Antibody Screening Solutions Made Easy - 11

Antibody Screening Solutions Made Easy * Intellicyt® Mouse IgG Type and Titer Kit: A Novel Solution to Expedite Antibody Discovery

* Simultaneous measurement of cell count
and cell viability:
Monitoring cell proliferation and cell health in
the original hybridoma or B-cell culture plate is
important in determining if the cells are healthy
enough for the RNA extraction needed for PCRmediated antibody cloning.
Data analysis of a 96-well Hybridoma Plate
Using the Mouse IgG Type and Titer Kit
Traditionally, high-content data requires complicated and time-consuming data analysis. The
mouse IgG Type and Titer kit leverages ForeCyt's
unique analysis algorithms to quickly analyze data
and to identify hits. In this study, a single 96-well
plate of mouse hybridoma cells were prepared by
limiting dilution. After ~two weeks, 20 ul of sample
from 72 clones (containing a mixture of cells and
supernatant) were transferred to an assay plate.
After incubation with assay reagents, samples
were acquired on the iQue Screener PLUS (Figure 3).

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The kit template automatically generated four isotype-specific standard curves, and all the sample
IgG isotypes were automatically quantified and
populated in heat maps and a data grid. Cell count
and cell health information was also automatically
populated in the same visualization format. Next,
a Panorama view of all IgG isotypes in the samples
wells were created using an overlay line graph. All
72 mouse hybridoma samples with IgG isotype
and quantity information are displayed in the line
graph for monitoring and quality- control of the
screening results at the single plate level. The total
time from data acquisition to the identification of
potential candidates for downstream processes
only takes minutes per plate.
Data Analysis of a Multi-plate B-cell
Screening Campaign
In this experiment 20 ul of supernatant from
five, 96 well plates of primary B-cell clones were
analyzed using the high sensitivity protocol

found in the Mouse IgG type and Titer kit (due
to the comparatively low levels of antibody
secretion in B-cell cultures; Figure 4). Standard
curves were generated from a separate standard
plate and ForeCyt used these isotype-specific
standard curves to auto-calculate the IgG
quantity in each sample. The data from the five
plates were populated in the data grid in the
Panorama feature. Profile maps identifying
specific hits at the screening level (all five plates)
were produced with user defined criteria (wells
that had greater than 0.1 ug/ml IgG1 and less than
0.1 ug/ ml of all other isotypes). Samples that met
the profile map criteria were ranked by IgG1
concentration using a Panorama line graph. The
data acquisition analysis and this whole screening
campaign was complete in about one hour.

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Antibody Screening Solutions Made Easy

Table of Contents for the Digital Edition of Antibody Screening Solutions Made Easy