We now show that the ability of defensins to bind to SP-D and
alter its functional activity appears to be confined to HNP-1–3. It
is of interest that HBDs and other some -defensins (e.g., HNP-4
and HD6) did not bind significantly to SP-D. The lack of binding
of HNP-4 may relate again to its sequence divergence from HNP-
1–3. The results obtained in the current and prior studies using
ELISA were broadly confirmed by SPR, at least for - and -defensins;
however, ELISA studies failed to detect the binding of
RC1 and RC2 to SP-D, whereas this activity was clearly evident in
our SPR studies. For technical reasons the ELISA had to be performed
with defensins bound to the plate. It is possible that binding
of RCs to plastic uses the same, presumably hydrophobic, components
of the molecule that would otherwise participate in its
binding to SP-D. In addition, having the defensins bound to the
plate prevents cooperative binding or self-association among the
defensins that could amplify binding to SP-D. SPR assays not only
provide an independent way to confirm (or not confirm) ELISA
results, they yield useful estimates of binding affinity, association
and dissociation binding constants, and molar binding ratios as
described in this study.