Bioaffinity Based Immobilization of Enzymes - Advances in Biochemical Engineering Biotechnology - page 208

which was retained the biotin bound secondary antibody. An immune complex
of the enzyme-antiglucose oxidase or antienzyme antibody followed by enzyme
was passed through a small column of the Reactigel in order to achieve immo-
bilization. In view of the remarkably high affinity of the avidin-biotin system,
antigen-antibody interactions could be conveniently disrupted for enzyme
desorption without affecting the matrix-secondary antibody interaction. The
glucose oxidase reactors prepared thus,when coupled to a flow injection analy-
sis system could be used for the sensitive and reproducible analysis of glucose.
2.4
Reusability of Immunoaffinity Supports
Relative stabilities of monoclonal and polyclonal antibodies may differ substan-
tially [20,47],yet their stability against various forms of inactivation are usual-
ly far superior than those of the antigenic enzymes. The severity of the elution
procedure permissible in the immunoaffinity purification of enzymes is there-
fore decided by its effect on the later rather than on the antibody. Since desorp-
tion of the immunoaffinity immobilized enzyme from the reactors or sensors is
undertaken only when it fails in its catalytic function,elution condition far
harsher than those employed in affinity purification of enzyme may be per-
missible. Presumably for this reason,non-specific elution procedures have been
generally applied with remarkable success for elution of enzymes immobilized
on antibody supports. These include 0.1 M phosphoric acid pH 2.0 [13],0.2 M
glycine buffer pH 2.3 [26],6.0 M urea in buffered saline,3.0 M guanidine HCl
or 0.1 M acetic acid [16] or 10 % dioxane pH 2.5 and 0.5 % (v/v) Triton X-,pH 7.0
[27]. Even while employing non-specific elution procedure for elution of enzy-
mes,the immunoaffinity supports have been used successfully for over 50 bin-
ding and elution cycles without any decrease of binding capacity [52] and for
over 200 cycles with a decrease of about half of the initial binding capacity [84].
A few instances of multiple reuse of immunoaffinity supports of bioreac-
tors/biosensor are also available. deAlwis et al. [13] eluted glucose oxidase from
a polyclonal antibody support in a flow injection analysis system at acid pH
and reloaded the enzyme for 10 cycles without any apparent loss in binding.
Similarly immunoaffinity bound urease and NADase could be eluted and fresh
enzyme bound to the reactor for 5 cycles with no decrease in binding [27]. The
binding capacity of the anti-transglutaminase antibody support remained un-
harmed after 4 elution and binding cycles [16]. In the last two studies it was also
demonstrated that specific binding of the antigenic enzyme to the appropriate
antibody support can be achieved directly and specifically from the crude
homogenates. This suggests that pure enzyme preparations may not be essential
for the immobilization of enzymes on immunoaffinity support.
Non-specific enzyme elution procedures may however gradually inactivate
and decrease the utility of antibody supports. For instance,exposure of antibody
supports to pH below 4.0 may decrease the affinity for the antigen and promote
susceptibility to proteolysis [52]. Improvement in resistance to proteolysis has
however been achieved by controlled modifications of matrix associated anti-
body with polyethylene glycol [85].