Bioaffinity Based Immobilization of Enzymes - Advances in Biochemical Engineering Biotechnology - page 206

Preparation of immunoaffinity adsorbents require reasonably pure antibodies
and usually salt/solvent fractions of antisera [28,33] or affinity purified polyclo-
nal/monoclonal antibodies [18,22] are employed.Antibody purification strategies
have been reviewed extensively [53,65] and do not fall with in the purview of
this article. It is however of interest to point out of a novel strategy employing
thermo-sensitive immunomicrospheres that appear highly effective in the large
scale purification of antibody from the sera of immunized animals [66,67].
Majority of the immunoadsorbents constructed for the purpose of enzyme
immobilization comprise of appropriate supports to which are attached the
antibodies using group specific reagents [23,26 - 28,67]. While such immuno-
adsorbents serve as efficient supports,random binding of the antibodies on to
the support may remarkably lower the ability of the antibody to bind the enzy-
me. For instance Ikura et al. [16] have shown that the binding capacity of the
anti-transglutaminase monoclonal antibody for the enzyme may be lowered to
about one fifth on random coupling to Affi-Gel10. Several attempts have there-
fore been made to favourably orient antibodies on the support matrices.
2.3.2.1
Favourable Orientation of Antibodies on Supports
The oligosaccharide chains of polyclonal antibodies are primarily [68] though
not exclusively [69] located in their Fc regions. Several investigations have been
therefore directed towards immobilizing antibodies via their carbohydrates
chains for improving the accessibility of the antigen binding sites. Although
Quash et al. [70] were the first to immobilize IgG through oligosaccharide moie-
ties,Prisyazhnoy et al. [71] made the first comparison between the rabbit anti-
mouse antibodies immobilized through the -SH and via oligosaccharides and
concluded that the latter exhibited a 3-fold higher antigen binding activity. Simi-
larly,anti-human IgG immobilized on hydrazide derivative via oligosaccharide
also exhibited superior binding activity towards the IgG [72]. Little et al. [73]
have however observed that the magnitude of increase in antigen binding activi-
ty depends upon the nature of antigen-antibody pair. In a more recent study
Fleminger et al. [74] immobilized carboxypeptidase and horse radish peroxi-
dase on amino and hydrazide derivative of Eupergit C via carbohydrate chains
and achieved antigen binding activity close to the theoretical value of 2 moles
antigen bound/mole immobilized antibody. Surprisingly,comparative increase
in binding activity was not observed when attempts were made to orient mono-
clonal antibodies by immobilizing them through their oligosaccharide chains
[75,76]. This may be related to the nature and extent of glycosylation of the
monoclonal antibodies [77].
An alternative strategy of oriented immobilization IgG appears to be their
binding to the matrices precoupled with protein A that recognizes exclusively
the Fc region [78]. Solomon et al. [17] prepared,purified and characterized
several mouse monoclonal antibodies recognising carboxypeptidase and com-
pared the properties of two carboxypeptidase preparations immobilized with
the help of mouse monoclonal antibody - m100 which was either randomly
coupled to the support or oriented favourably on protein A-Sepharose [18].