Bioaffinity Based Immobilization of Enzymes - Advances in Biochemical Engineering Biotechnology - page 203

clonal antibody population is however more of a rule than exception,although
this can be advantageous in some situations. While methods exist for the
fractionation of polyclonal antibodies recognising different epitopes of a single
antigen or those of differing in affinity for a single epitopes [32,33],handling of
large volumes of antisera may be problematic especially if the required antibody
constitutes only a minor fraction of the total antibody population. For most
enzyme immobilization applications,however,heterogeniety of the antibody
population may not cause any serious problem provided they do not comprise
of inhibitory and/or labilizing antibodies. Formation of active site recognising
and hence inhibitory antibodies is quite likely if an animal is immunized with a
native enzyme [17,34,35],although several reports describing the non-inhibi-
tory nature of the antisera raised against several enzymes are available [14,24,
28,36 - 39]. Some plausible explanations offered for the absence of inhibitory
antibodies in the antisera include: the active site acting as blind spot for the
immune system,steric hinderance by high affinity antibodies recognising adja-
cent locations of active site directed antibodies and continued accessibility of
the active site in the complex formed between active site recognising antibodies
and the enzyme [22].
Some ingenious techniques for the prevention of the formation of active site
directed polyclonal antibodies have also been described in the recent years.
Fusek et al. [21] immunized pigs with active site blocked chymotrypsin pre-
pared by treating the enzyme with diisopropylphosphofluoridate. The non-in-
hibitory antibodies were isolated from the sera of immunized animals using
chymotrypsin coupled to Sepharose via its active site as the affinity adsorbent.
More recently Stovicova et al. [26] raised non inhibitory anti-trypsin antisera in
pigs by immunizing them with trypsin complexed with its specific inhibitor
antilysin. The IgG fraction isolated from the sera of the immunized animals on
coupling to Sepharose support yielded an immunosorbant that immobilized
trypsin without decreasing its catalytic activity.
The potential of polyclonal glycoenzyme glycosyl recognizing antibodies in
the immobilization of glycoenzymes has also been demonstrated. It was envis-
aged that since enzyme glycosyls participate rarely if at all in catalytic function
[40],glycosyl recognising antibodies may not be inhibitory and hence useful in
the immobilization of enzymes. Convenient procedures are available for raising
antiglycosyl polyclonal antibodies against glycoproteins and glycoenzymes [29,
41,42]. Briefly the strategy involves preparation of neoglycoproteins comprising
of the oligosaccharides of the enzyme in question and a polypeptide region of a
simple protein like BSA. Animals are immunized against the neoglycoproteins
and their antisera passed through affinity column of a suitable support to which
the enzymes are coupled. Only the antibodies exhibiting affinity towards the
enzyme glycosyls are retained in the column and they are eluted using appro-
priate chaotropic agents or by change in pH [29]. Glycosyl recognising polyclonal
anti-invertase antibodies were found to be non-inhibitory towards the enzyme
and more effective in the immobilization of the enzyme than those recognising
the polypeptide domains [29,30].
To my knowledge there exists a single report describing the direct compari-
son of the relative effectiveness of monoclonal and polyclonal antibodies in the