Sartorius_May21_NeutAntiMakingTheir - 16

Antibody Internalization

Ab1a

Ab2

Ab3

Controls

Ab1b

Ab4

Ab5

Controls

Figure 5: Screening test of Abs for internalization. The pH-sensitive reagent is suitable for high-throughput antibody internalization
assays in a real-time, live-cell analysis system.

six different commercially available anti-CD71
antibodies into HT1080 fibrosarcoma cells. We

same clone from two different sources).
We found that three antibodies (Ab1a, Ab2,

labeled the anti-CD71 with our pH-sensitive

and Ab1b) gave internalization signals that we

reagent before adding to cells in 96-well plates.

detected at low concentrations (< 0.05 μg/ml)

We then captured the internalization signal in a

(Figure 5). Reassuringly, Ab1a and Ab1b gave

live-cell analysis system every 30 minutes over

similar internalization responses. Antibodies

12 hours using a 10X magnification.

3, 4, and 5 were internalized more weakly and

The plate view in Figure 5A shows clear

only at higher concentrations. From the control

positive and negative control responses

responses, we calculated a mean Z' value of

in column 11 and 12, with concentration-

0.82 (two plates: 0.75, 0.87), indicating high

dependent responses for each antibody across

robustness for this microplate assay.

the two plates (antibodies 1a and 1b are the
16 |

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These data show our method is suitable


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