Gram-negative bacteria yielding similar results following cloning of
capsular and protein vaccine-induced antimeningococcal hmAbs
from ICA-enriched plasmablasts.
2 Materials
Refer to storage instructions for all reagents. Ensure safety guidelines and proper waste disposal procedures are followed.
2.1 Bacterial Growth,
Fixing, and Staining
1. Frozen stocks of pneumococcal and meningococcal strains.
2. Brain-Heart Infusion (BHI) broth supplemented with 0.5%
yeast extract (BHI-YE): Suspend 14.8 g of BHI powder and
2 g of yeast extract in 383.2 mL of deionized water. Sterilize at
Fig. 1 Schematic illustration of the experimental procedure. Quasi-presentation of secreted IgG is achieved by
exploitation of the surface expression of CD45 on plasmablasts. An anti-CD45 streptavidin conjugate antibody
is then used as a link between the CD45-expressing plasmablast and the anti-human IgG (conjugated to biotin)
that will capture IgG secreted into the immediate milieu of the plasmablast. This captured IgG can then bind to
a cognate antigen, if present, on the bacterial surface
Isolating Bacterial Pathogen-Specific Monoclonal Antibodies
11
capsular and protein vaccine-induced antimeningococcal hmAbs
from ICA-enriched plasmablasts.
2 Materials
Refer to storage instructions for all reagents. Ensure safety guidelines and proper waste disposal procedures are followed.
2.1 Bacterial Growth,
Fixing, and Staining
1. Frozen stocks of pneumococcal and meningococcal strains.
2. Brain-Heart Infusion (BHI) broth supplemented with 0.5%
yeast extract (BHI-YE): Suspend 14.8 g of BHI powder and
2 g of yeast extract in 383.2 mL of deionized water. Sterilize at
Fig. 1 Schematic illustration of the experimental procedure. Quasi-presentation of secreted IgG is achieved by
exploitation of the surface expression of CD45 on plasmablasts. An anti-CD45 streptavidin conjugate antibody
is then used as a link between the CD45-expressing plasmablast and the anti-human IgG (conjugated to biotin)
that will capture IgG secreted into the immediate milieu of the plasmablast. This captured IgG can then bind to
a cognate antigen, if present, on the bacterial surface
Isolating Bacterial Pathogen-Specific Monoclonal Antibodies
11