care to compare production levels between conditions, and even more between

laboratories.

8.3.2.1

Immuno-Based Assay

The single-radial immunodiffusion assay (SRID) is another method that is often used

for viral quantification. It is also used for the quantification of other kinds of proteins.

The principle holds on the embedment of specific antibodies in agarose gel in which

target proteins will later diffuse (Figure 8.3). In the case of viral preparation, polyclonal

antibodies are preferred to enhance the potential interaction between viruses and an-

tibodies through their attachment to different epitopes. Holes of calibrated size (com-

monly 3 to 4 mm diameter) are punched in the agarose where the viral suspension is

deposited at various dilutions. Viral components migration stops its diffusion while

forming a precipitation ring when the network of antibodies/virus are too large to pass

through the agarose mesh. The quantification assay is then based on a calibrated reagent

with a known content of viral antigens while the amount of antigen is correlated to the

size of the precipitation rings. As for hemagglutination assay, such quantification

method is highly variable due to operator experimentation (holes preparation, mea-

surement of precipitation rings) and to reference reagent lot to lot variability.

Nevertheless, in some cases (influenza vaccine), the SRID assay stays the only assay

validated by the FDA to release vaccine lots. In this specific case, great effort has been

dedicated by research teams to replace this assay with alternative quantification ap-

proaches, which could overcome the problematics of reference reagent supply.

The ELISA, or enzyme-linked immunosorbent assay, uses a similar principle

with improved control of the antigen/antibody’s interactions. In that case, the anti-

bodies or the antigens are coated on a planar surface, generally in a 96-well plate

allowing for high throughput analysis. The read-out is the presence of attachment/

interaction between a specific antibody and the protein of interest. Indeed, non-attached

analytes will be washed out by several buffer rinses. ELISA was developed for several

kinds of viruses and generally allows to reach a lower limit of detection and quantifi-

cations than SRID assays. The reproducibility is also much better than the SRID.

Nevertheless, for now, it is not sufficient to replace the SRID. Indeed, as an example, for

the influenza vaccine lot qualification, the use of polyclonal antibodies raised in sheep

after inoculation of influenza viruses demonstrate the specificity of the antigens to-

wards a global immune response. Whereas, in the case of ELISA, the most common

antibodies used are monoclonal antibodies produced at a high rate in cell culture and

which target a single epitope of the viruses [12] or receptor-based assays [23].

Biosensors have been developed for multiple applications in the diagnosis field.

Some of these technologies were used in the bioproduction field. One of these

techniques for example is the surface plasmon resonance (SPR). It was originally

applied to investigate virus/host interactions [24]. In the viral quantification field, it is

to be compared/included in the immuno-based assay category as it was first applied by

antibodies grafting onto sensor surfaces. SPR does present some advantages over

ELISA, as it does not required for detection of a fluorescence or HRP label (and thus a

secondary antibody). The interaction between the analyte (here viruses or antigens)

and the ligand (here the antibodies) is taking place within a liquid solution on the

biosensor surface. The detection of the interaction is based on the physical

Analytics and virus production processes

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