UNIT 2.5 Production of Monoclonal Antibodies

 

Unit Introduction

Highly specific antibodies can be obtained by fusing immune B cells from the spleen with tumor cells to produce hybridomas, each of which will then secrete a single antibody. The desired antibody-producing hybridoma can be identified by a screening process. If this hybridoma is subjected to a cloning process in which clones are selected, such that all progeny are derived from a single cloned parental cell, a monoclonal antibody is obtained. Monoclonal antibodies have high specificity and can be produced in large quantities. Thus, these biological reagents have been used extensively as probes in a wide range of systems including the characterization of novel cell-surface and soluble proteins and carbohydrates, as enzyme catalysts, and for targeting in immunotherapy (see Commentary).

This unit describes the production of monoclonal antibodies beginning with protocols for immunization (Basic Protocol 1) and cell fusion and selection (Basic Protocol 2). An Alternate Protocol describes cell fusion and one-step selection and cloning of hybridomas utilizing a semisolid methylcellulose-based medium (ClonaCell-HY, a registered trademark of StemCell Technologies, Inc.; http://www.stemcell.com). Methods are provided for screening primary hybridoma supernatants for antibodies of desired specificity (Support Protocol 1), establishment of stable hybridoma lines (Support Protocol 2), cloning of these B cell lines by limiting dilution to obtain monoclonal lines (Support Protocol 3), recloning of hybridoma cells in semisolid medium (Support Protocol 4), and preparation of cloning/expansion medium (thymocyte-conditioned medium; Support Protocol 5). Figure 2.5.1 summarizes these stages and notes the protocols in this and subsequent units in which they are detailed. Selection and cloning of hybridomas using the techniques described in Basic Protocol 2 require a major commitment of time and labor. However, the Alternate Protocol reduces the overall time necessary to produce monoclonal antibodies by 18 to 20 days. When successful, the monoclonal antibody may be an extremely valuable reagent that will be available in large quantities.

Figure 2.5.1 Stages of monoclonal antibody production, with references to the Basic, Alternate, and Support Protocols in this unit (as well as subsequent units) that describe the steps.

Submission of monoclonal antibodies to the American Type Culture Collection (ATCC) for distribution to the scientific community is encouraged. Moreover, the ATCC serves as a repository for cell lines should the line be lost in the investigator's laboratory due to unforeseen circumstances.

NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must conform to governmental regulations for the care and use of laboratory animals.

NOTE: All solutions and equipment coming into contact with living cells must be sterile, and proper aseptic technique should be used accordingly.

NOTE: All culture incubations are performed in a humidified 37°C, 5% CO₂ incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO₂ to maintain pH 7.4.

 

Basic Protocol 1: Immunization to Produce Monoclonal Antibodies

A wide variety of antigen preparations have been used successfully to produce monoclonal antibodies (see Critical Parameters for discussion of antigen preparation). The following protocol provides an immunization schedule for the production of most antibodies, although several different schedules can be used. In this protocol, emulsified antigen is injected intraperitoneally into the species of choice. A booster injection is administered 10 to 14 days after the primary immunization. Three days after the booster injection, the animals' spleens are ready for cell fusion (Basic Protocol 2).

 Materials

• Antigen
• Complete Freunds adjuvant (CFA; Sigma)
• Animal: pathogen-free mouse, hamster, or rat (Armenian hamsters from Cytogen Research are recommended; see Critical Parameters for discussion of animal choice and unit 1.1)
• Incomplete Freunds adjuvant (IFA; Sigma), optional
• 1- to 2-ml glass syringes with Luer-Lok tips, sterile
• 3-way stopcock
• 20- and 22-G needles, sterile

• Additional reagents and equipment for handling and restraint of animals (unit 1.3) and intraperitoneal injection (unit 1.6)

CAUTION: CFA is an extremely potent inflammatory agent, particularly if introduced intradermally or into the eyes. Profound sloughing of skin or loss of sight may occur. Self-injection can cause a positive TB skin test and lead to a granulomatous reaction. Use gloves and protective eyewear when handling CFA.

1.	Prepare antigen using 2 × 10^6 to 5 × 10^7 cells or 1 to 50 µg protein or peptide per animal to be immunized in normal saline. p type = annotation The antigen may be in several different forms depending on the desired property of the MAb and the method of screening (see Critical Parameters for discussion of antigen preparation and screening assays). If cells are the immunogen, wash three times in serum-free medium before immunization. Plan the immunization of several animals (enough for several fusions) so that primed and boosted animals will be ready 3 days before fusion (see Basic Protocol 2). p type = annotation To minimize the risk of introducing a pathogen into the rodent colony, screen cells for pathogens by antibody-production assay (unit 1.1).
2.	Draw up antigen into a sterile 1- to 2-ml glass syringe with a Luer-Lok tip. Connect syringe to a 3-way stopcock.
3.	Completely resuspend CFA to disperse the Mycobacterium tuberculosis bacilli, which settle to the bottom of the container with time. Draw up a volume of CFA equal to the antigen volume in a syringe and connect to the antigen-containing syringe.
4.	Emulsify antigen and CFA by discharging antigen into CFA, then discharging back and forth until a thickened mixture results. Test whether the emulsion is stable—a stable emulsion will not disperse when a drop of it is placed in water. p type = annotation See unit 2.4 for further discussion of immunization. Figure 2.4.1 illustrates the double-syringe device.
5.	Transfer all of the CFA/antigen emulsion to one syringe and remove the other syringe and stopcock. Attach a sterile 20-G needle to the syringe containing the emulsion.
6.	Inject emulsion intraperitoneally into the animal using <0.2 ml/mouse, 0.5 to 1 ml/rat, or 0.2 to 0.4 ml/hamster. p type = annotation Be careful not to force the syringe plunger, since excessive pressure may dislodge the needle and spray the emulsion. Introduce the needle through the skin and tunnel the needle between the skin and peritoneal wall before entering the peritoneal cavity at a site distant from the dermal puncture site. Twirl needle before withdrawal to minimize leakage. p type = annotation Rats are generally anesthetized (unit 1.4) whereas mice and hamsters can be manipulated with one hand and do not require anesthetic.
7.	Boost animal after 10 to 14 days with approximately the same dose of antigen as in step 5. If cell fusion is planned for 3 days after boosting, immunize with antigen alone in aqueous solution, or intact cells in suspension. If a fusion is not immediately planned, boost the animal with antigen emulsified in IFA (which does not contain Mycobacterium tuberculosis bacilli). p type = annotation Do not use CFA for the booster immunizations as this will cause intense inflammation and increased anti-TB antibody response. p type = annotation Blood can be collected from a tail bleed (unit 1.7) after the first intraperitoneal boost to obtain antibodies from the blood as a positive control in subsequent detection assays. p type = annotation If desired, antibody titers can be assayed by ELISA (unit 2.1) or immunoprecipitation (unit 8.3), 7 to 10 days after the primary and booster immunizations.

 

Basic Protocol 2: Cell Fusion and Selection of Hybridomas

While animals should be immunized as soon as the decision has been made to produce a monoclonal antibody and the antigen prepared, do not perform cell fusion until the screening assay (Support Protocol 1) has been perfected. Artifactual results that may arise from conditioned media must be identified before cell fusion, because after a fusion there is only a finite amount of time available to assay for the desired monoclonal antibody.

Prior to cell fusion, the partner (myeloma) cell line is expanded and a booster injection of antigen is administered to the primed animals. On the day of fusion, the spleens are harvested. Spleen cells and partner cells are washed, harvested, and mixed. Cell fusion is performed at 37°C in the presence of polyethylene glycol (PEG). The resulting pellet is harvested and plated into tissue culture plates. After incubation with hypoxanthine, aminopterin, and thymidine (HAT) medium and feeding over ~2 weeks, the hybridomas are ready for screening (Support Protocol 1).

 Materials

• SP2/0-Ag14 myeloma cell line (drug-marked, nonsecretory; ATCC #CRL 1581)
• Complete DMEM-10 and -20 media (appendix 2A) with 10 mM HEPES and 1 mM sodium pyruvate
• Primed animal; mouse, hamster, or rat (10 to 14 days after primary immunization; (see Basic Protocol 1)
• Complete DMEM medium (appendix 2A), serum-free
• 50% polyethylene glycol (PEG), sterile
• Ammonium chloride solution (see recipe)
• Complete DMEM-20/HEPES/pyruvate/HAT (or HT) medium (see recipe)

• 175-cm² flasks
• Fine-mesh metal screen
• 50-ml conical polypropylene centrifuge tubes
• Beckman TH-4 rotor or equivalent
• 96-well flat-bottom microtiter plates

• Additional reagents and equipment for animal euthanasia (unit 1.8), spleen removal (unit 1.10), and counting cells and assessing cell viability by trypan blue exclusion (appendix 3B)

 Prepare myeloma cells (1 week before fusion)

1.

One week before fusion, begin expansion of SP2/0-Ag14 myeloma cell line (the fusion partner cell line) in complete MEM-10/HEPES/pyruvate (see Critical Parameters). By the day cell fusion is to be performed, the following total number of myeloma cells must be available (in multiple 175-cm^2 flasks containing 100 ml each), depending upon the source of the primed animal: mouse spleen, 1 × 10^8 cells in two or three flasks; hamster spleen, 2 × 10^8 cells in three or four flasks; and rat spleen, 5-10 × 10^8 cells in ten flasks. p type = annotation Two mouse or hamster spleens, or one rat spleen, will provide enough cells for the fusion (see step 7).

