Biomedical Engineering Reference
In-Depth Information
the loading wells (Fig.
4c
, lane 4) while reduced conditions allowed quantum dots
to run through the gels (Fig.
4c
, lanes 2, 3, and 5). Some amount of antibodies did
transfer in nonreduced conditions for biotin-streptavidin, IgG-quantum dot com-
plexes because the reducing agents in the running buffers and the gel caused the
light chain to dissociate in the same manner as for the covalent conjugation.
However, since all bands were much stronger in the biotin-streptavidin method in
general, bands for the non-reduced condition were correspondingly stronger. Bands
in non-DTT-treated antibody lanes (i.e., Fig.
4c
, lanes 7, 9, and 10) show the reduc-
tion process in greater detail since reduction agents in the running buffers reduced
the antibodies less effi ciently than DTT-treated conditions (Fig.
4a
, lanes 2, 3, 5, 6,
and 8).
Based on these data and the qualitative models introduced above that describe
the different putative binding scenarios for antibodies directly covalently conju-
gated to quantum dots and for antibodies bound to quantum dots via biotin and
streptavidin (Fig.
3b
), we derived the average number of functional IgG conjugated
to quantum dots. We use the term “functional antibody” to describe the amount of
Fc light chain, which includes a part of the target protein-binding epitope that is
physically oriented outward from a quantum dot and presumably able to interact
with its ligand. Molecularly, roughly, the fi rst 110 amino acids at the amino-terminal
end of both heavy and light chains form the variable V regions which contain highly
variable segments called complementary-determining regions. The pairwise asso-
ciation of V regions from both heavy and light chains is what actually forms the
antigen-binding site. As such, only a partial fragment bound to the quantum dot
would be functional. Furthermore, because of the structure of the antigen-binding
site, a partial fragment covalently bound to the quantum dot oriented with the light
chain facing the nanoparticle would almost surely prevent ligand binding. Since it
is the Fc light chain portion of the antibody that actively binds to proteins, quantify-
ing the amount of light chain fragments not directly bound to quantum dots and
oriented outward gives a good approximation of the functional activity of antibody-
quantum dot complexes.
To determine the number of functional IgG bound to quantum dots, we measured
the density of the 25-kDa light chain bands and compared them to controls of known
antibody concentrations. Using image analysis software that measures the band
density of electrophoresis gels (ImageQuant TL, GE Healthcare), we fi tted curves
to known concentrations of unconjugated IgG to obtain standard curves of IgG band
densities (Fig.
4a and b
; all r
2
³ 0.89). Using these curves, we then determined the
concentration of IgG bands associated with covalently bound IgG and 2:1 and 1:1
IgG:quantum dot molar ratio streptavidin-biotin conjugation conditions (Fig.
5c, d
).
Finally, we calculated the number of functional antibodies bound to the quantum
dots for each condition (Fig.
5e
).
For covalently conjugated IgG, we calculated that on average there is much less
than one antibody molecule (0.076 + 0.014) per quantum dot. In other words, adding
10 mL of antibodies directly conjugated to quantum dots is equivalent to adding
0.455 mL from a 0.5 mg/mL stock. This suggests that covalently conjugated anti-
bodies have low amounts of functionally available antibodies and are of inadequate
