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

infection. Then, a weak green fluorescence was observed, in contrast to the red native
fluorescence of cells. The titer could be determined from the eighth day, as single
infected cells with strong green fluorescent cytoplasms cou Id be easily distinguished
(Table 3).
LabeUing of infected cells was observed from the second day when applying the
15DIIC9 monoclonal antibody. However, considering the low intensity of the
fluorescence during the first days of infection, the titer could be calculated precisely
first from the seventh day post-infection and onwards (Table 3).
Discussion
ln the present work a production of LDV specific monoclonal antibodies was
obtained using infected BF2 cells as antigen. LDVs may be purified from fish
nodules (Schnitzler, Riisen-Wolff & Darai, 1990) or, with relatively low yields, from
infected cell cultures (Robin & Berthiaume, 1981). In both cases antigens would
mainly consist of viral structural proteins. Infected cells represent more complex
antigens, containing a mixture of cellular and viral epitopes which may be
advantageous when producing monoclonal antibodies for use in early infection
diagnosis, as detection of non-structural viral proteins may then be important. This
might have been the case in the present study, as three of the monoclonal antibodies
produced enabled antigen detection as soon as two or three days post-infection.
The suitability of the immunogen used here was confirmed by the LDV -specific
reactivity of immune sera after absorption with acetonic extracts of BF2 cells.
However, due to the relative abundance of antibodies against BF2 cellular antigens, it
was supposed that an important proportion of immunized mouse spleenocytes, and
subsequently hybridomas, would be specific of cellular antigens. The hybridoma
precloning limited the risk of obtaining mixed antibodies, sorne possibly reacting
with BF2 cell antigens, masking antibodies specific for LDV antigens. Additionally
the hybridoma selection was accurate due to the direct visualization of fl uorescence
which enabled an identification of even subtle and faint green fluorescent dots, and
also to compare reactivities against LDV infected and non-infected BF2 cells. The
direct immunofluorescence assay (HF A) was easy to perform due to the application
of a protocol using microculture plates as described by Stitz el al. (1988).
Cell culture conditions were improved throughout the experiments as it was observed
that LDV -infected cells had a tendancy to detach and be lost during HF sample
treatment. Wells were therefore treated with poly-D-Iysin previously to BF2
inoculations. After incubation, virus suspensions were replaced by fresh medium and
added methyl cellulose. This procedure proved efficient as infected cells remained at
the weil bottom until formaldehyde fixation and during immunofluorescent assay.
il appeared that II A2 was the most suitable monoclonal antibody due to its
reliability in identifying infected cells at low magnification. This permitted a rapid
examination of microculture wells. However, other monoclonal antibodies are also
suitable, in particular l5DIIC9, which produced a pin-point cytoplasmic
fluorescence being recognizable as soon as Iwo days post-infection.
Titer estimation by observation of cytopathic effects on BF2 cells did not give a
precise LDV titration since it was difficult to interpret these observations after the
seventh day post-infection. Compared to the observation of cytophathic effects,
indirect immunofluorescent assay was far more sensitive since it was possible to
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