 Boost primed animal (3 days before fusion)

2.	Three days before fusion, boost primed animal(s) according to step 7 of Basic Protocol 1.

 Prepare reagents and split myeloma cells (1 day before fusion)

3.	One day before fusion, prepare all reagents and media, particularly 50% PEG.
4.	One day before fusion, split SP2/0-Ag14 myeloma cells (from step 1) into fresh complete DMEM-10/HEPES/pyruvate medium. p type = annotation Vigorous growth of the SP2/0-Ag14 cells is generally required for good fusion.

 Check myeloma cells and prewarm reagents (day of fusion)

5.

Use an inverted microscope to check the SP2/0-Ag14 myeloma cells to make sure they are growing vigorously (refractile and not pyknotic), they are not contaminated (no obvious bacteria or fungi), and there are enough cells for the fusion. p type = annotation It is better to postpone the fusion than to perform an ill-advised fusion, since the entire selection and screening effort will take ~3 weeks.

6.

Prewarm the following in a 37°C water bath: Three 400- and three 600-ml beakers, each containing ~100 ml H2O 20 ml sterile complete serum-free DMEM 5 ml sterile 50% PEG solution.

 Harvest spleen and prepare cells

7.	Sacrifice boosted animal(s) (unit 1.8) and aseptically harvest spleen(s) (unit 1.10). p type = annotation Do not use anesthetics for sacrifice. Instead, use cervical dislocation for mouse, or CO2 asphyxiation for mouse, hamster, or rat to avoid introducing an anesthetic into the bloodstream and therefore into the cultures.
8.	Transfer spleen to a sterile 100-mm-diameter petri dish filled with 10 ml sterile complete serum-free DMEM. p type = annotation Perform all subsequent steps in a laminar flow hood.
9.	Tease spleen into a single-cell suspension by squeezing with angled forceps or by chopping with fine-tipped dissecting scissors. Remove debris and disperse cells further by passage through a fine-mesh metal screen.
10.	Transfer spleen cell suspension to a sterile 50-ml conical centrifuge tube and fill with sterile complete serum-free DMEM. p type = annotation Do not use protein- or HEPES-containing medium because the PEG will precipitate proteins and HEPES can be toxic to cells during fusion.
11.	Centrifuge 5 min in TH-4 rotor at 1500 rpm (500 × g), room temperature, and discard supernatant.
12.	Lyse red blood cells (RBC) by resuspending pellet in 5 ml ammonium chloride solution. Let stand 5 min at room temperature.
13.	Add 45 ml sterile complete serum-free DMEM, and centrifuge as in step 11.
14.	Resuspend pellet in 50 ml sterile complete serum-free DMEM. Centrifuge as in step 11. Repeat DMEM addition and centrifuging once (each repeat is a wash).
15.	While spleen cells are being washed, separately harvest the SP2/0-Ag14 myeloma cells (from step 5) by transferring the cells to 50-ml conical centrifuge tubes. Centrifuge as in step 11. Resuspend myeloma cells in DMEM and pool all cells into one 50-ml conical centrifuge tube. Wash myeloma cells three times as in step 14.
16.	Separately resuspend the spleen and myeloma cells in 10 ml complete serum-free DMEM. Count cells and assess viability in each cell suspension using a hemacytometer and trypan blue exclusion (appendix 3B); there should be nearly 100% viability of both suspensions.
17.	On basis of cell counts (from step 16), calculate the amount of complete DMEM-20/HEPES/pyruvate needed to plate cells at ~2.5 × 10^6 total cells/ml. Prewarm this amount of complete DMEM-20/HEPES/pyruvate in 37°C water bath. Prepare 96-well flat-bottom plates by labeling them sequentially: one plate is required for each 10 ml of final cell suspension.

 Perform cell fusion

18.	Mix SP2/0-Ag14 myeloma and spleen cells at a 1:1 ratio in a 50-ml conical centrifuge tube. Fill the tube with complete serum-free DMEM. p type = annotation Other cell ratios work. Successful fusions have been performed with a ratio of myeloma/spleen cells as low as 1:20.
19.	Centrifuge cell mixture 5 min at 500 × g, room temperature.
20.	While cells are in the centrifuge, prepare three 37°C double-beaker water baths in the laminar flow hood by placing a 400-ml beaker (from step 6) containing 100 ml of 37°C water into 600-ml beaker containing 75 to 100 ml of 37°C water. Place the tubes of prewarmed 50% PEG solution and complete serum-free DMEM (from step 6) into two of the 37°C water baths in the hood.
21.	Aspirate and discard supernatant from the mixed-cell pellet (from step 19).
22.	Perform the cell fusion at 37°C by placing the tube containing the mixed-cell pellet in one of the double-beaker water baths in the laminar flow hood.
23.	Using a 1-ml pipet, add 1 ml prewarmed 50% PEG to the mixed-cell pellet drop-by-drop over 1 min, stirring the cells with the pipet tip after each drop. Stir for an additional minute.
24.	Using a clean pipet, add 1 ml prewarmed complete serum-free DMEM to the cell mixture drop-by-drop over 1 min, stirring after each drop. Repeat once with an additional 1 ml of prewarmed complete serum-free DMEM.
25.	With a 10-ml pipet, add 7 ml prewarmed complete serum-free DMEM drop-by-drop over 2 to 3 min. p type = annotation Macroscopic clumps of cells should be obvious at this point.
26.	Centrifuge 5 min at 500 × g, room temperature.
27.	While the cells are in the centrifuge, rewarm the beaker water baths to 37°C and place in the hood. Place prewarmed complete DMEM-20/HEPES/pyruvate (from step 17) in the beaker water bath.
28.	Discard the supernatant (from step 26). Place tube in the beaker water bath.
29.	With a clean 10-ml pipet, forcefully discharge 10 ml prewarmed complete DMEM-20/HEPES/pyruvate to the cell pellet.
30.	Repeat step 29 until the total volume of prewarmed complete DMEM-20/HEPES (calculated in step 17) is added. If necessary, allow clumps to settle and disrupt with the pipet tip. Further warming of cell suspension is no longer required. p type = annotation If the total volume exceeds 50 ml, gently aspirate and transfer to another sterile container such as a tissue culture flask.
31.	Gently aspirate 10 ml of cell suspension with a 10-ml pipet. Add 2 drops (100 to 125 µl) of suspension to each well of a 96-well flat-bottom plate (continue until entire suspension is plated). Incubate overnight in a humidified 37°C, 5% CO2 incubator. p type = annotation Vigorous pipetting of the cell suspension should be avoided at this point, as the newly formed hybrids are unstable. Moreover, the vigorous addition of cells to the wells with repeating micropipettor is not advised. Use a pipet aid and hold the 10-ml pipet at a 45° angle with the tip 1 to 2 cm above the well, bracing the pipet with a finger from the opposite hand. To avoid introducing contaminants, do not hold hands above the plate. A steady, even flow of drops from the pipet will allow the most efficient delivery of cell suspension or medium to the wells. Use a fresh pipet to withdraw additional cell suspension. p type = annotation As an optional step to minimize fibroblast overgrowth, permit the fibroblasts in bulk-fused cell suspension to adhere overnight to tissue culture flasks before seeding the 96-well plates. p type = annotation Many investigators select their hybridomas under bulk conditions—i.e., they incubate large numbers of cells per well in larger plates or flasks. This makes feeding easier, but allows fast-growing hybridomas to overgrow the others. Since nonproducing hybridomas tend to grow faster, especially in the hamster-mouse fusions, hybridomas are isolated initially in multiple small wells in this protocol. The primary hybridomas tend to be monoclonal. This is especially important when screening procedures are used that require differential reactivities, e.g., to different cell lines by flow cytometry analysis or to different antigen preparations. In those cases, multiple hybridomas per well will obscure the reactivity of the MAb of interest.

