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and purified before adsorbing to the plastic ELISA

thorough functional analysis of a smaller set

plate. Extracting antigens from the membrane

of antibody candidates using microscopy

often leads to disruption of conformational

(Doerner et al., 2014).

epitopes that can be important targets for

Like FACS, microscopy techniques also require

therapeutic antibodies. Finally, to minimize

labeling each antibody with a fluorescent tag,

background signal, ELISA requires multiple

which must be separated from the free label via

wash steps to remove unbound antibodies and

a column or wash step because analysis requires

detection reagents, resulting in long, labor-

robust isolation of internalized antibodies

intensive screening workflows.

from those outside the cells. To aid isolation

Also, although ELISA can provide data on the

of the positive signal, researchers often resort

immunoglobulin G (IgG) titer, it offers no reflec-

to perturbing techniques, such as washing

tion on how the therapeutic antibody candi-

cells, using blocking dyes, and reducing the

dates affect the health of the test cells. i.e. how

temperature to slow cellular activity. However,

quickly or efficiently they induce death. Thus,

cells can be lost during washing steps, and the

candidates that appear to be productive based

associated reductions in temperature perturb the

on ELISA screening may be carried forward into

cellular environment.

the next step of the production process even

A further drawback to almost all the

though they are unideal candidates in terms of

techniques used for antibody screening is

function or vigour.

that they only enable end-point analysis,

Microscopy techniques, in contrast to FACS,

which means that multiple experiments are

are useful for single-cell analysis, as well as

required to follow an antibody attribute, such as

for such localization and temporal studies as

internalization, over time.

antibody internalization and live-cell imaging

The best approach to address all the

for monitoring individual cell behavior.

limitations of traditional antibody screening

Microscopy techniques, however, have limited

is to combine data from advanced screening

throughput due to data acquisition time, and

methods; using advanced methods,

they have limited multiplexing capabilities.

researchers can choose their candidates

Thus, for antibody discovery, some other

based on a thorough evaluation of all relevant

method, preferably high-throughput, is

characteristics, such as IgG titer, cell health,

generally used for the initial selection and

internalization, and their associated kinetics

screening of large libraries, followed by a more

early in their discovery process.

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