Human Anti-Staphylococcus aureus Recombinant Antibody (clone STAU-229) (CAT#: PABS-0225)

Figure 1 In vitro blocking of binding mediated by IGHV1-69-encoded mAbs.

Biolayer interferometry was used to detect whether the mAbs blocked binding of hemoglobin to NEAT2. Biotinylated hemoglobin was loaded onto streptavidin-coated biosensor tips before association with either IsdB alone, IsdB mixed with mAb, mAb alone, or kinetic buffer. Error bars represent standard deviations. P values were determined by an unpaired t test. This experiment was performed at least 3 independent times.

Bennett, M. R., Dong, J., Bombardi, R. G., Soto, C., Parrington, H. M., Nargi, R. S., ... & Crowe Jr, J. E. (2019). Human VH1-69 gene-encoded human monoclonal antibodies against Staphylococcus aureus IsdB use at least three distinct modes of binding to inhibit bacterial growth and pathogenesis. MBio, 10(5), e02473-19.

Figure 2 In vitro inhibition of growth of S. aureus (d to f) mediated by IGHV1-69-encoded mAbs.

(d to f) S. aureus heme-dependent in vitro growth curves were performed in 96-well plates over 32 h. S. aureus strain Newman was subcultured at a 1:200 dilution into RPMI medium with ethylenediamine-N,N'-bis(2-hydroxyphenylacetic acid) and normalized to an OD600 of 1.0.

Bennett, M. R., Dong, J., Bombardi, R. G., Soto, C., Parrington, H. M., Nargi, R. S., ... & Crowe Jr, J. E. (2019). Human VH1-69 gene-encoded human monoclonal antibodies against Staphylococcus aureus IsdB use at least three distinct modes of binding to inhibit bacterial growth and pathogenesis. MBio, 10(5), e02473-19.

Figure 3 IGHV1-69-encoded mAbs reduce bacterial burden in vivo.

Seven-week-old female BALB/c mice were inoculated retro-orbitally with a suspension of S. aureus strain Newman at an OD600 of 0.4. Mice were given IGHV1-69-encoded full-length wild-type IgG LALA Fc variant (L234A/L235A) IgG antibodies by the intraperitoneal route. The hearts, livers, and kidneys of the infected mice were harvested after 96 h. Statistical significance was evaluated by analysis of variance (ANOVA) using multiple comparisons. All other comparisons, including comparison of LALA mAbs to the isotype control, were not significant.

Bennett, M. R., Dong, J., Bombardi, R. G., Soto, C., Parrington, H. M., Nargi, R. S., ... & Crowe Jr, J. E. (2019). Human VH1-69 gene-encoded human monoclonal antibodies against Staphylococcus aureus IsdB use at least three distinct modes of binding to inhibit bacterial growth and pathogenesis. MBio, 10(5), e02473-19.

Figure 4 Repertoire analysis of somatic variants of IGHV1-69-encoded mAbs.

Deep sequence analysis of antibody variable genes expressed in donor PBMCs revealed somatic variants for the heavy chains of STAU-229. These somatic variant heavy chain antibodies were expressed recombinantly with the original light chain and purified. (a and b) Neighbor-joining tree analysis of mAb clonal variants by Geneious. The germ line gene sequences of IGHV1-69*01 or IGHV1-69-2*01 were used as the outgroup sequences. (c and d) Each variant mAb was tested for binding to IsdB, and the half-maximal effective concentration (EC50) for binding was determined. Biolayer interferometry was used to determine the on and off rates of each variant antibody for binding to NEAT2, and the calculated KD is graphed.

Bennett, M. R., Dong, J., Bombardi, R. G., Soto, C., Parrington, H. M., Nargi, R. S., ... & Crowe Jr, J. E. (2019). Human VH1-69 gene-encoded human monoclonal antibodies against Staphylococcus aureus IsdB use at least three distinct modes of binding to inhibit bacterial growth and pathogenesis. MBio, 10(5), e02473-19.