyrophosphate, 200 mM NaF, 2 mM ATP, and 200 mM Phosphate pH7.5) 3. Vortex and then centrifuged for 30 min at the top speed of the microfuge. 4. The supernatant was transferred to a tube containing mAb and 20 µ l 1:1 protein A sepharose slurry, and the tube was rotated end-over-end overnight at 4 oC. 5. The immunoprecipitate was then washed at least 6 times with MES-Salt buffer(20 ml MES pH 6.8, 20 mM NaCl, 1 mM EDTA) for 5 min. each. 6. The beads were pelleted for 2 min. at a microfuge, resuspended into 2 x SDS sample buffer, boiled for 5 min. at 90 oC sand bath. 7. The slurry was pelleted again, keep and supernatant. 8. Run SDS-PAGE. (P.C., 01/30/95)

Notes: IgG heavy chain will run at 50kd IgG light chain will run at 22kd Whole IgG runs at 150kd Need to worry about detergents used to lyse cells inhibiting Ab binding. Need to worry about immunocomplex not being soluble. Need to work in zone of equivalence (Ag:Ab ratio). Instead of 2° GAMIgG can use whole Staph. aureus, Protein A, Protein G which binds to IgG1 better than Protein A, Prot.A/Prot.G mix (Pierce Immunochemicals), or antimouse Affigel. Can release antigen from immunocomplex by 0.1M Glycine pH 2.5 acid shock, or high salt (3M NaSCN) shock, then passage through Costar Spinex 0.22m m filter unit into Eppendorf. Can increase titer of 1° mAb by passing 9E10 media onto Sepharose CNBr beads, this binds 5 or more Fc portions of the IgG effectively making it an IgM. Can increase titer of 1° mAb by adding equal volume of saturated NH4SO4 dropwise to spinning 9E10 media at roomtemp, pelleting the precipitate at 2500g for 5min, resuspending the pink (RPMI) pellet in dH20, repeating the precipitation.

To make saturated NH4SO4: boil water, add NH4SO4 until crystals fall out of solution, cool to RT, pH to 7.0 with NH4Cl, filter through 3MM paper. Can grow 9E10 cells in 2% FCS to lower contaminating Ig.

Microscale antibody-exchange affinity-purification for indirect immunofluorescence and epitope selection of phage clones. This protocol takes advantage of the fact that antibodies transfer rapidly between antigen samples attached to separate solid supports. It allows one to affinity purify small amounts of antibody using less than a picamole of antigen. This is great for doing immunofluorescence with dirty antibodies that are in short supply or whose antigens are in short supply, or for quickly screening expression clones for epitope selection.

For immunofluorescence: 1. Immobilize your antigen on a 1 cm2 chip of nitrocellulose. For example, spot blot 1-10 ng of native Dicty dynein in 10 µ l P100 buffer onto a nitrocellulose chip. This is your source blot. Make one source blot for every coverslip you wish to stain. 2. Let nitrocellulose dry. Mark the protein side with a pencil. Block in 5% milk/PBS for 30 minutes. Source blots can be stored in blocking solution in the fridge if NaN3 is added to 0.02%. 3. Incubate antigen source blot with antibody in PBS/0.1%BSA for 4 hours to overnight. Handle blots with blunt forceps. Use a dilution of antibody that gives good results with a Western blot. The wells in a 24-well plate are good for this if you have several samples. 4. Wash source blots 3 times 5 minutes with PBS/0.5% Tween 20. 5. Prepare coverslip sample for indirect immunofluorescence (see relevant section in labman). Place 50 µ l PBS on fixed, blocked coverslip. Place washed source blot, antigen/Ab complex down, on top of coverslip. Incubate at 37 degrees, 1 hour, in humid chamber. The antibody on the source blot will rapidly equilibrate with the antigens on the fixed cells on the coverslip. Proceed with washing and secondary antibody as per immunofluorescence protocol. 6. Source blots can be re-used if washed extensively and stored in PBS/0.1% BSA/0.02% NaN3. Re-incubate with antibody each time.

