Sartorius_May21_NeutAntiMakingTheir - 10

Antibody Internalization

when engineering and producing mAbs

their fluorescence properties, it cannot be used

as therapeutics. Engineering antibodies to

to perform high-throughput, time-dependent

optimize their biological potencies during

studies on individual cells because the cells

the discovery phase can address many of

must be sorted into single colonies before any

these challenges. However, these attempts to

further analysis can be performed (Doerner

optimize one attribute can have profound and

et al., 2014).

unintended consequences on other antibody

ELISA, on the other hand, is adaptable to

attributes. For instance, optimizing an antibody's

high-throughput screening (Saeed et al., 2017),

specificity may negatively affect activity

and thus has historically been used to screen

(Tiller and Tessier, 2015).

hybridomas and other libraries for antibody

One way of simultaneously optimizing

binding to each of its targets. ELISAs are

multiple antibody properties is by using

performed by coating a single target antigen

mutagenesis to produce large screening

onto the wells of assay plates, followed by the

libraries. However, large screening libraries

addition of individual samples from an antibody

necessitate a thorough in vitro, high-

library (for instance, from hybridoma or phage

throughput screening method to quickly

display). Antibodies that bind to the immobilized

identify the most suitable drug candidates

antigen are detected by a color change due to an

for further development early in their

indirect enzyme/substrate reaction.

discovery process.
Workflows for antibody screening commonly

In addition to its adaptability to highthroughput screening, ELISA is rapid, consistent,

include fluorescence-activated cell sorting

and relatively easy to analyze (Saeed et al., 2017).

(FACS), enzyme-linked immunosorbent assays

However, ELISA has several disadvantages

(ELISA), and microscopy (such as confocal)

that can limit its successful use in a modern

techniques. Yet, these in vitro antibody screening

antibody screening lab. First, primary screens

methods have several drawbacks: (1) they can

that test binding to a single antigen often require

be labor intensive with limited throughput,

subsequent secondary and sometimes even

(2) they do not allow direct, head-to-head

tertiary screens with control antigens to confirm

comparisons of antibodies, and (3) they need

their specificity and cross-reactivity. Second, ELISA

large amounts of reagents.

is not the best method for screening antibodies

For instance, even though FACS can be used

that bind to cell surface antigens because these

to sort hundreds of thousands of cells based on

antigens are extracted from the cell membrane

10 |


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