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Data from MAB Profiling ELISA
Plate Layout for MAB Profile
Example of MAb Profile
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Characterisation using monoclonal antibodies
Panels of monoclonal antibodies (MAbs) have been produced against all
seven serotypes of FMDV. MAbs against types O, A, C and SAT 2 have been
used to identify at the amino acid level the antigenic sites on the virus
that are responsible for neutralisation.
Using panels of MAbs against a particular serotype it is possible to obtain a MAb Profile of a field strain by measuring the ability of MAbs to bind the virus in ELISA. Field strains with similar profiles from the same geographic region are assumed to be related.
The absolute specificity of each MAb for its particular epitope allows precise characterisation of viruses in terms of the epitopes they possess.
This is particularly useful to monitor strains during the adaptation required to produce a new vaccine strain from a field strain. Provided a sufficiently extensive panel of MAbs is used, the finding that the MAb profile of the vaccine strain and the field strain are identical suggests that the two strains are antigenically unchanged.
MAb profiling is not yet used to assess field strains for their similarity to vaccine strains in terms of the potential protection that the vaccine strain might confer as:
• The relative importance of different antigenic sites
in terms of protection is not known.
• Only minor changes are required to affect significantly
the binding of MAbs whereas binding of polyclonal
sera (as in natural immunity) is usually only affected
by major alterations in antigenicity.
• Wider panels of MAbs against a variety of epitopes
on each major antigenic site are required before
the full spectrum of antigenicity of an FMD virus
can be assessed. Such diverse panels are not
yet available.
• The antigenic diversity of FMDV means that panels
of MAbs would probably be required for each of
the main vaccine strains within a serotype.
Despite these difficulties progress is being made in terms of relating MAb profiles to protection and it is likely that MAbs will ultimately be widely used and may even replace the use of polyclonal sera in the strain characterisation ELISA.
MAb profiling technique
It is beyond the scope of this module to describe in detail how MAb profiles are obtained. In principle, the following steps are followed:
1 Obtain a panel of MAbs to a well characterised
reference strain of FMDV, ensuring that the MAbs
recognise a range of different epitopes on the virus,
preferably from several distinct antigenic sites.
2 Titrate each MAb against the parental strain in a
'trapping' ELISA using as coating antiserum
either a polyclonal serum or another MAb
which is known to bind to a site different from
that recognised by the MAb used as detector.
Use an anti-mouse antiserum conjugated with
horseradish peroxidase as the conjugate.
3 Select a dilution of each MAb giving approximately
70-80% of maximal signal (an optical density (OD)
of approximately 1.5).
4 Titrate field viruses using polyclonal trapping and
detecting sera and determine a saturating dilution
where the antigen is just present in excess.
5 React each MAb with each field virus using the MAb
profile plate layout at the dilutions
determined above.
6 Data is analysed as described in the data from MAb profiling ELISA.
In principle, the OD reading for each MAb against each field virus is
expressed first as the percentage reactivity compared with the polyclonal
detector system (to standardise for the amount of each field virus that
is trapped on the plate). This figure is then expressed as percentage
reactivity compared with the reaction between the MAb and the parental
strain.
7 Profiles are generally represented as histograms
(Example of MAb Profile).
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