Anti-HIV type 1 Polyclonal antibody[HRP]

C-X-C chemokine receptor type 4 (CXCR4) is involved in several intractable disease processes, including HIV infection, cancer cell metastasis, leukemia cell progression, rheumatoid arthritis, asthma and pulmonary fibrosis. Thus, CXCR4 represents a promising drug target and several CXCR4 antagonizing agents are in preclinical or clinical development. Important parameters in drug lead evaluation are determination of binding affinities to the receptor and assessment of their stability and activity in plasma or blood of animals and humans. Here, we designed a microtiter plate-based CXCR4 antibody competition assay that enables to measure inhibitory concentrations (IC50 values) and affinity constants (K-i values) of CXCR4 targeting drugs. The assay is based on the observation that most if not all CXCR4 antagonists compete with binding of the fluorescence-tagged CXCR4 antibody 12G5 to the receptor. We demonstrate that this antibody-competition assay allows a convenient and cheap determination of binding affinities of various CXCR4 antagonists in living cells within just 3 h. Moreover, the assay can be performed in the presence of high concentrations of physiologically relevant body fluids, and thus is a useful readout to evaluate stability (i.e. half-life) of CXCR4 ligands in serum/plasma, and even whole human and mouse blood ex vivo. Thus, this optimized 12G5 antibody-competition assay allows a robust and convenient determination and calculation of various important pharmacological parameters of CXCR4 receptor-drug interaction and may not only foster future drug development but also animal welfare by reducing the number of experimental animals.

The membrane proximal external region (MPER) of HIV-1 gp41 contains epitopes for at least four broadly neutralizing antibodies. Depending on solution conditions and construct design, different structures have been reported for this segment. We show that in aqueous solution the MPER fragment (gp160(660-674)) exists in a monomer-trimer equilibrium with an association constant in the micromolar range. Thermodynamic analysis reveals that the association is exothermic, more favorable in D2O than H2O, and increases with ionic strength, indicating hydrophobically driven intermolecular interactions. Circular dichroism, C-13(alpha) chemical shifts, NOE, and hydrogen exchange rates reveal that MPER undergoes a structural transition from predominately unfolded monomer at low concentrations to an alpha-helical trimer at high concentrations. This result has implications for antibody recognition of MPER prior to and during the process where gp41 switches from a pre-hairpin intermediate to its post-fusion 6-helical bundle state.