 Monitor and feed cells

32.	After one day of incubation, check wells under an inverted microscope. If seeded with the appropriate number of cells, there should be a nearly confluent monolayer of highly viable cells on the bottom and obvious clumps of cells.
33.	Add 2 drops complete DMEM-20/HEPES/pyruvate/HAT to each well with a 10-ml pipet (see step 31). Place in humidified 37°C, 5% CO2 incubator. p type = annotation Use a separate pipet for each microtiter plate and keep the same covers with each plate to ensure that each plate remains a separate unit and to avoid spreading contamination. It cannot be overemphasized that it takes practice and meticulous attention to possible sources of contaminants to keep these plates sterile during the subsequent 2-to 3-week feeding and monitoring schedule. p type = annotation If plates become contaminated, discarding them is advised. Alternatively, contamination in one or two wells may be treated by aspirating the contents of the contaminated well with a sterile Pasteur pipet attached to a vacuum flask, rinsing the well with 70% ethanol, and wiping with a sterile cotton swab. Wash twice with ethanol. Finally, blot the well dry with the sterile cotton swab and blot the appropriate area of the cover with a sterile cotton swab soaked in 70% ethanol. Do not open contaminated plates while other plates are in the hood.
34.	On days 2, 3, 4, 5, 7, 9, and 11, aspirate half the volume of each well using a sterile, short Pasteur pipet attached to a vacuum flask, holding pipet at a 45° angle and touching tip to surface of supernatant at the point where the liquid meets the opposite wall of the well. Feed the cells by adding 2 drops complete DMEM-20/HEPES/pyruvate/HAT from a 10-ml pipet (see steps 31 and 33) to each well, and return to humidified 37°C, 5% CO2 incubator. p type = annotation Use a separate Pasteur pipet for each plate to minimize spreading contamination. Since the frequency of successful viable hybridoma formation is £10^–5, when HAT is added, profound cell death should be apparent at days 2 and 3 and the remaining viable cells should not be readily apparent until they have expanded. By day 7 to 9 for mouse-mouse fusions, day 11 for rat-mouse fusions, and day 14 for hamster-mouse fusions, clusters of hybridoma cells should become visible under the inverted microscope. If profound cell death is not apparent on days 2 and 3, check the medium containing HAT and the parental cell line by incubating an aliquot of the parental myeloma line with the medium containing HAT. p type = annotation The feeding schedule is not rigid except for the first 4 days, when it is necessary to remove the toxic products of cell death. Thereafter, feedings will depend on the actual number of cells deposited in the wells, efficiency of fusion, and appearance and growth of hybridomas. Do not allow wells to become yellow (acidic) for more than a day. Examine plates daily, even if cells are not scheduled to be fed, and feed plates if acidic wells are noted.
35.	On day 14, repeat feeding as outlined in step 34 except use complete DMEM-20/HEPES/pyruvate/HT to feed cells. Return to 37°C, 5% CO2 incubator. p type = annotation Cells do not require more than one change of complete DMEM-20/HEPES/pyruvate/HT. After this change, the aminopterin (from prior addition of HAT medium) is apparently diluted out enough so that the cells can survive without additional HT.
36.	On day 15 and subsequently, feed wells as noted using complete DMEM-20/HEPES/pyruvate without HAT or HT. The hybridomas are ready for screening when most of the wells containing growing cells demonstrate 10% to 25% confluence and when those with denser populations turn yellow within 2 days after feeding (see Support Protocol 1). p type = annotation If the screening assay requires a [^3H]thymidine incorporation assay (appendix 3D), be aware that the large amount of thymidine in complete DMEM-20/HEPES/pyruvate with HAT and HT will serve as a cold-label inhibitor of [^3H]thymidine incorporation. At least 3 to 4 changes of complete DMEM-20/HEPES/pyruvate without HT are required to dilute out excess thymidine.

 

Alternate Protocol: Cell Fusion, Selection, and Cloning of Hybridomas Using a Semisolid Medium (Clonacell-HY)

Traditionally, monoclonal antibody development has involved selecting hybridomas in suspension cultures, identifying cultures that produce antibodies specific for the targeted antigen, and cloning the specific antibody-producing hybridoma(s) by at least one round of culture under limiting-dilution conditions (as described in Basic Protocol 2 and Support Protocols 1 to 3). This approach is laborious and time consuming and may result in the selection of identical, duplicate clones. This section describes protocols for the use of a methylcellulose-based medium system, ClonaCell-HY, for cloning and selection of mouse hybridomas. Performing hybridoma selection and cloning simultaneously in ClonaCell-HY reduces the time and reagents necessary to obtain a monoclonal hybridoma producing antibody against the antigen of interest. Cultures do not need feeding or maintenance during the selection process. This approach also allows all daughter cells to remain together during the selection process, decreasing the number of clones that need to be tested for antibody production. Selection of duplicate hybridomas, a common occurrence with hybridoma cloning in suspension cultures, is avoided. An additional advantage is that smaller, slow-growing clones, which can easily be lost due to overgrowth by larger, faster-growing hybridomas in traditional liquid suspension culture, remain physically separated in semisolid medium from the larger, faster-growing clones, and can thus be isolated and screened separately.

NOTE: All solutions and media should be prewarmed to 37°C unless otherwise indicated.

 Materials

• Myeloma cell line (e.g., SP2/0, X63Ag8.653; available from ATCC)
• ClonaCell-HY Monoclonal Antibody Production Kit (StemCell Technologies, Inc.) containing:
  • Medium A—ClonaCell-HY Pre-Fusion Medium and Hybridoma Expansion Medium, 500 ml
  • Medium B—ClonaCell-HY Fusion Medium, 500 ml
  • Medium C—ClonaCell-HY Hybridoma Recovery Medium, 100 ml
  • Medium D—ClonaCell-HY Hybridoma Selection Medium containing HAT, 90 ml
  • Medium E—ClonaCell-HY Hybridoma Growth Medium containing HT, 500 ml
  • Polyethylene glycol—ClonaCell-HY PEG Solution, pretested for cell fusion, 1.5 ml
• Immunized mouse, 1 to 4 days after final antigen boost (Basic Protocol 1)
• 3% (v/v) acetic acid
• Liquid nitrogen (optional)
• Fetal bovine serum (FBS) containing 20% (v/v) DMSO

• 15- and 50-ml conical polypropylene centrifuge tubes
• 100-mm petri dishes
• Fine-mesh metal screen
• Low-speed tabletop centrifuge
• 3-ml and 12-ml syringes
• 25- and 75-cm² tissue culture flasks
• 16-G blunt-ended hypodermic needles
• 96- and 24-well tissue culture plates
• Cryotubes (e.g., Nunc)
• Liquid nitrogen freezer (Dewar flask and canes to accommodate cryotubes; optional)

• Additional reagents and equipment for determining cell viability by trypan blue exclusion (appendix 3B), animal euthanasia (unit 1.8), spleen removal (unit 1.10), preparing a single-cell suspension of splenocytes (unit 3.1), counting cells using a hemacytometer (appendix 3), assaying for antigen production from hybridoma clones by ELISA (unit 2.1), flow cytometry (Chapter 5), or immunoblotting (unit 8.10), and cryopreservation of cells (appendix 3G)

 Prepare myeloma cells

1.	Culture the parental myeloma cells in 25-cm^2 tissue culture flasks with Medium A (Pre-Fusion Medium from ClonaCell-HY kit) for at least 1 week prior to fusion to ensure they are well adapted to this medium. Seed cells at a density of ~5 × 10^4 cells/ml and passage every 2 days. Suggested maximum cell density is 4 × 10^5 cells/ml. p type = annotation The parental myeloma cells must not secrete any of their own immunoglobulin chains. They should be mycoplasma-free and efficiently fuse to form stable hybridomas that continuously secrete specific monoclonal antibodies. Parental myeloma cells that meet these criteria (such as SP2/0 and X63Ag8.653) are widely available.
2.	Calculate the cell growth rate at every passage (appendix 3A). The day before the fusion, count the viable cells and split cells so that there will be at least 2 × 10^7 parental myeloma cells available the next day. p type = annotation The recommended cell density for fusion is 2 × 10^5 cells/ml. Only 100 ml of these cells will be needed, but 200 ml should be cultured to ensure an adequate supply.
3.	Harvest the required number of parental myeloma cells in a 50-ml conical polypropylene centrifuge tube. Centrifuge 10 min at 300 × g, room temperature or 37°C, and remove the supernatant. Wash three times, each time by adding 30 ml of Medium B (Fusion Medium), centrifuging again as before, and removing the supernatant. Resuspend final pellet in 25 ml Medium B. p type = annotation This step may be performed simultaneously with or subsequent to spleen cell preparation (steps 6 to 10) to ensure that the myeloma cells do not sit for an extended period of time. p type = annotation It is important to remove all the serum adhering to the cells by washing with serum-free Medium B. If the serum is not removed, the PEG will not fuse the cell membranes and the fusion frequency will drop drastically.
4.	Count live cells using a viability stain (appendix 3B). p type = annotation Viability of parental myeloma cells should be >95%.
5.	Calculate the volume of cell suspension that contains 2 × 10^7 cells, to be used in step 11. Place cells at room temperature or 37°C.

 Harvest spleen and prepare spleen cells

6.	Sacrifice immunized animal(s) (unit 1.8) and aseptically remove spleen(s) (units 1.8 & 1.10). Place spleen in a sterile 100-mm petri dish containing 5 ml Medium A (Pre-Fusion Medium). p type = annotation IMPORTANT NOTE: The ClonaCell-HY kit has been optimized for use with mouse hybridomas. Hybridomas from other species have not been tested. p type = annotation Do not use anesthetics for sacrifice. Instead, use cervical dislocation or CO2 asphyxiation to avoid introducing anesthetic into the bloodstream and therefore into the cultures. p type = annotation It is important to collect blood from the animal to obtain serum as a source of antibodies for a positive control in subsequent screening assays. Blood can be collected from a tail bleed (unit 1.7) after the first intraperitoneal boost (see Basic Protocol 1, step 7) or from the heart at the time of spleen harvest. To collect blood from heart use a sterile Pasteur pipet and place blood into a 1.5-ml microcentrifuge tube. Let blood stand at room temperature with the cap off to allow blood to clot. After 30 min, remove the blood clot with a sterile Pasteur pipet and place tube at 4°C. The next day, centrifuge tube for 15 min at 400 × g, 4°C or room temperature. Collect serum and add sodium azide to a final concentration of 0.1%. Store at –20°C
7.	Disaggregate the spleen into a single-cell suspension (unit 3.1). Transfer the spleen to a fine-mesh metal screen placed on top of a 50-ml conical centrifuge tube, and use the plunger of a 3-m1 syringe to gently grind the cells out of the spleen. Rinse the screen with Medium B to help cells pass through the screen. Pipet the cells up and down in the tube with a 10-ml pipet to break up lumps. Try not to cause the solution to foam. p type = annotation Only the spleen membrane should be left in the screen. See unit 3.1 for additional discussion of the above procedure. Other spleen disaggregation methods may also be used.
8.	Centrifuge cell suspension 10 min at 400 × g, room temperature or 37°C, and remove supernatant. Wash the splenocytes three times, each time by adding 30 ml Medium B, centrifuging 10 min at 400 × g, room temperature or 37°C, and discarding the supernatant. Resuspend the final cell pellet in 25 ml Medium B. p type = annotation It is important to remove all the serum adhering to the cells by washing with serum-free Medium B. If the serum is not removed, PEG will not fuse the cell membranes and the fusion frequency will drop drastically.
9.	Prepare a 1/10 dilution of cells in 3% acetic acid, e.g., by mixing 10 µl of cell suspension with 90 µl of 3% acetic acid. Count cells in this diluted sample using a hemacytometer (see appendix 3A).
10.	Calculate the volume of cell suspension that contains 1 × 10^8 cells, to be used in step 11. Place cells at room temperature or 37°C until fusion.

