Bioaffinity Based Immobilization of Enzymes - Advances in Biochemical Engineering Biotechnology - page 205

antibodies derived from rabbits until 20 weeks of priming were shown to be of
low or intermediate affinity compare those isolated after longer durations [57,
58]. Emomoto et al. [59] have more recently demonstrated that binding to the
enzyme of antibodies from primary response is far more sensitive to pH and
ionic strength alterations than those from the secondary response indicating
significant differences in the nature of binding.
2.3
Immunoaffinity Enzyme Immobilization Strategies
A variety of approaches have been adopted for the immobilization of enzymes
with the help of antibodies. These range from simple complexing of enzymes
with their antibodies to form insoluble complexes to enzyme immobilization on
matrices with oriented antibodies.
2.3.1
Immobilization Without Solid Support
Enzyme-antibody complex formation represents the simplest among the im-
munoaffinity immobilization procedures and the immunocomplexes can be
readily formed simply by mixing of the enzyme solution with the antibody or
even antiserum. Interestingly neither pure enzyme nor pure antibody may be
required for the formation of immunocomplexes. Several early [14,36,39] and
some recent studies [22,24,28] indicate high retention of catalytic activities by
various enzymes in the immunocomplexes and marked stability enhancement
against various forms of inactivation. The small particle dimensions of the
enzyme-antibody complexes may however lead to their compact packing and
consequently to slow flow-rates in the column reactors. Their usefulness can be
however remarkably enhanced by entrapping the complexes in a polymeric
matrix [60,61].
2.3.2
Construction of Immunoadsorbents
Majority of immunoaffinity enzyme immobilization studies employ specific anti-
bodies coupled to appropriate porous/non-porous solid supports in order to help
facilitate ready masstransfer,heat transfer,etc. and offer good flow characteristics.
The matrix associated antibody generally acts as a large spacer contributing
remarkably to the accessibility of the immobilized enzyme. A repository of useful
information on the preparation of immunoaffinity supports is available from the
studies aimed at developing immunoaffinity adsorbents for enzyme purification.
Since the first deployment of immobilized antibody in enzyme purification [62],
a large number of variations and adaptations for the improvement of the strategy
have appeared. Excellent reviews are available on the subject [63,64]. More recently,
Ehle and Horn [65] and Desai [52] reviewed comprehensively the work in the area
including that on the choice of antibodies,preparation of immunosorbents and
optimal conditions for binding and elution of enzymes from the support.