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Non-Enzymatic Biological Catalysis 261
analogue for a chemical reaction, a protocol was devised for the generation
of monoclonal (i.e. single, homogeneous) catalytic antibodies, as shown in
Figure 12.7.
First of all the transition state analogue is attached to a protein such as
bovine serum albumen in order to generate a sizeable immune response. The
‘hapten’ thus formed is injected into a mouse or rabbit and the immune
response triggered. The antibody-secreting cells are then isolated, and these
cells are immortalised by fusing them with myeloma (cancer) cells. The immortalised
antibody-secreting cells are then diluted and screened for catalytic
activity, usually by a colorimetric assay. The most active cell lines are
then selected, allowing the monoclonal antibodies to be puriWed and analysed
kinetically.
The Wrst catalytic antibodies were generated using phosphonate ester transition
state analogues for the tetrahedral intermediate involved in ester hydrolysis
reactions. For example, the phosphonate ester shown in Figure 12.8 acts as a
transition state analogue for the hydrolysis of the corresponding ester. Haptens
based on this transition state analogue elicited antibodies capable of catalysing
the hydrolysis of this ester at rates 10 3 --10 5 -fold greater than the rate of
uncatalysed ester hydrolysis.
Subsequently haptens have been designed for the more challenging amide
hydrolysis reaction. Using a phosphonamidate analogue shown in Figure 12.9,
a catalytic antibody 43C9 has been elicited that is capable of catalysing the
amide hydrolysis reaction shown. Antibody 43C9 accelerates the hydrolysis of
this amide by a factor of 10 6 at pH 9.0, and the Michaelis–Menten kinetic
Reaction S [S] P
Transition state analogue S'
S'
immune
response
S'
hapten
Fuse with
myeloma cells
Isolate antibodysecreting
cells
S
P
screen for
catalytic activity
culture & dilute cell mixture
Figure 12.7 Isolation of catalytic antibodies.