 Combine myeloma cells and splenocytes for fusion

11.

Add 2 × 10^7 parental myeloma cells and 1 × 10^8 viable splenocytes (as calculated in steps 5 and 10, respectively) to a 50-ml conical centrifuge tube and centrifuge 10 min at 400 × g, room temperature or 37°C. Aspirate supernatant. p type = annotation Complete removal of the supernatant is essential to avoid dilution of PEG in the next step.

 Perform cell fusion by one of the two following methods

 Method A

12a.	Break up the cell pellet obtained in step 11 gently by tapping the bottom of the tube. Add 0.5 ml of polyethylene glycol (PEG) solution from the ClonaCell-HY kit dropwise to the pellet using a 1-ml pipet. Centrifuge the mixture 3 min at 133 × g, 37°C or room temperature (37°C is preferable for this centrifugation but not essential), then aspirate off all of the PEG (during this procedure, not all cells will pellet; some will clump in the PEG; do not aspirate the clumped cells). Work quickly since cells must not be exposed to PEG for too long.
13a.	Carefully add 5 ml of Medium B dropwise to the pellet while gently swirling the tube to resuspend the cells.
14a.	Slowly add 5 ml Medium C (Hybridoma Recovery Medium from the kit) to the solution. Continue to swirl the tube.
15a.	Transfer the cell suspension to a 75-cm^2tissue culture flask containing 40 ml Medium C (total culture volume will be 50 ml). Incubate 16 to 24 hr at 37°C in 5% CO2 atmosphere. p type = annotation There will still be clumps of cells at this point, which will disaggregate overnight. Be gentle; fused cells are very fragile immediately after fusion.

 Method B

12b.	Slowly add 1 ml polyethylene glycol (PEG) solution from the ClonaCell-HY kit to the pellet obtained in step 11 dropwise over a period of 1 min, without stirring, using a 1 ml pipet. When all of the PEG has been added gently stir the cells continuously with the pipet tip for 1 min.
13b.	Add 4 ml medium B to the fusion mixture dropwise over a period of 1 min, continuously stirring with the pipet tip for a total of 4 min. Slowly add 10 ml Medium B. Incubate 15 min in a water bath at 37°C.
14b.	Slowly add 30 ml of Medium A over a period of 1 min. Centrifuge the cells 7 min at 400 × g, 37°C or room temperature. Discard the supernatant, add 40 ml Medium A, then centrifuge again as before and discard the supernatant to ensure all PEG is removed.
15b.	Slowly resuspend the cell pellet in 10 ml Medium C. Transfer the cell suspension to a 75-cm^2 tissue culture flask containing 40 ml Medium C (total culture volume, 50 ml). Incubate 16 to 24 hr at 37°C in 5% CO2 atmosphere. p type = annotation IMPORTANT NOTE: Incubating cells up to 24 hr in liquid medium allows fused cells time to go through one cell cycle and begin expressing the enzyme HPRT, which is necessary for cell survival during HAT selection. Freshly fused cells are also very fragile. Waiting a day before mixing these cells with methylcellulose (see below) will improve their survival.

 Select and clone hybridoma cells

16.	On the day of the fusion, place the frozen Medium D from the ClonaCell-HY kit (Hybridoma Selection Medium containing HAT) at 4°C and leave overnight to thaw. Before using, shake vigorously to thoroughly mix contents and leave on bench to warm up to room temperature. p type = annotation Do not thaw Medium D in a 37°C water bath, as the methylcellulose can be pulled out of solution causing the medium to become lumpy.
17.	Transfer the fused cell suspension (step 15a or b) into a 50-ml conical centrifuge tube and centrifuge 10 min at 400 × g, room temperature or 37°C. Remove supernatant. Resuspend cells in Medium C for a total volume of 10 ml. p type = annotation It is critical that the total volume in this step not be greater than 10 ml. Include any additional cytokines or growth factors in this 10 ml volume prior to adding to Medium D.
18.	Transfer the 10 ml of cell suspension into the 90 ml of Medium D. Mix thoroughly by gently inverting the bottle. Once mixed, let sit for 15 min at room temperature or 37°C to allow the bubbles to rise to the top. p type = annotation It is not advisable to warm the bottle in a 37°C water bath. Instead, place the bottle in a 37°C incubator.
19.	Using a 12-ml syringe and 16-G blunt-end needle, aseptically plate out 9.5 ml of the cell suspension from step 15a into each of ten 100-mm petri dishes. Tilt the plates to level the mixture and try not to introduce air bubbles. Incubate dishes at 37°C in a 5% CO2 atmosphere. Do not disturb plates for 10 to 14 days. p type = annotation Methylcellulose (present in Medium D) is a viscous solution and cannot be accurately dispensed using pipets due to adherence of the medium to pipet walls. p type = annotation The incubator must be well humidified. It is advisable to put the plates in a separate plastic container together with an open 100-mm petri dish containing 10 ml sterile distilled water. Open and close the incubator door carefully to avoid shaking. p type = annotation IMPORTANT NOTE: Handling plates prior to the recommended 10- to 14-day incubation time may result in “runny” colonies. Identical daughter cells may break apart and spread across the plate, rather than remain in a tight colony. This will make it more difficult to identify single colonies to pick for cloning.

 Harvest clones

20.	At a time point 10 to 14 days after plating, examine the plates for the presence of colonies visible to the naked eye (a typical fusion will produce 1000 or more colonies over the ten plates). p type = annotation Recloning in semisolid medium (Support Protocol 4) is recommended if colonies are not well distributed in the semisolid Medium D at this step, or if it is difficult to distinguish between individual colonies. This should be done after cells have been harvested and established in Medium E (see subsequent steps).
21.	Remove isolated colonies (usually 500 to 1000 colonies are picked) from the plates with sterile pipet tips using a micropipettor set to 10 µl. Pipet each clone into an individual well of a 96-well tissue culture plate containing 200 µl Medium E (Growth Medium containing HT). Incubate the plates at 37°C in a 5% CO2 atmosphere 1 to 4 days without feeding. p type = annotation Usually by the fourth day, each well has a high cell density and the medium has begun to turn yellow. p type = annotation It is a good idea to pick clones of different sizes, as slower-growing clones (i.e., smaller colonies) are often very good antibody producers. Such slow-growing hybridomas are often missed in other hybridoma screening procedures.
22.	Transfer 150 µl of the supernatant from each hybridoma well to a separate well on a new 96-well plate and analyze by an assay system appropriate for the antigen involved; e.g., ELISA (unit 2.1), flow cytometry (Chapter 5), or immunoblotting (unit 8.10).
23.	Add 150 µl of fresh Medium E to each hybridoma-containing well of the original 96-well plate.
24.	Gently resuspend the hybridomas that showed a positive response in step 22. Transfer 100 µl of the resuspended positive hybridoma to each of two wells of a 24-well plate containing 1 ml of Medium E.
25.	When cells have grown to a suitable density (~4 × 10^5 cells/ml), freeze the cells from one well by centrifuging the culture at 400 × g, room temperature, remove the medium using a pipet and add ice-cold FBS/20% DMSO for a concentration of 4 × 10^5 cells/ml. Transfer cells and medium to a cryotube, and place at –70°C, –135°C, or in liquid nitrogen (also see appendix 3G). Expand the remaining positive clones (duplicate, identical wells) in 25-cm^2 tissue culture flasks containing 5 ml of Medium A and 5 ml of Medium B. p type = annotation Individual hybridomas may differ in their optimal cell density for growth. The culture should not be too sparse, or there will be insufficient numbers for recovery of frozen cells. If the culture is allowed to grow to too high a density, the medium will become acidic and cell viability may decrease. Suggested density is 5 × 10^4 cells/ml, with a maximum density of 4 × 10^5 cells/ml.
26.	Keep a sample of cells in Medium E, in case they do not adapt well to the mixed medium.
27.	When cells have grown to a suitable density (~4 × 10^5 cells/ml), pipet 5 to 10 ml of the cell culture into 30 ml Medium A. Adjust the volume to ensure that the final cell concentration is 1–4 × 10^4 cells/ml.
28.	Maintain expanded hybridomas in 100% Medium A at a concentration of 5 × 10^4 to 5 × 10^5 cells/ml. p type = annotation More aliquots of cells can be frozen at this point in order to secure the supply of hybridoma. p type = annotation Recloning in semisolid medium (Support Protocol 4) is recommended if hybridomas have been in continuous culture for a long period of time and are secreting antibody, but monoclonality or stability of the hybridoma needs to be confirmed.

