Bioaffinity Based Immobilization of Enzymes - Advances in Biochemical Engineering Biotechnology - page 204

immobilization of an enzyme. Ikura et al. [16] observed that activity of guinea
pig liver transglutaminase affinity bound on monoclonal antienzyme antibody
support was higher than that bound to matrix precoupled with polyclonal anti-
bodies. The authors however have not reported if the polyclonal antibody pre-
paration used contained inhibitory or labilizing antibodies. More detailed com-
parison is however available in case of non-enzyme proteins- bovine and human
serum albumins. After investigating the effects of a variety of factors on the
adsorption equilibrium between immobilized polyclonal and monoclonal anti-
bodies and the antigens it was concluded that the former showed a homoge-
neous affinity of Langmuir type while the later was heterogeneous. It was also
observed that binding on immunoadsorbants prepared using polyclonal anti-
bodies may be relatively stronger and their dissociation more difficult due to the
binding of the antigen to more than one kind of antibodies [43,44]. In instances
where a single enzyme has the possibility of interacting with more than one
molecule of antibody as is the situation during soluble [22] or insoluble immu-
nocomplex formation [15,28],non inhibitory polyclonal antibodies may offer
considerable advantage. In such situations polyclonal antibodies recognising
more than one epitope of the enzyme may fix the native conformation of the
later and hence confer greater stability against denaturation [22,28]. It is now well
recognized that enzymes attached to support via multiple covalent [45] or non-
covalent associations [46] exhibit remarkably higher stability than those attached
via fewer linkages. Additive stabilising effects of some monoclonal antibodies have
also been demonstrated recently [47]. Monoclonal antibodies may have shorter
half lives [48] and as compared to the polyclonals they may be more labile [20].

2.2
Selection of the Antibodies

Animals immunized with enzymes may respond by producing a spectrum of
antibodies differing not only in their specificities towards various epitopes but
also with respects to their affinities. The dissociation constants of antigen and
antibody complexes have been shown to vary between 10 -3 -10 -14 dm -3

[49]. Antibodies of intermediate avidities are considered optimal for preparative
affinity chromatography in order to ensure adequate binding specificity and
good recovery of the bound enzyme/protein antigen [50,51] and those in the
range of 10 -6 -10 -10 mol

mol ◊

dm -3 are taken as particularly useful [52]. Antibodies
with relatively higher affinities may be better suited for the immobilization of
enzyme,as in case of immunodiagnostic analysis where essentially irreversible
binding is required. Monoclonal antibodies exhibiting affinities falling within
the required ranges can be readily isolated by immunoaffinity purification from
the chosen hybridoma clones [53,54]. Reports describing fractionation of the
polyclonal antibodies based on their affinity towards the antigenic determinants
are also available [33,55,56]. Such fractionation is however of only limited value
if the desired antibody population does not constitute a major fraction of those
present in the antiserum.
Some attempts have also been made to obtain polyclonal antibodies of de-
sired affinity from the sera of immunized animals. For instance,polyclonal

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