pluriBead®

FAST, EASY, GENTLE!

Application Example

B cell isolation with pluriBead®
and stimulation of antibody
secretion

pluriSelect products in this flyer
CD19 S-pluriBead® anti-hu
S-pluriBead® Mini Reagent Kit

11-01900-10
70-50010-11

pluriSelectpluriSelectSpring Valley, CA 91977
USA

Deutscher Platz 5c04103 Leipzig
Germany

Phone: 619-928-9265
support.usa@pluriselect.com
sales.usa@pluriselect.com

Phone: +49 341 333858-0

USA

www.pluriselect.com

Worldwide

support@pluriselect.com
sales@pluriselect.com

V 1.1

B cell isolation with pluriBead® andstimulation of antibody secretion
Sample volume

50 ml whole blood (with EDTA)

Isolation method

300 µl CD19 S-pluriBead® anti-Hu

Yield~3.5 x 106 B lymphocytes

Vitality>99% (trypan blue staining)

Purity~97% B lymphocytes

Purity determination

Anti-CD20 FITC antibody staining
followed by FACS analysis

Fig. 1: Purity of isolated B lymphocytes

Introduction
pluriBead® allows for positive non-magnetic cell isolation fromany sample material. In this example, we use CD19 S-pluriBead®
for the isolation of B lymphocytes. Find the complete isolation
protocol on our website or in the manual.

250 cells/well each

Anti-influenza nucleocapsid-specific memory B cells

500 cells/well each

Anti-tetanus toxoid
specific memory B cells

Non-activated cells(500 cells/well each)

Negative control

Fig. 2: ELISpot analysis ofanti-Ig-specific cells
in duplicatesFig. 3: ELISpot analysis of antigen-specific memory B cells
in duplicates, 105 cells/wellwww.pluriselect.com/manuals-protocols.html

Frequency determination of antigen-specificmemory B cells
After target activation, frequency of antibody-secreting plasma
cells (ASCs) was determined via ELISpot. Isolated B cells had beenstimulated for 5 days with a polyclonal activation mixture. Afterwards, they had been grown on a plate coated with the specificantigen. After 24 hours incubation, the assay had been processed.
Spots (shown in fig. 2 and fig. 3) had been counted and evaluatedwith a specific software (AID ELISpot6.0 iSpot). In the adjacentdiagram, the blue numbers reflect the count of antigen-specificspots per well.

Results
CD19 S-pluriBead® Mini Kit allowed for isolating B lymphocytes
from human whole blood sample with a purity of ~97%. The
isolated targets showed a vitality of >99% after trypan bluestaining. They had been successfully grown in cell culture as well
as differentiated from antibody secreting plasma cells (ASCs).
ASCs proved suitable for analysis of antigen-specific memory B
cell response via ELISpot assay. Results had been comparable tothose gained from a reference method (Dynabeads® CD19 PanB).
For reference method, see publication: Bussmann B. M., Reiche S., Bieniek B.,
Krznaric I., Ackermann F., Jassoy C.: Loss of HIV-specific memory B cells as apotential mechanism for the dysfunction of the humoral immune response
against HIV. Virology 2010; 397(1):7-13)

Proliferation factor(Growing of B lymphocytes into ASCs
after activation)

Activation efficiency

3-fold(Percentage of B lymphocytes that had
been differentiated into ASCs)

32.2%

Influenza nucleocapsid-specific memory B cells

0.03%

Tetanus toxin-specific memory B cells

0.05%

Table 1: ELISpot analysis of B cell