 

Support Protocol 1: Screening Primary Hybridoma Supernatants

The vast majority of wells will not contain the desired antibody or may contain nonproducing hybridomas. The purpose of screening is to discover which wells (<1% to 5%) contain hybridomas that secrete the antibody of desired specificity (note that the antibody is not yet monoclonal.) Screening should be performed when most of the growing wells demonstrate 10% to 25% confluence when viewed with an inverted microscope or when some of the denser wells begin to turn yellow within 2 days after feeding. This point may be reached 10 to 14 days after a mouse-mouse or rat-mouse fusion, and 14 to 21 days after a hamster-mouse fusion. Although the most dense wells can be screened first and the less dense wells when they become more dense, this duplicates the effort required and is not recommended. Thus, the wells are fed and 2 days later, aliquots of the supernatants are tested in the screening assay for the presence of the desired antibody.

 Additional Materials (also see Basic Protocol 2)

• Growing hybridomas (Basic Protocol 2)
• Additional reagents and equipment for ELISA (unit 2.1) and indirect immunofluorescence (unit 5.3)

1.	Estimate the number of wells with growing hybridomas using an inverted microscope. Determine whether it will be more efficient to screen all wells or only the wells that contain hybridomas.
2.	Allow hybridomas in the viable wells to grow in a humidified 37°C, 5% CO2 incubator without feeding for ³2 days. p type = annotation This is usually enough time to build a saturating titer of antibody in the culture supernatant.
3.	Remove 100 µl from each well to be tested and use in a screening assay, such as an ELISA or indirect immunofluorescence (see Critical Parameters). p type = annotation A micropipettor with disposable sterile tips is convenient. Use a new pipet tip with each well. If all wells are screened, a multichannel pipet is convenient for transfer to another 96-well assay plate. p type = annotation Keep track of the plate number and well by its row letter and column number for each sample. This is frequently the origin of the MAb's name.
4.	After harvesting one entire plate, feed wells with fresh complete DMEM-20/HEPES before harvesting the next plate (see step 33 of Basic Protocol 2).

 

Support Protocol 2: Establishment of Hybridoma Lines

Once candidate hybridomas are identified (see Support Protocol 1), they are expanded and fed, then the cells are both frozen and cloned either by limiting dilution (see Support Protocol 3) or with semisolid cloning medium (see Support Protocol 4). Unfortunately, this must be done for all candidate lines before the monoclonal antibody specificity is fully characterized. This additional work ensures that viable antibody-producing hybridomas are available after the screening process. To limit the amount of extra work involved, select the twenty best candidate wells. All twenty should be frozen and limiting-dilution plates set up while the supernatants are checked.

 Additional Materials (also see Basic Protocol 2)

• Growing hybridomas (Basic Protocol 2 or Alternate Protocol)
• Cloning/expansion medium (Support Protocol 5)
• 24-well microtiter plates

• Additional reagents and equipment for cryopreservation of cells (appendix 3G)

1.	When the growing hybridoma is 25% to 50% confluent in the 96-well plate (master well), expand the candidate hybridoma to a well in a 24-well plate by resuspending the cells in the master well with a sterile pipet (a micropipettor set at 100 µl is convenient). Transfer the entire contents of the master well to a well in the 24-well plate. p type = annotation Sufficient numbers of cells will remain to serve as backup to the expanded cells. p type = annotation If a small number of cells are transferred, they may not expand. However, if there are fibroblasts in the master well, the hybridoma cells should be transferred as soon as possible to avoid fibroblast overgrowth in the well (if necessary, transfer to another 96-well plate). When transferring cells from a well with fibroblasts, be careful not to scrape the bottom of the well (which will loosen the fibroblasts).
2.	Feed cells in the master well with 3 drops complete DMEM-20/HEPES/pyruvate from a 1-ml pipet (see step 33 of Basic Protocol 2). Incubate cells in humidified 37°C, 5% CO2 incubator.
3.	Use a fresh pipet and feed cells in 24-well plate with 1 to 1.5 ml cloning/expansion medium. Incubate 2 to 3 days in humidified 37°C, 5% CO2 incubator. p type = annotation Remember that there are only two wells in the world that contain this hybridoma and that each is a backup to the other in case of contamination or other undesirable circumstance. Therefore, once established, each is treated as an individual entity and fed with media from different bottles and different pipets.
4.	When cells in the 24-well plate are 25% to 50% confluent (2 to 3 days), they are ready to be used in Support Protocol 3 (cloning by limiting dilution). p type = annotation Repeat steps 1 to 3 as necessary when cells are 25% to 50% confluent in master well.
5.	After taking cells for limiting-dilution cloning, transfer remainder of cells in the 24-well plate to a 4-ml sterile capped tube and feed the cells in the 24-well plate with complete DMEM-20/HEPES/pyruvate.
6.	Centrifuge the cells in the 4-ml tube 5 min at 500 × g, room temperature. Keep supernatant for further characterization of the antibody and as a control, and freeze cell pellet (appendix 3G). p type = annotation The supernatant usually contains sufficient antibody that could be reassayed in the original screening test and/or in any confirmatory test. The cell line is not established until stable clones can be identified, frozen, and successfully thawed. If the limiting-dilution plate does not yield an antibody-producing line, the original cells from the 24-well plate can be thawed, seeded back into a 24-well plate, grown overnight, used to seed another series of limiting-dilution plates, and refrozen for safekeeping.

 

Support Protocol 3: Cloning by Limiting Dilution

Monoclonal antibodies are secreted by the progeny of a single cell that can produce only a single antibody (assuming a nonsecretory fusion-partner line). Cloning is required to ensure that the problems of polyspecificity are avoided and the risk of overgrowth by nonproducing cells minimized. Although cloning can also be performed by the soft-agar technique, clones derived by this technique must be adapted to liquid culture before the supernatants can be tested (Coffino et al., 1972). Since cloning by limiting dilution allows direct testing of the supernatants, this method is advantageous. Cloning in semisolid medium is described in the Alternate Protocol and Support Protocol 4.

 Additional Materials (also see Basic Protocol 2)

• Candidate hybridoma line (Support Protocol 2)

1.	Resuspend the candidate hybridoma line from step 4 of the Support Protocol 2 in their wells and count and assess viability of a small aliquot (50 µl) of cells using a hemacytometer and trypan blue (appendix 3B).
2.	Prepare 10 ml of cells at 50 viable cells/ml and 10 ml at 5 viable cells/ml in cloning/expansion medium. p type = annotation The degree of dilution is usually very large and thus, serial dilution may be needed.
3.	Seed a 96-well plate with the cell suspensions at 200 µl/well. Incubate 7 to 10 days in a humidified 37°C, 5% CO2 incubator. p type = annotation There will be enough to seed half of the wells in the plate with each dilution at final concentrations of 10 cells/well and 1 cell/well.
4.	Determine which dilution was optimal for monoclonal growth by determining the number of wells that show growing hybridomas. p type = annotation Usually hybridoma growth is obvious by macroscopic visualization of the well bottoms. A microplate reading mirror (Flow Laboratories or Dynatech) is useful to avoid the necessity of holding the plate above one's head. p type = annotation Poisson statistics indicate that if <22% of the wells have growing cells (the proportion expected if the cells were seeded at 0.3 per well and had a cloning efficiency of 100%), then 88% of these wells have only one clone. However, primary hybridomas generally have a poor “plating efficiency,” and thus more cells need to be seeded in each well to derive a reasonable number of growing clones. Use the wells seeded with 1 cell/well if there are “growing” wells and, if necessary, the wells seeded with 10 cells/well. The frequency of antibody-producing clones is dependent on the time after the initial testing that the cloning plates were set up and the MAb species. Generally, the longer the wait before plating the primary hybridomas and the more phylogenetically distant from the mouse the source of spleen cells, the less frequent the positive clones.
5.	Inspect wells for monoclonality with an inverted microscope before feeding cells, looking for tight single clusters of cells as evidence of monoclonal growth. Polyclonal growth is evidenced by more than one cluster of cells; if possible, do not use these wells.
6.	Use the screening assay that was used in the initial identification of the master well (see Critical Parameters) to test the monoclonal wells for desired antibody activity on day 7 to 14. Use an aliquot of the original hybridoma supernatant (from step 6, Support Protocol 2) as a positive control. p type = annotation Mouse-mouse hybridomas could be checked as early as 7 days after plating, whereas hamster-mouse hybridomas may require up to 14 days for growth. If any of the wells begin to turn yellow, they should be tested. Since the wells were not fed, any well with growing cells should have readily detectable antibody if the cells produce the desired MAb. Wait 2 days before testing if the wells were fed.
7.	When the desired clone is identified, expand and freeze the well as for the primary hybridoma in Support Protocol 2.
8.	Reclone one of the positive hybridoma clones as in steps 1 to 3; seed two new 96-well plates at 0.3 cells/well (60 viable cells in 40 ml cloning medium).
9.	Repeat steps 4 to 6.
10.	Expand and freeze the recloned hybridoma as in Support Protocol 2.
11.	Wean the recloned cells to complete DMEM-10/HEPES/pyruvate by splitting the cells 1:2 every day (for 3 days) with complete DMEM-10/HEPES/pyruvate. At this point, the desired hybridoma should be stable as a cell line. p type = annotation Freeze multiple vials on different days with different aliquots of freezing medium (appendix 3G). Thaw representative vials and check cells for growth and supernatant for MAb activity in appropriate assay. The hybridoma can then be used for ascites production and for large-scale production of hybridoma supernatants (unit 2.6). The isotype of the MAb can now be determined (unit 2.2). p type = annotation Even recloned hybridomas have an unstable phenotype, especially some hamster-mouse hybridomas, which may require additional recloning. Prolonged cultures in vitro may result in loss of MAb production. To minimize this problem, frozen aliquots of cells known to produce the MAb are necessary and should be verified as sources of viable cells. p type = annotation An occasional cloned hybridoma will not tolerate complete DMEM-10/HEPES/pyruvate and will require higher concentrations of FCS. It may be necessary to wean cells first to DMEM-15 before weaning to DMEM-10. Mycoplasma contamination should be considered (see appendix 3E).

