Sartorius_May21_NeutAntiMakingTheir - 12

Antibody Internalization

Antibody Internalization Reagent Principles
Outside cell:
pH ~7.4

Antibody
Internalization
Reagent

Mix Antibody
Internalization
Reagent with
test antibody

Inside cell:
pH ~7.0-7.2

Add labeled test antibody
to cells and read on iQue3
(iQue3 VBR, BR, BR HD)

Early
endosome:
pH ~6.3

Late
endosome:
pH ~5.5

Lysosome:
pH ~4.7

Figure 2: The pH-sensitive fluorescent probe principle. A novel pH-sensitive fluorescent probe enables one-step, no-wash labeling
of isotype-matched antibodies. A fluorescent signal is generated as internalized antibody is processed into the acidic endosome and
lysosome pathway.

A Simple Way of Labeling Antibodies

Fab fragments conjugated to a pH-sensitive

for Addressing Challenges with

fluorescent probe (Nath et al., 2016). This type

FACS, ELISA, and Microscopy

of novel reagent enables a generic, one-step,

One of the challenges presented above with

no-wash labeling protocol for all isotype-

FACS, ELISA, and microscopy techniques is

matched, Fc-containing test antibodies when

the requirement for wash steps to minimize

optimized for use on specific instruments.

background signal. Among other undesirable

Figure 2 shows how this reagent works: labeled

effects, such as increased time to results, this

antibodies are added to cells, and a fluorogenic

need for wash steps also increases reagent

signal is produced as the Fab-Ab complex is

requirements (and cost) and makes cell loss

internalized and processed via acidic (pH 4.5-5.5)

inevitable. However, an advanced method for

lysosomes and endosomes.

labeling cells could reduce reagent requirements,

Antibodies of interest are quickly and

as well as simplify protocols when analyzing

effectively labeled, with low reagent

antibody internalization.

requirements, by incubating in growth media

ABI Assays Made Simple, With No-Wash Labeling

with this novel, pH-sensitive dye (Figure 3).

and Low Reagent Requirements via a Novel,

Cells are then added to 384-well plates, along

Ph-Sensitive Reagent

with the dye-conjugated antibodies, and

The key to this simple, no-wash protocol is a

incubated again. This reagent, when used with

novel reagent composed of Fc-region targeting

an advanced flow cytometry platform, provides

12 |

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