Mouse Anti-HIV Recombinant Antibody (clone YZ23) (CAT#: PABL-136)

Figure 1 Heterologous HIV neutralization by mAbs to E-gp120.

A, aligned sequences of the immunodominant 307–325 region epitope (HXB2 numbering) of the gp120 immunogen (strain MN) and clinical HIV isolates tested for mAb neutralization.., identities; #, unidentified residues. B, neutralization of heterologous CCR5-dependent, subtype C strain ZA009 by anti-E-gp120 mAbs. Shown are IC50 values for seven neutralizing mAbs obtained from assays conducted at increasing mAb concentrations. The following 10 mAbs did not neutralize the virus: YZ20, YZ24, SK-T01, SK-T02, SK-T03, SK-T04, 2F2, 7H3, 1F4, and 5E11 (IC50>20 μg/ml). C, representative strain ZA009 neutralization data at varying mAb YZ23, 3A5, and SK-T03 concentrations.

Nishiyama, Y. Planque, S. Mitsuda, Y. Nitti, G. Taguchi, H. Jin, L. Symersky, J. Boivin, S. Sienczyk, M. Salas, M. Hanson, C.V. Paul, S. (2009). Toward Effective HIV Vaccination. The Journal of Biological Chemistry, 284, 30627-30642.

Figure 2 Competitive inhibition of mAb YZ23 binding to immobilized gp120 (A) and mAb YZ18 binding to immobilized E-gp120 1a (B) by synthetic 421–435 and 292–306 peptides.

Binding was determined by ELISA. mAbs are 5 μg/ml. Plotted are % values of binding (mean ± S.D. of duplicates) in the absence (A490 for YZ23, 0.768 ± 0.035; YZ18, 0.836 ± 0.028) or presence of increasing competitor peptide concentrations. Values are corrected for nonspecific binding observed in diluent instead of the mAbs. A noninhibitory control peptide is shown in each panel.

Nishiyama, Y. Planque, S. Mitsuda, Y. Nitti, G. Taguchi, H. Jin, L. Symersky, J. Boivin, S. Sienczyk, M. Salas, M. Hanson, C.V. Paul, S. (2009). Toward Effective HIV Vaccination. The Journal of Biological Chemistry, 284, 30627-30642.

Figure 3 Specific binary reactivity of mAb YZ23 with probes of the 292–306 and 421–433 peptide regions determined by ELISA.

A, structure of E-288–306 3, 292–306 peptide 4, E-421–423 5a, biotinylated E-416–433 6a, the control shuffled (Sh) sequence probes 7 and 8, and control E-VIP 9. Probes 5b and 6b are BSA-conjugated E-421–433 and E-416–433, respectively. E1 and E2 denote the electrophilic phosphonate groups. Bt indicates 6-biotinamidohexanoyl. B, mAb YZ23 binding to 292–306 peptide 4 and E-421–433 5b determined by ELISA (mean ± S.D. of duplicates). Control mAb SK-T03 binds an irrelevant gp120 epitope. C, Fab YZ23 binding to 292–306 peptide 4 and E-421–433 5b determined by ELISA (mean ± S.D. of duplicates). Control Fab SK-T03 binds an irrelevant gp120 epitope. Inset, silver-stained Fab YZ23. D, scFv YZ23 binding to 292–306 peptide 4 and E-421–433 5b determined by ELISA (mean ± S.D. of duplicates). Control pHEN2 is the extract of bacteria harboring the vector devoid of the scFv purified by the same procedure as scFv YZ23. Inset, silver-stained (lane 1) and anti-c-Myc-stained (lane 2) SDS-electrophoresis lanes of the purified scFv YZ23.

Nishiyama, Y. Planque, S. Mitsuda, Y. Nitti, G. Taguchi, H. Jin, L. Symersky, J. Boivin, S. Sienczyk, M. Salas, M. Hanson, C.V. Paul, S. (2009). Toward Effective HIV Vaccination. The Journal of Biological Chemistry, 284, 30627-30642.

Figure 4 Specific binary reactivity of mAb YZ23 with E-288–306 and E-421–433 determined by denaturing electrophoresis.

A, streptavidin-peroxidase-stained reducing SDS-electrophoresis lanes showing reaction mixtures of mAb YZ23 (75 μg/ml) incubated for 6 h with E-288–306 3, the control E-VIP probe 9, E-421–433 5a, and control shuffled Sh-E-421–433 peptide 7 (10 μM). Also shown are the corresponding control mAb reactions (IgG CRL1689). B, time course of E-288–306 and E-421–433 binding by mAb YZ23. mAb YZ23 (150 μg/ml) treated with E-288–306 3 or E-421–433 5a (10 μM) for varying lengths of time was subjected to SDS-electrophoresis and the mAb-peptide adducts were detected as in A. Band intensities quantified by densitometry are shown in duplicate. Biotin quantification was by comparison with a standard curve constructed using biotinylated BSA. Bmax is the extrapolated maximum binding obtained by fitting the data to the first-order equation (r2 for E-288–306- and E-421–433-binding curves, 0.97 and 0.99, respectively). C, ternary complexation of mAb YZ23 with E-288–306 and E-421–433. mAb YZ23 was reacted with E-288–306 3 alone (3 h, left bar) or E-288–306 3 (3 h) followed by E-421–433 5a (8 h, right bar). Peptide adducts were detected, and reaction stoichiometry was determined as in B.

Nishiyama, Y. Planque, S. Mitsuda, Y. Nitti, G. Taguchi, H. Jin, L. Symersky, J. Boivin, S. Sienczyk, M. Salas, M. Hanson, C.V. Paul, S. (2009). Toward Effective HIV Vaccination. The Journal of Biological Chemistry, 284, 30627-30642.