 

Support Protocol 4: Recloning in Semisolid Medium (Clonacell-HY)

Recloning is recommended if hybridomas have been in continuous culture for a long period of time and are secreting antibody, but monoclonality or stability of the hybridoma needs to be confirmed.

Recloning is also recommended if the Alternate Protocol was followed and colonies are not well distributed in the semisolid medium (Medium D) or the cell density is too high, making it difficult to distinguish between individual colonies. In this case, recloning is recommended after the cells have been harvested and established in Medium E.

 Additional Materials (also see Alternate Protocol)

• Growing hybridomas (Basic Protocol 2 or Alternate Protocol)

1.	Culture the hybridomas in 10 ml of Medium E to a maximum cell density of 2 × 10^5cells/ml. Prepare a cell suspension at a density of 200 cells/ml in Medium A. p type = annotation Hybridomas can be cultured directly into Medium E from a frozen sample.
2.	Mix 9.5 ml Medium D and 0.5 ml hybridoma suspension (containing 100 cells) in a tube. Let sit at 37°C for 15 min.
3.	Using a 12-ml syringe and 16-G blunt-end needle, aseptically plate out the 10 ml of suspension into one 100-mm petri dish. Tilt the plates to level the mixture and try not to introduce air bubbles. Incubate dishes at 37°C in a 5% CO2 atmosphere. Do not disturb plates for 10 to 14 days. p type = annotation See Alternate Protocol, step 19 for more details.
4.	At a time point 10 to 14 days later, examine the plates for the presence of colonies visible to the naked eye. p type = annotation Assuming a viability of 50% to 80%, there will be 50 to 80 colonies in the dish.
5.	Remove 15 to 20 colonies from the plate with sterile pipet tips using a micropipettor set to 10 µl. Pipet each clone into an individual well of a 96-well tissue culture plate containing 200 µl of Medium E. Incubate the plates at 37°C in a 5% CO2 atmosphere for 7 to 10 days.
6.	Continue with the Alternate Protocol, steps 22 to 27.

 

Support Protocol 5: Preparation of Cloning/Expansion Medium

Many investigators add feeder cells (i.e., peritoneal washout cells, splenocytes, or thymocytes) to produce conditioned media that seem to enhance hybridoma growth and cloning. The direct addition of irradiated, freshly isolated cells to wells, however, sometimes results in contamination. Therefore, cell-free, sterile-filtered supernatants of all suspensions are recommended to enhance hybridoma cloning efficiencies. The following procedure describes the preparation of a cell-free thymocyte-conditioned medium from mice. After sacrificing several mice and removing each thymus, a single-cell suspension is prepared and grown for several days. The supernatant is harvested, filter sterilized, and stored at –20°C.

 Materials

• Mice
• Complete DMEM-20/HEPES/pyruvate/HAT (or HT) medium (see recipe)

• 75-cm² tissue culture dishes
• 50-ml conical polypropylene centrifuge tubes
• 0.45-µm filters

• Additional reagents and equipment for thymectomy (unit 1.9) and preparation of single-cell suspension from thymus (unit 3.1)

1.	Sacrifice 5 or 6 mice, avoiding use of anesthetics (see annotation to step 7 of Basic Protocol 2). p type = annotation Mice (e.g, BALB/c) should be 4 to 6 weeks old. Obtain pathogen-free mice from a reliable supplier that screens for mycoplasma contamination.
2.	Aseptically remove the thymus (unit 1.9). Tease thymus into a single-cell suspension as in steps 8 to 11 of Basic Protocol 2. Resuspend cells in 20 ml of complete DMEM-20/HEPES/pyruvate medium.
3.	Add 10 ml thymus cells to a 75-cm^2 flask. Add complete DMEM-20/HEPES/pyruvate medium to final amount of ~20 ml medium per thymus (maximum 60 ml cell suspension/flask). Incubate flask 4 to 5 days in upright position in a humidified 37°C, 5% CO2 incubator.
4.	Transfer suspension to a sterile 50-ml conical centrifuge tube. Centrifuge suspension 5 min at 1000 × g, room temperature, and harvest supernatant.
5.	Filter sterilize supernatant through 0.45-µm filter. Freeze at –20°C in 10-ml aliquots.
6.	Thaw and use at 10% to 20% final concentration in desired medium. p type = annotation An alternative to feeder cells and thymocyte-conditioned medium is the use of a source of IL-6 (plasmacytoma growth factor), such as liposaccharide-stimulated P388D1 supernatant (unit 6.6).

sect1 type = reagents

 

Reagents and Solutions

p type = annotation

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see appendix 2A; for suppliers, see appendix 5.

 Ammonium chloride solution

• 0.02 M Tris×Cl, pH 7.2 (appendix 2A)
• 0.14 M NH₄Cl

 Complete DMEM-20/HEPES/pyruvate/HAT (or HT)

To complete DMEM-20 medium (appendix 2A) containing 10 mM HEPES and 1 mM pyruvate, add 100× HAT (hypoxanthine/aminopterin/thymidine) or 100× HT supplement to 1× final. Store at 4°C for up to 1 month.

100× HAT and 100× HT supplement are available commercially (e.g., Sigma).

 50% polyethylene glycol (PEG)

Autoclave 10 g PEG 4000 (Merck or ATCC) in a Wheaton glass bottle and cool. Before it solidifies (at ~55°C), add 10 ml complete serum-free DMEM. This makes enough for ~20 fusions. The solution may be kept at room temperature for several months; it will become alkaline but this does not affect its performance.

Be sure not to use protein-containing medium because PEG precipitates proteins.

sect1 type = commentary

 

Commentary

 

Background Information

Since the original description of a technique to produce monoclonal antibodies (MAb) of defined specificity (Köhler and Milstein, 1975), MAbs have proven to be powerful tools to analyze a myriad of biological systems. As a testimony to the broad utility of this technique, the original authors noted above received the Nobel Prize for medicine in 1984. Furthermore, a search of Medline (online medical literature database) for 1988 to 2006 shows that more than 175,604 citations referred to MAb.

To produce MAb, a suitable host is immunized with an antigen and antibody-secreting B cells are immortalized by the fusion of the host immune B cells with a nonsecretory, drug-marked myeloma cell line. Since the unfused normal B cells cannot survive long in an in vitro culture, they derive immortality by fusion to a partner tumor cell line. The tumor line is resistant to the purine analog 6-thioguanine because of deficiency of hypoxanthine-guanine phosphoribosyl transferase (HGPRT). This deficiency results in lethal sensitivity to aminopterin, which blocks de novo synthesis of purines. The normal B cell is not sensitive to aminopterin when hypoxanthine and thymidine are supplied; salvage pathways utilizing HGPRT are necessary for survival. Thus, only hybridomas (normal B cells fused to tumor cell) will survive selection in HAT. If a hybridoma that produces the antibody of interest is identified and subjected to a cloning procedure that results in monoclonality (i.e., derivation of progeny cells from a single cell), a MAb of desired specificity is produced.

Although monoclonal antibodies are powerful tools, it cannot be overemphasized that a major commitment is necessary to identify a MAb of interest. The work is tedious and labor intensive. Moreover, in some cases, specific polyclonal antisera, which are generally easier to produce, may be as suitable as—and in some cases superior to—MAb. For example, specific antisera against defined synthetic peptides that will immunoprecipitate cell-surface antigens of interest can be made. Such antisera frequently will be useful in immunoblots whereas MAb, unless directly screened for such a purpose, may not recognize determinants on denatured antigens. Nevertheless, if the desired MAb is produced, this reagent has a wide variety of uses, particularly for the characterization of novel molecules, and as a specific antagonist or agonist of ligand-receptor interactions. Moreover, large quantities of the MAb can be readily purified.

There is a large body of literature on MAb production. More specific elaboration of the methods described here are discussed by Goding (1986) and Harlow and Lane (1988). Furthermore, many reports describing refinements of the basic technique are frequently published in the Journal of Immunological Methods. With general basic skills in animal handling, tissue culture, and screening assays, the novice should be able to produce MAb using the protocols in this unit. The major obstacle, as with any screening effort, is identification of the MAb of desired specificity. Because the technique produces many MAb– secreting hybridomas, the temptation exists to keep all or selected hybridomas even though they are not producing the MAb of initial interest. A large number of hybridomas secreting antibodies to unique antigens may be isolated, but the extra effort required is considerable. Nevertheless, it is a strategy that may prove useful while the attempt to identify the desired MAb proceeds. Many antibodies described in the literature are such by-products.