For epitope selection of expression clones 1. Grow up dense IPTG-induced (clonal!) phage plates and lift expressed fusion proteins onto nitrocellulose filters as usual. 2. Block phage filters in 5% milk/PBS for 30 min. Incubate with the antibody for 1-4 hours at the same dilution used to screen the phage library. Wash filters in PBS. These are your source filters. 3. Run a curtain SDS gel of a sample of purified or enriched antigen. Blot the gel onto nitrocellulose and mark the protein side with a pencil line across the entire width of the blot. Block in 5% milk/PBS for 30 minutes and cut into strips. 4. Incubate each source filter to be tested with a strip of curtain gel. Place the source filter and the blot strip with protein sides facing each other in a seal-a-meal bag. Add

just enough PBS/0.1%BSA to wet both blots. Seal the bag. DO NOT ADD ANTIBODY AT THIS POINT! 5. Incubate blots together for 1-4 hours. Then remove blot strips and wash extensively in PBS. Do not mix strips in washing solution or secondary antibody solution, as antibody may transfer between strips. Incubate the strips in the appropriate secondary antibody for 1 hour. Wash and develop strips. 6. If a phage clone encodes a truly positive cDNA, the corresponding strip will be positive as well. Negative strips indicate false positive phage clones. 1/95, Trivinos (From Hammarback and Vallee ,1990, JBC 265:12763)

Washington University School of Medicine Department of Pathology Center for Immunology Immunization Protocol for Monoclonal Antibody Production Antigens: Proteins: Cells:

10-100 µg 0.5 - 5.0 x107

Adjuvant: Complete Freunds Adjuvant None Schedule: Day 0 14 24 35 42 45

Manipulation Primary Immunization Boost #1 Boost #2 Boost #3 Bleed and test for antibody Boost #4

Adjuvant CFA IFA IFA IFA

Site s.c. s.c. s.c. i.v.

IFA

i.v.

66+ 69

3-4 week rest followed by i.v. or i.p. boost fusion 3d after i.v. boost or 4d after i.p. boost

Saline/PBS

i.v.

--

i.v.

Back to Hybridoma Center Protocol for Cell Fusion Click here for cell fusion recipies 1. Healthy Sp2/0 cells should be rapidly growing by this time. Sp 2/0 cells should be started about two weeks before the cell fusion. Every two days, they should be centrifuged at a 64.4 xg on the IEC clinical centrifuge (in 50 ml tubes) for two minutes. The media should be removed and the cells resuspended in fresh media. This ensures that only the healthiest cells will fall to the bottom of the tube. Centrifugation at higher speeds or for longer time periods should be avoided at this step, because that causes weak or old Sp2/0 cells to precipitate also, and is contra-indicative of what you want to accomplish. 2. Begin the cell fusion by centrifuging two 50 ml or eight 10 ml flasks of Sp2/0 cells that have been transferred to fresh media one to two days previously, at a setting of not more than 3 for not more than 3 min in an IEC clinical centrifuge. Decant the media. You need a ratio of 1:5 healthy, rapidly growing Sp2/0 to spleen cells. This number of flasks will provide about the correct number. 3. Remove two spleens from the mice selected above, and place them in washing media. Tease apart the spleens. Don't be afraid to take two pair of tweezers, or tweezers and syringe tip and shred the spleens. Then remove cells by perfusing media through the spleens and also by disrupting portions of the spleen with the tweezers. Then remove the remaining large chunks of spleens with tweezers, and place the cells in a 50 ml tube. Centrifuge at a setting of 3 in IEC clinical centrifuge for 3 minutes. 4. Using warm (37oC) washing media, transfer all the cells to one 50 ml tube, bring the final volume to about 25 to 30 mls, mix by vortexing (THIS IS THE LAST TIME YOU WILL VORTEX ANYTHING in this procedure ) , and centrifuge at a setting of 5 for 3 minutes. The objective here is to pack the cells tightly so that their cell membranes are touching. You also need a large surface area, and to prevent layering of the large Sp2/0 cells on the bottom and spleen cells on top. The best tubes to use are the widest, flattest bottomed ones (our 50 ml conical tubes work fine). The worst tubes are the narrow, very conical bottomed tubes (our 15 ml culture tubes). If it is necessary to use 15 ml tubes, use round bottom ones only! 5. Add one ml of warm (37o) 50 % PEG (m. wt. 1500) (source and type) over one minute, gently swirling the tube at 37oC. If you add the PEG with a 1 ml pipette and gently stir the cells, you will probably obtain many more hybridomas. After the PEG is added, cap the tube, gently swirl a couple more times, then centrifuge at a setting of 5 for 3 minutes (minimum). This is the critical step. Cells must be compacted and stuck together for cell fusion to

