Contribution à l'étude de virus de mollusques marins apparentés ...

days, according to Reed and Muench's formula (Reed & Muench, 1938), by
observation of cytopathic effects, which were rounded and vacuolated hypertrophied
cells (Midlige & Malsberger, 1968). Parallely, titers of the same LDV suspensions
were caJculated by indirect immunofluorescence (IIF) using the II A2 and the
15D II C9 monoclonal antibodies.
3. Immunization
Nine Biozzi mice were immunized by intraperitoneal injections of LDV infected cell
homogenate (200 Ill, 10 432 TCIDsolml) prepared as described above. Three injections
were performed with one week intervals.
Ten days after the last injection, the serum anti-LDV titer of each mi ce was assayed
as follows : sera were epurated with BF2 cells acetonic powder prior to an indirect
immunofluorescent assay as described in hybridoma screening protocols. The mouse
showing the highest titer for LDV was injected a fourth time three days before the
fusion.
Production of hybridomas
The myeloma cellline P3-X63-Ag8-653 (653) was cultured in RPMI-1640 medium
(Gibco) containing 1 mM glutamine, 100 mgll penicillin G, 100 mgll streptomycin
sulfate and 10% FCS. Spleen cells were fused with the myeloma line by a method
adapted from French, Fischberg, Buhl & Scharff (1986). 1.3.10 8 splenocytes were
mixed with myeloma cells at a ratio of 4: 1, centrifuged (200 g, 10 min, 20°C) and 1
ml of 40% polyethylene glycol-1540 was added to the cell pellet dropwise during 30
seconds while stirred gently. After one minute at 37°C, cells were pelleted by
centrifugation (200 g, 1 min 30, 20°C) and incubated for two minutes at 37°C. The
pellet was then diluted to 15 ml with serum free RPMI-1640. The tirst 6 ml were
added during tive minutes. Cells were pelleted (150 g, 10 min), resuspended in
RPMI-1640 (15% FCS) and distributed (2.5.10 4
; 5.10 4 or lOS cells/well) into
microculture plates containing mice macrophages as feeder cells (5.1 0 3 Balb/C
peritoneal exudate cellslO.2 ml/well). After 24 hours, hybridomas were selected by
addition of 100 III hypoxanthine-aminopterin-thymidin moditied RPMI-1640 (HAT
2X : 10 mM hypoxanthin, 0.04 mM aminopterin, 1.6 mM thymidine). Seven days
after the fusion, 100 III culture supematant per well was replaced by 100 III HAT 1 X
medium.
Hybridoma screening
For each hybridoma culture supematant, three parallel screening assays were
performed to estimate the relative antibody reactivity against BF2 cells (negative
control assay), and against LDV early (3 days) and delayed early (9 days) infected
BF2 cells (positive assays). Indirect immunofluorescence (HF) was performed
directly on infected and non-infected BF2 ceU cultured in 96 weUs microculture
plates foUowing a procedure derived from Stitz, Hengartner, Althage & Zinkemagel
(1987). Cells were tixed with 3% formaldehyde diluted in immunofluorescence
phosphate buffer (PBS, Diagnostics Pasteur), (100 ill/weil, 30 min., 20°C), followed
by ceU membrane permeabilization by Triton X-IOO (3% in PBS) (Peeples, 1987).
Cells were washed once (PBS, 100 ill/weil), incubated with hybridoma culture
supematants (50 IlVweU, Ih, 20°C) and then washed twice (PBS, 100 ill/weil, 3 min.,
20°C). Each weU was added a fluorescein conjugated antimouse Ig (Diagnostic
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