Since the original publication of monoclonal antibody generation (Köhler and Milstein, 1975), a wide variety of methods have been developed and published to fuse, grow, select, and clone hybridomas. However, many of these approaches have limitations in that they are time consuming and require many manipulations. In addition, a problem often encountered is that some hybridomas will overgrow others prior to cloning. Often these faster-growing cells do not synthesize antibodies (Kennett et al., 1980), resulting in a failure to obtain the desired hybridomas.

A solution to these limitations is the development of a system that reduces the time and manipulations necessary for hybridoma selection and cloning, and that prevents the overgrowth of slow-growing colonies by faster-growing colonies. One such system, which offers these advantages, involves simultaneous cloning and selection of hybridomas using agar-, agarose-, or methylcellulose-based semisolid medium. Performing the selection and cloning in semisolid medium instead of liquid suspension culture eliminates the possibility of overgrowth of potentially valuable slow-growing clones by fast-growing clones. In addition, the number of clones to be screened for secretion of a specific antibody is minimized. Because the daughter cells of each hybridoma stay together in a single colony, each positive clone identified is unique, and isolation of duplicate identical clones is avoided. The time, manipulations and reagents required are also minimized, as selection and cloning are performed at the same time and medium changes are not required.

Agar, agarose, and methylcellulose have all been used as semisolid media for the growth of monoclonal antibody–producing hybridomas. Methylcellulose has been shown to be superior to other semisolid media for the cloning of both established and newly derived hybridomas (Davis et al., 1982).

 

Critical Parameters

There are several major factors, other than technical, that should be considered before any MAb production project is begun.

While several fusion partners are available, the SP2/0-Ag14 myeloma is a good general-purpose cell line. The best sources of the cell line are ATCC or a laboratory actively producing hybridomas. Some sublines do not fuse well, perhaps due to some genetic variation or to undetected contaminants such as mycoplasma.

The SP2/0-Ag14 cells should grow in suspension with minimal adherence to the tissue culture flask. The cells should not be overgrown and the medium should not become acidic (yellow). Healthy cells will be refractile and none of the cells should be pyknotic when viewed with an inverted microscope. In general, the cells should be diluted (split) into fresh medium (³1:5 if the cells will be used on the following day) when the cells in an undisturbed flask form a monolayer on the bottom of the flask (1–2 × 10⁶ cells/ml). If the cells do not appear healthy even when grown at lower densities, mycoplasma contamination should be suspected.

Freeze multiple large aliquots of cells (appendix 3G) so that only a few days of expansion will be necessary for future fusions. If planned correctly, a primed animal can be boosted, cells thawed on the same day, and a fusion performed 3 days later.

 

Choice of animal for immunization

Basic Protocol 1 on immunization outlines the production of MAb by in vivo immunization and concentrates on the use of rodents for immunization. Specialized work may require the use of splenic injections, in vitro immunizations, or human monoclonals; these techniques are much more difficult to implement successfully and are not recommended for the novice. Other protocols designed to eliminate unwanted MAb against particularly immunogenic epitopes involve the use of cytotoxic agents immediately after the initial immunization with the unwanted antigens and should be considered only after some experience has been gained (Sharpe et al., 1985).

Four animal species (mouse, rat, hamster, and rabbit) can be used for MAb production. The spleen cells from a given species must be able to produce stable MAb-producing hybridomas with a rodent myeloma cell line. Although several rat and mouse tumor cell partners exist and all have been successfully used, the SP2/0-Ag14 mouse cell line from ATCC is recommended because it is a drug-marked, nonsecretory myeloma that does not constitutively produce either light or heavy chains. Therefore, hybridomas derived from fusion to SP2/0-Ag14 will not make chimeric MAb. This cell line also forms stable hybridomas with mouse, rat, and hamster B cells. Rabbit MAb have recently been described (Raybould and Takahashi, 1988) but little collective experience has accumulated.

It is technically more difficult to produce stable MAb-producing hybridomas from animals that are phylogenetically distant from the mouse. First attempts at producing MAb should use either the mouse or rat. Of the two, the mouse is the best choice for most xenogeneic antigens, such as human antigens, because many more antibodies have been produced in the mouse and thus, MAb to defined antigens are available commercially. Isotype matched MAb are obtained easily, which can be used as controls in the assays of interest. Moreover, the mouse is easier to handle, anti-Ig reagents specific for each mouse Ig isotype are more commonly available, and generally mouse MAb are easier than rat MAb to purify. If a mouse is to be immunized, the best choice is a BALB/c mouse, because the hybridomas resulting from fusion to SP2/0-Ag14 will be entirely of BALB/c origin and thus should grow in a BALB/c host for ascites fluid production. Moreover, BALB/c spleens are generally larger than spleens from other mouse strains.

In contrast, immunization of mice would not be appropriate for most mouse antigens unless an allotypic difference is known. Fortunately, the rat is a reasonable choice for many mouse antigens because rat MAb will frequently recognize framework determinants on mouse proteins. Most of the commonly available rat strains can be used, although the authors' laboratory usually employs Lewis rats. If rat MAb are produced, the mouse anti-rat MAb, MAR 18.5 (ATCC) is useful, as this MAb recognizes only rat light chains and binds protein A, and thus is easy to purify. Moreover, the hybridoma produces high titers of MAb in culture supernatants.

Although there are mouse MAb that recognize many major, functionally important human cell-surface and soluble antigens, it has proven difficult, despite considerable effort, to produce rat MAb to several of the homologous mouse antigens. It is possible that these mouse molecules are not antigenic in rat. Recently, it has been shown that some of these mouse molecules may be more antigenic in hamsters, presumably due to the phylogenetic distance involved (Schreiber et al., 1985). However, hamster MAb are more difficult to produce because of fibroblast overgrowth and instability of hybridomas. Thus, hamsters are not an ideal choice for the novice until experience with several fusions is acquired. The Armenian hamster strain is the best suited for hybridoma production because the hybridomas are more stable and fibroblast overgrowth is less than with other available hamster strains. Unfortunately some Armenian hamster MAb are nonreactive with standard, commercially available anti-hamster immunoglobulin antibodies. However, many hamster MAb are reactive with protein A and/or with the mouse anti-rat MAb, RG7/7.6, which cross-reacts with hamster chains (Sanchez-Madrid et al., 1983). Finally, there are few serologic reagents available for hamster pathogens and thus it is wise to quarantine hamsters—and the hybridomas derived from them—in case there are occult infections that could be spread to other rodents (unit 1.1).

The sex of the host animal does not appear to be important. It is usually advisable to immunize young adult animals because the immunization schedule may be prolonged. Fusions of spleens from older hamsters (>6 months old) tend to have more fibroblasts.

 

Antigen preparation and immunization

Many types of antigen preparations have been used successfully including whole cells, partially purified lymphokines and cytokines, solubilized cell membranes and protein bands isolated from SDS-polyacrylamide gels (unit 8.4). The nature of the antigen preparation to be used is dependent on several factors, particularly the ease in preparing the antigen, the screening assay, and the desired specificity and property of the MAb.

Although it is desirable to immunize with a purified antigen to increase the frequency of hybridomas secreting the desired antibodies, in contrast to polyclonal antisera production, this is not a major requirement.

It is important to note that MAb are quite specific. It is possible to immunize with impure antigens or with multiple antigens, and with a highly specific screening assay, pick the MAb that identifies a specific antigen. In many instances, this is exactly the protocol used. For example, animals are usually immunized with whole cells to derive MAb that recognize a specific cell-surface antigen. The major advantage to whole-cell immunization is that the proteins will be in their native conformation and thus, the MAb produced will recognize these antigens in their native form. The major disadvantage to whole-cell immunization is the production of MAb to many other antigens, particularly those that have been previously produced (since they tend to be the most commonly made); immunization of rats with whole mouse T cell suspensions will produce ³25% anti-Thy-1 MAb (W. M. Y., unpub. observ.). Nevertheless, collective experience with whole-cell immunizations suggest that although the antigen preparation may be impure, it is important to consider the nature of the antigen relative to its native form.

Because protein purification frequently denatures molecules and synthetic peptides usually do not achieve native conformations, immunization with synthetic peptides and gel-purified proteins generally has resulted in the production of MAb that recognize the antigen in its denatured form. While such MAb may be useful for immunoprecipitation and immunoblot studies, often they are not useful for flow cytometry analysis of cell-surface antigens (units 5.3 & 5.4) or functional assays that require binding of the antigen in its native conformation.

There are many immunization protocols used to produce MAb. The major requirements appear to be a primary immunization with an adjuvant and fusion 3 to 4 days after the boost. However, there are notable exceptions. For example, a single primary immunization and fusion 4 to 5 days later have been successfully used to produce MAb against cell-surface antigens (Logdberg et al., 1985). It is generally agreed that a successful fusion requires the presence of activated B cells.

Most injections can be given intraperitoneally with good results. Many investigators prefer to boost intravenously with antigen without adjuvant. While this can be done via tail vein in the mouse, this vein is inaccessible in the rat or Armenian hamster and intravenous injections are generally not used in these animals. Be aware that intravenous injections may occasionally result in fatal systemic reactions.

Many investigators screen their immunized animals for serum antibody titers before a final boost and fusion. This depends on the purity of the antigen and an assay that is not influenced by serum. However, many MAb have been successfully produced against cell-surface antigens without screening the sera before fusion even if whole cells are used to immunize.

