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To directly radiolabel an anti-hepatoma mAb fragment HAb18 F(ab’)2 with 99mTc …
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Biology Articles » Biophysics » Medical Biophysics » Direct technetium-99m labeling of anti-hepatoma monoclonal antibody fragment: a radioimmunoconjugate for hepatocellular carcinoma imaging » Discussion
Discussion - Direct technetium-99m labeling of anti-hepatoma monoclonal antibody fragment: a radioimmunoconjugate for hepatocellular carcinoma imaging
Great efforts have been made to develop a method that can be
used for the direct labeling of mAbs with 99mTc[16].
Earlier studies involved the incubation of mAbs with stannous
phthalate tartrate solution for up to 21h at room temperature, which
was named “pretinning” method. Clinical success with this method
has been claimed by the author[23].
One
aim of our study was to further evaluate the role of stannous as a
reducing agent in the direct labeling of mAb F(ab’) 2 with 99mTc.
The difference between the “pretinning” method and this method
is that we use GH instead of phthalate-tartrate as transfer ligand
and stabilizer to avoid Sn or Tc-c olloid formation. To do this, we
investigated the effect of the quantity of Sn/ GH on the labeling
time and efficiency. When the molar ratio of Sn/GH to mA b F(ab’)2
was constant, we found that there was no obvious difference on the
number of-SH between the reduction time of 20min and 30min or even
longer[ 24]. The whole labeling process can be
accomplished within 1.5h. Hnatowich et al. reported that
labeling efficiency in the case of the stannous ion-reduced a
ntibodies was generally in excess of 70%[12], however, in
our method mol ar ratio of Sn/GH to mAb was an important parameter
to obtain good labeling re sults, and molar ratio of 40∶1
or higher were needed to get labeling effici ency of more than 80%
(Table 1). The low percentage of free 99mTcO4
and radiocolloid in each sample implied that pH 5.3 and GH are the
opti mal pH value and transfer ligand. Under this condition, the
labeled mAb HAb18 F( ab’)2 keeps its immunoreactivity.
Autoradiography of SDS-PAGE had only one m igration of component
identical to that of native HAb18 F(ab’)2 determination
by staini ng with Coomassie brilliant blue R250 (Figure 2), which
demonstrated that S n/GH reduction is mild and does not destroy
interchain bridges in mAbs. Labeli ng efficiency of 2% in control
experiments using unreduced HAb18 F(ab’)2 indicated
that there was no exchange with the low affinity sites and also
demonstrated tha t reduction of disulfides is a necessary initial
step in 99mＴc
direct labeling of antibodies. The bond between SH and Tc is
stronger than that of N -Tc or O-Tc which was verified by the
challenge assay of 99mTc-HAb18 F(ab’)2 in
the presence of EDTA. We found that EDTA even at a molar rat io of
10000∶1
failed to remove a significant amount of 99mTc, this is
in agreement with the results of Rhodes et al[8].
But L-cys at 625∶1
remove one-tenth of the label (Figure 5). Despite such insta bility
of the label, there was no in vivo evidence of release of
pertechneta te due to no thyroid imaging observed in the whole
imaging process (Figure 7) . Tumor localization of 99mTc-HAb18
F(ab’)2 was successfully dem onstrated in a human
tumor/nude mouse xenograft model. Biodistribution and ima ging
results showed the highest tumor uptake at 24h post-injection. Where
as kidney levels were foun d to be higher in the whole process.
Accumulation of radioactivity in the kidney may be the result of
retention of this metallic radionuclide by the kidney pro ximal
tubule[25], the possible release of 99mTc-labeled
cysteine and gluta thione[26]stemming from the
radioimmunoconjugate catabolism, and the r elative amount of 99mTc-GH.
A technique has been used in patients to block renal tubule uptake
of 99mTc-anti-CEA Fab’ fragments by amin o acid
infusion[27].
In
conclusion, a radioimmunoimaging conjugate for hepatoma detection
was prepare d by direct labeling mAb HAb18 F(ab’)2-with 99mTc
using stannous/g lucoheptonate as reducing agent. Although the
labeling efficiency is not satisf actory to some degree, it has
several advantapes: simple, easy and quick, besid es, the labeled
mAb fragment retains its immunoreactivity. Biodistribution and
imaging studies reveal that this conjugate is useful for the
detection of hepato ma.
ACKNOWLEGDGMENTS The authors are grateful to Dr. Wang Jing
for mice imaging and the Department of Nuclear Medicine of Shaanxi
People’s Hospital for their support of this project.
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