The interaction between vitamin K-dependent protein S and the C4b-binding protein (C4BP) was studied using surface plasmon resonance and genetic engineering. The affinity, as well as association and dissociation rates of the complex, was measured for human and bovine protein S at five different calcium concentrations. The binding to C4BP of six protein hybrids containing different parts of coagulation factor IX and protein S was studied in the absence and presence of calcium. The results show that dissociation of the human protein S-C4BP complex is extremely slow in the presence of > or = 10 microM calcium (k(off) = 7 x 10(-6) s(-1)) and the association rate constant is k(on) = 7 x 10(4) M(-1) s(-1). Human and bovine protein S were found to bind to human C4BP with the same affinity, K(D) = 0.1 nM, but the rates of association and dissociation were higher for the bovine protein S (k(on) = 2 x 10(5) M(-1) s(-1), k(off) = 2 x 10(-5) s(-1)). In the absence of calcium, the affinity for C4BP was reduced by a factor of 65 for human protein S and by a factor of 40 for bovine protein S. The decreased affinity could be mainly attributed to an increased off-rate (12-17-fold), while the on-rate decreased 3-4-fold. The studies using chimeric proteins show that the portion of protein S that is responsible for binding to C4BP is fully contained in the two laminin-G-type repeats, which are homologous to the sex hormone binding globulin (SHBG). All hybrids that contain the laminin-G-type repeats bind to C4BP with the same affinity as recombinant protein S, whereas hybrids lacking these repeats show no detectable binding to C4BP. The present data also suggest that the effect of calcium on the C4BP-binding properties is mediated by calcium binding site(s) in the laminin-G-type repeats.
The relation between domain structures of recombinant monoclonal antibody fragments and their reaction kinetics was studied for the first time using a novel biosensor based on surface plasmon resonance technology. The association and dissociation rate constants of Fab, Fv and single domain (VH fragment) anti-lysozyme antibodies were determined and compared to the intact monoclonal antibody. Fab and Fv fragments showed similar reaction kinetics and had affinity constants of 6 x 10(9) M-1 and 25 x 10(9) M-1, respectively. The single domain antibody had significantly different reaction kinetics compared to the fragments consisting of paired heavy and light chain domains. The VH domain had both a higher dissociation and a lower association rate constant, which resulted in an affinity constant approximately 250 times lower than the Fab fragment. This rapid evaluation of antibody reaction kinetics should prove to be an important selection parameter when comparing antibody fragments for their utility in therapeutic or other applications.
SUMMARYIn vitro antibody responses to a synthetic immunogen, consisting of both a B cell [V3 loop of gp120 from human immunodeficiency virus type 1 (HIV-1)] and a T-helper epitope (15 amino acids of tetanus toxoid) was studied. The in vitro activation was performed by primary and secondary in vitro immunizations, using lymphocytes obtained from uninfected, seronegative donors. Analysis of the in vitro immune response demonstrated an antigen-specific isotype switch, which was dependent on the presence of antigen-specific T-helper cells, CD40 ligation and antigen. Antibody libraries were constructed from cells derived directly from the naive donors, or from primary or secondary in vitro immunized B cells. Five libraries were displayed on filamentous phage and selected for anti-V3-specific Fab fragments, using a selection approach that linked recognition and phage replication. A panel of 19 recombinant antigen-specific Fab, representing different phases of the humoral in vitro immune response, were sequenced, expressed and analysed using a biosensor. Recombinant Fab fragments derived from cultures on day 12 exhibited an increase in affinity of close to two orders of magnitude compared to those obtained from cells primary immunized for 7 days. This study provides the first evidence that an antigen-specific in vitro immune response can yield high-affinity immunoglobulinG antibodies.
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