 

Screening assays

The cell-fusion protocol outlined in this unit should yield hybridomas in ~50% of the wells. The purpose of the screening assay (Support Protocol 1) is to exclude all hybridomas that are unlikely to produce the MAb of interest and yet include all likely candidates. In that regard, a reliable assay that will detect a few false positives but no false negatives will help decrease the number of hybridomas from several hundred to ~20. Depending on the difficulty of the assay and the availability of the antigen, all wells can be screened for reactivity or—especially if most wells do not contain hybridomas—the wells can be screened first for visual evidence of growing hybridomas. Visual screening can be problematic since it requires inspection with an inverted microscope and a significant amount of bookkeeping to record which wells contain hybridomas (and which do not).

Screening assays should be perfected before the fusion is done. The assay should be rapid, with results available within 1 to 2 days, reliable, sensitive, and simple to perform in large numbers (hundreds to thousands of wells). In the protocol described here, the assay must be performed with 100 to 150-µl volumes. Alternatively, the supernatants from several wells could be pooled to screen fewer samples. One clever screening approach is to pool 50 µl from each well in each horizontal row and then separately pool 50 µl from wells in each vertical column of a single plate. The well containing the desired hybridoma can be pinpointed by the positive row and column. Because each supernatant is tested twice, the percentage of false positives is diminished. This technique should not be used for low-sensitivity assays as the supernatant is diluted significantly. Many different screening assays and techniques have been described. ELISA (unit 2.1) and radioimmunoassay (RIA) have been popular because of the above considerations.

Because MAb are exquisitely specific, those which recognize the same antigen may bind to slightly different epitopes and thus have different functional properties. For example, if a MAb to a cell-surface antigen that inhibits ligand-receptor interaction is required, flow cytometry analysis (units 5.3 & 5.4) may not be the best choice for the initial screening assay because a MAb that binds well by flow cytometry analysis may not be functionally active and vice versa. However, if the desired property of the MAb could be improved by a change of the heavy chain, it is possible to isolate isotype-switch variants that may improve the usefulness of a particular MAb. Such studies have been performed with isotype-switch variants of anti-CD3 MAb (van Lier et al., 1987). Nevertheless, the ideal screening assay should identify MAb with the desired property, not just the desired specificity.

 

Troubleshooting

The production of MAb is a prolonged procedure. Success requires the optimization of several steps. The antigen preparation and immunization protocol must be adequate; this can be checked by serum titers after immunization. If the spleen is collected too early or too late after the last boost or if the number of individual rounds of immunization is too low, the frequency of activated antigen-specific B cells may be too low, thus decreasing the chance that hybridomas producing the desired antibodies will be generated or detected. Generally, the longer the time interval between injections, the higher the affinity of the antibodies produced, because fewer antigens are present a long time after an immunization. Therefore, only the cells with the highest affinity for the antigen will still be in circulation. The use of proper antigen dose, adjuvant, and immunization schedule is particularly important for antigens that are only weakly immunogenic or available in very small doses. If no hybridomas are produced, then the immunization protocol may not have produced activated B cells, an unlikely situation if an adjuvant was used. Alternatively, hybridoma formation and growth may be inadequate, particularly if the cells (spleen or fusion partner) were contaminated with mycoplasma or if the lot of fetal bovine serum (FBS) does not support hybridoma growth. Mycoplasma contamination can cause hybridoma death during HAT selection because these organisms degrade thymidine at a fast rate. Obtain the fusion partner from a reliable source, either a lab that is successfully producing hybridomas or from ATCC. Freeze aliquots of these cells as soon as possible. Obtain animals from specific pathogen-free suppliers and cage them in uncontaminated rooms. The FBS lot can be tested by assaying the cloning efficiency of stable hybridoma lines and using a lot that supports high-efficiency cloning.

If hybridomas are produced at the anticipated level but the desired MAb is not found, assay the master wells for other antibodies that should react with the immunogen. For example, if whole cells were used as an immunogen and the original assay was a functional one (e.g., inhibition of proliferation), several wells should be screened for antibodies that recognize cell-surface antigens by flow cytometry (units 5.3 & 5.4); under these conditions, 25% to 50% of growing wells will produce antibodies that recognize cell-surface antigens. This result would prove that MAb were produced but that the desired specificity was not found. Unfortunately, this is a typical result and the source of the major commitment in time and labor that MAb production requires. The solution to the problem is to perform more fusions and screening. It is useful to consider alternatives to the immunization, choice of animals, and screening procedure.

During the cloning procedures, there are several problems that may be encountered. If none of the cloned hybridomas secrete the desired MAb, either the hybridoma was not producing the MAb initially or the clones have lost the ability to produce the MAb. An aliquot of supernatant from an expanded master well can be tested in confirmatory assays. If none of the clones produce the MAb of interest, the frozen master-well cells should be carefully thawed, reseeded in multiple cloning plates, and then refrozen. Beware that mycoplasma contamination will affect cloning efficiency adversely.

If no useful hybridomas are produced following the Alternate Protocol, there are several possible explanations. Failure to grow myeloma cells in optimal conditions will reduce the number of viable myeloma cells available for fusion. Myeloma cells should be passaged every 2 days in Medium A at least a week prior to fusion. The concentration of cells should not exceed 4 × 10⁵ cells/ml. Inefficient removal of serum from these cells prior to fusion, as well as low PEG concentration resulting from failure to completely remove the supernatant in step 6, will greatly affect the fusion outcome. Additionally, cell death will occur if cell exposure to PEG is longer than the recommended time. Note that hybridoma viability will be reduced if the cells are cultured longer than the 10 to 14 day incubation.

Difficulty in distinguishing individual colonies when performing the Alternate Protocol's harvest procedure could be due to low viscosity of Medium D. Since the viscosity of Medium D changes exponentially with the volume of liquid medium added, adding 100 µl extra will cause a dramatic decrease in the viscosity. Handling the plate earlier than the 10- to 14-day incubation may unsettle newly forming colonies and break them apart, resulting in dispersal and overlap of individual colonies. If this happens, it is still possible to harvest colonies, but it may be necessary to perform an additional cloning step to ensure that monoclonal hybridomas are obtained (see Support Protocol 4).

If low numbers of clones are produced, this could be a result of not picking a representative sample of colonies from the plates. Both small and large colonies should be selected, as the smaller colonies are often good antibody producers. Mycoplasma contamination and a poor immune response, as discussed previously, are additional factors.

 

Anticipated Results

With the cell-fusion protocol outlined in this unit (Basic Protocol 2), hybridoma growth should be apparent on the days mentioned and ³50% of the master wells will contain hybridomas. Depending on the purity of the antigen used and the immunization protocol, £1% to 5% of the wells will contain the desired hybridoma. If the screening assay demonstrates many positive wells, it is likely that these are false positives.

Cloning by limiting dilution of the primary hybridomas (Support Protocol 3) should yield ³10 to 50 “growing” wells/plate when seeded as described. The second cloning under more stringent conditions should yield ³10 wells when seeded at 0.3 cells/well. Remember that the cloning efficiency of the primary hybridomas is relatively poor. If no viable clones are derived, seed additional wells at higher density and/or use a source of IL-6 (plasmacytoma growth factor; Nordan and Potter, 1986; Bazin and Lemieux, 1989).

The Alternate Protocol described in this unit will produce 1000 or more monoclonal colonies distributed over ten plates. At a time point 10 to 14 days after fusion and plating in Medium D, hybridoma colonies should be visible to the naked eye. Colonies will be monoclonal. 90% to 100% of colonies transferred from the plate containing Medium D to individual wells of a 96-well plate containing Medium E will grow. However, growth rate and the time at which individual wells will be ready for screening will vary between wells. Some colonies will be larger than others, and some will grow more quickly than others in Medium E.

 

Time Considerations

Traditional methods of selecting and cloning monoclonal antibody-producing hybridomas (Basic Protocol 2) in suspension cultures can require up to 7 weeks of culture. This includes:

• Fusion: 1 day
  
• Select fused cells: 14 days
  
• Screen supernatants: 1 day
  
• Clone: 14 days
  
• Screen individual supernatants: 1 day
  
• Reclone (optional): 14 days
  
• Screen supernatants: 1 day
  
• Expand hybridomas: time varies.
  

Fusion, cloning, and screening typically require a full day of work each, and frequent (daily or every-other-day) manipulations and feeding are required during the selection of fused cells. Initial immunization to final cloning may require 2 to 3 months if the MAb is produced and identified in the first fusion. Usually several fusions are required, and thus a fusion every 3 weeks may be necessary. During much of this time, especially during the 3 weeks after fusion, daily or every-other-day tasks are required. An entire day of work is required when the actual cell fusion and screening assays are performed. Feedings require 5 to 10 min/plate.

Simultaneous cloning and selection of hybridomas in semi-solid medium using the ClonaCell-HY method requires less than 3 weeks of culture. This includes:

• Fusion: 1 day
  
• Select and clone: 14 days
  
• Harvest monoclonal hybridomas: 4 days
  
• Screen supernatants: 1 day
  
• Expand hybridomas: time varies.
  

Fusion, harvesting of clones, and screening typically require a full day work each. During the selection and cloning in Medium D, cultures do not require manipulation or feeding, although it is important to check that there is water remaining in the water dish inside the culture container (Alternate Protocol, step 19).