occur. This step will also ensure that the PEG comes to the surface while the cells are compacted at the bottom of the tube. Then remove as much PEG as possible with a pipette. (CAUTION: PEG is toxic to cells, and should be removed promptly. This is not the step to "take a break.") 6. Add 8 to 10 mls of warm (37oC) washing media over a period of 2.5 to 3 minutes, again gently swirling the tube. Do not disturb the cell pellet. Add the media as gentle drops. Remember that the cells are very fragile. After addition of the media, cap the tube, gently swirl it another time or two, then centrifuge at a setting of 5 for 3 minutes. Remove the media. This step can be repeated once more, to ensure that all the PEG is removed, but this appears to be an optional step. 7. Finally, using a large tipped pipette, such as a 5 ml disposable pipette, and warm complete media (containing HT media, for the very best results) transfer the cell pellet to a 50 ml flask. Do not disrupt the cell pellet or use a syringe!. Hybridomas will grow out of the clumps of stuck-together cells. Allow the cells to grow in a 50 ml flask in the presence of CO2, HT media, and the unfused cells, for 4 to 24 hours. It seems that the cells are too fragile to handle immediately after fusion, but large numbers of hybridomas have been obtained when cells were plated on the same day as the cell fusion. Cold Spring Harbor (1989) does not include the overnight incubation prior to plating. 8. Add HAT media to the 50 ml of media in the flask (50x HAT is used at a ratio of 1 ml/100 ml of media; 500x HAT is used at a ratio of .1 ml/100 ml of media). Plate the 50 mls into the center 60 wells of 5 96 well plates WITH A WIDE TIPPED PIPETTE (300 wells) - DO NOT use a syringe and needle! Cells should cover the bottoms of the wells. This is important to maintain the CO2 level at a high enough level, and possibly the hybridomas obtain growth hormones from the unfused spleen cells. The complete media supplemented with fetal bovine serum should support the growth of 50 cells/well, but ours does not appear to do this. The first thing one would consider, is that the fetal bovine serum content is too low, but our media supports the growth of Sp2/0 and established hybridoma cells very well. The problem here appears to be the CO2 content. Therefore, high cell density maintains the CO2 at a higher level, and more hybridomas survive. 9. After 4 to 5 days, add complete media supplemented with HT to the wells. 10. After 1 week, test the wells with hybridomas against the antigen by an ELISA. 11. Expand positive hybridomas into 24 well plates. You MUST use spleen feeder cells and HT media. If many wells are positive, hybridomas may be combined into expansion wells. CELLS MUST BE RE-EXPANDED EVERY TWO TO THREE DAYS. Biochemistry book recommends that hybridomas be cloned immediately, in order to prevent overgrowth of the desired cells, by non-secreting cells that may be present. Summary: The mouse is immunized six times. After the spleen is removed, the splenocytes are obtained and fused with myeloma cells using PEG. The hybrids are plated into 96 well tissue culture plates and observed for colony growth.

OUTLINE Fusion is an important procedure to produce monoclonal antibodies (MoAb). PROTOCOL 1. prepare the culture of myeloma cells and maintain the concentration of the cells at the level of less than 10^6 cells/ml (IMDM + 10% FCS/FBS). Just before the fusion wash 3 times in IMDM (serum free!), 1200 rpm, 5min, 37ѓC. Count cells. 2. sacrifice the mouse and dissect the spleen. 3. tease the spleen in ice-cold IMDM (serum free!); pass through the nylon filter; wash 3 times in IMDM (serum free!), 1200 rpm, 5min, 37ѓC. Count cells. 4. mix 10^8 spleen cells (in 25 ml of IMDM [serum free]) with 2x10^7 myeloma cells (in 25 ml of IMDM [serum free]) in Falcon-50 ml conical tube. 5. centrifuge at 1200 rpm, 5 min, 37ѓC. 6. aspirate the supernatant (SN) with a Pasteur pipette (all till the last drop!!!) 7. gently break the pellet by tapping of the bottom of the tube. 8. place the tube on 37ѓC water bath; add slowly, dop by drop 1 ml of pre-warmed (37ѓC) 50% PEG 1500 (polyethylene glycol, Roche, Germany) continously stirring the cells with the pipette tip (this procedure lasts for a period over 1 min) 9. add 1 ml of pre-warmed to 37ѓC IMDM continously stirring the mixture (this procedure lasts for a period over 1 min). 10. add 3 ml of pre-warmed to 37ѓC IMDM continously stirring the mixture (this procedure lasts for a period over 3 min). 11. add 10 ml of pre-warmed to 37ѓC IMDM continously stirring the mixture (this procedure lasts for a period over 1-2 min). 12. let to stay for 5 min on the 37ѓC water bath. 13. centrifuge at 1200 rpm, 5 min, 37ѓC; aspirate the SN. 14. resuspend the cells in IMDM-FCS(10%v/v) HTx100 (1%v/v) - AJ(5%v/v) - IL-6, 0.5x10^6 cells/ml (calculation figured out from the number of spleen cells the fusion was started from); let to stay in incubator, 37ѓC, pCO2 7% for 2 hours in a big flasc. 15. add aminopterin; distribute on 96-wells cell culture