Bioaffinity Based Immobilization of Enzymes - Advances in Biochemical Engineering Biotechnology - page 207

While no attempts were made to compare the binding capacity of matrices,
enzyme immobilized on either support were shown to exhibit full catalytic
activity,improvement in stability,and no alteration in m ,V max and K i values.
The preparation obtained by direct covalent coupling of carboxypeptidase to
support however exhibited relatively lower activity. It was also observed that
orientation of the antibody on protein A supports may not after all enhance its
enzyme binding activity. The author reasoned that even the most accessible
antibody molecule may not bind to more than one enzyme molecule due to the
large dimensions of the latter.
Favourable orientation of antibodies also appears possible on immobilized
metal ions supports. Hale and Beidler [80] observed that an innate histidine-rich
sequence located in the C-terminal portion of the Fc region,well-conserved in
every antibody classes investigated from several species,binds strongly to the
Co 2+ -IDA resin. Thus,antibodies bound on the supports are oriented with their
combining site directed away from the resin and facilitate maximum antigen
binding [81].
The need for the minimization of non-specific adsorption and optimization
of the binding of the enzymes to antibody support has also been addressed in
some studies. DeAlwis et al. [13],and deAlwis and Wilson [82] suggested the use
of Fab' fragments instead of the intact antibody. As the immobilization of anti-
body via its side chain amino groups involves the risk of modifying or blocking
the antibody binding sites,coupling of the Fab' fragments through the hinge
region thiol groups on supports activated with 2,2,2-trifluoroethanesulfonyl
chloride [82] or maleimide [71] was also proposed. At pH 6.0 most amino acid
side chain amino groups of protein are protonated and hence unavailable for
reaction with tresyl activated supports. Coupling therefore occurs preferentially
and predominantly via the thiol groups [82].
2.3.3
Use of Secondary Antibodies
Several investigators have also utilized secondary antibodies in addition to the
primary antibodies for immunoaffinity enzyme immobilization. Two strategies
were employed by deAlwis et al. [13] for the immunoaffinity immobilization of
glucose oxidase in a flow injection system using an immunological reactions.
The first of these involved immobilization of human IgG on activated controlled
pore glass followed by the binding of enzyme-anti IgG conjugate. Alternatively,
a monoclonal antiglucose oxidase antibody immobilized on the support pre-
coupled with Fab' fragment of antimouse IgG was used as an immunoadsorbent
for the preparation of immobilized glucose oxidase. The anti IgG-glucose oxi-
dase conjugate used in the first procedure comprised of a mixture of those in
which a single enzyme molecule was linked to one,two or even three IgGs [83].
Their binding onto the IgG support was apparently multipoint and gave a more
stable immobilized preparation. The second approach on the other hand yielded
a far more versatile support capable of binding all IgGs. Both types of bio-
reactors were effectively used for the measurement of glucose concentration in
undiluted sera. deAlwis and Wilson [20] immobilized avidin on Reactigel on