Generation of Hybridoma Cell Lines

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Monoclonal Antibody Core Facility
Department of Neuroscience
Generation of Hybridoma Cell Lines
Our hybridoma facility will generate hybridoma cell lines against a given antigen and is
subdivided into four phases:
Phase I: Immunization of Mice and Serum Screening
(approx. 6 to 10 weeks)
Immunization of four (4) mice (Balb/c) with antigen(s) provided by the investigator to stimulate
antibody production. Typically, ELISA will be used to determine the highest titer mice and these
will be used for cell fusion.
Notes:
1. Approxinatley 2 mg of antigen is required for immunization. An immunogen can be
provided as powder or in solution, though the immunogen must be soluble (no gel) for IV boosts
at the end of the schedule.
2. The recommended concentration is ≥1 mg/ml (PBS).
3. An additional 4 mg of antigen in liquid for plate coating is required for screening by
ELISA.
4. The screening strategy needs to be discussed with the core prior to the initiation of a
monoclonal experiment. It is important that a screening strategy be defined beforehand which
will result in the efficient detection of potentially useful hybridomas. Antibody-producing
hybridomas need to be tested in the assay for which they will be used (i.e. if antibodies are to be
used in Western blots, they need to be screened by Western blots) as soon as reasonably possible
in the screening strategy.
Phase II: Fusion and Screening of Hybridoma Supernatant
(approx. 3-5 weeks)
An antibody-producing mouse is selected for fusion of spleen cells with myeloma cells.
Distribution of fusion product into several 96 well culture plates. Screening of all wells
containing fusion products will be assayed by ELISA, utilizing the antigen(s) provided by the
investigator. The antibody secreting wells (up to 48 positives) with the higher titer (as
determined by OD measurement) are grown in 24-well plates and retested by ELISA. Up to
eight positive wells will be amplified and frozen for parental hybridomas. The investigator will
be notified one or two days prior to supernatant harvest. Approximately 1 ml of supernatant will
be given to the investigator for evaluation via their own specific assay. Results of the assay are
required within 72 hours to insure that interesting hybriodmas are not lost to contamination or
other unforeseen events.
Notes:
The Core can not guarantee that a mAb will be produced that will work in the
investigators application
.
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Standard Operating Procedure: Generate Hybridoma Cell Line
Monoclonal Antibody Core Facility
Department of Neuroscience
Phase III: Cloning
(approx. 4-6 weeks)
Two of the best hybridomas will be selected and cloned by the limiting dilution method and
screened by ELISA. Clones with the desired titer and specificity will be expanded, and the
remaining antibody containing media (1 ml from up to 5 positive clones) from each of the subclones will be forward to the investigator for evaluation in the investigator’s specific assays.
Positive clones with the highest titer (as determined by OD measurement) are temporarily frozen
in duplicate vials. The investigator has two weeks to analyze and select the desired clones for recloning.
Phase IV: Re-cloning
(approx. 4-6 weeks)
A clone (as chosen by the investigator from Phase III) will be re-cloned by limiting dilution
technique and plated into five 96 well plates to generate a stable cell line. The growing cells are
then screened for secretion of antigen specific antibody by ELISA. The media samples from up
to 5 positive antibody-secreting clones (100-150 µl) will be given to the investigator for their
evaluation. At this stage, the positive clones are transferred to a 24-well plate and kept in culture
for 5 days, allowing the investigator to select a single clone for final expansion and subsequent
cryopreservation. Otherwise, all 5 clones are expanded and temporarily frozen in duplicate vials.
The investigator has 30 days to select final clone(s) for expansion and cell storage.
Additional Information:
Use myeloma cells that have:
 lost the ability to synthesize hypoxanthine-guanine-phosphoribosyltransferase (HGPRT).
This enzyme enables cells to synthesize purines using an extracellular source of
hypoxanthine as a precursor. Ordinarily, the absence of HGPRT is not a problem for the cell
because cells have an alternate pathway that they can use to synthesize purines. However,
when cells are exposed to aminopterin (a folic acid analog), they are unable touse this other
pathway and are now fully dependent on HGPRT for survival.

lost the ability to synthesize any antibody molecules of their own (so as not to
producehybridoma producing two kinds of antibody molecules). This enzyme enables cells to
synthesize purines using an extracellular source of hypoxanthine as a precursor. Ordinarily,
the absence of HGPRT is not a problem for the cell because cells have alternate pathway that
they can use to synthesize purines.
However, when cells are exposed to aminopterin (a folic acid analog), they are unable to
use this other pathway and are now fully dependent on HGPRT for survival.
Standard Operating Procedure: Generate Hybridoma Cell Line
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Monoclonal Antibody Core Facility
Department of Neuroscience
1. The first property is exploited by transferring the cell fusion mixture to a culture medium called HAT medium because it contains:

hypoxanthine

aminopterin

the pyrimidine thymidine
The logic:

Unfused myeloma cells cannot grow because they lack HGPRT.

Unfused normal spleen cells cannot grow indefinitely because of their limited life span.
However,

Hybridoma cells (produced by successful fusions) are able to grow indefinitely because
the spleen cell partner supplies HGPRT and the myeloma partner is immortal.
2. Test the supernatants from each culture to find those producing the desired antibody.
3. Because the original cultures may have been started with more than one hybridoma cell, you
must now isolate single cells from each antibody-positive culture and subculture them.
4. Again, test each supernatant for the desired antibodies. Each positive subculture - having been
started from a single cell - represents a clone and its antibodies are monoclonal. That is, each
culture secretes a single kind of antibody molecule directed against a single determinant on a
preselected antigen.
5. Scale up the size of the cultures of the successful clones.
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Standard Operating Procedure: Generate Hybridoma Cell Line
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