Biology Reference
In-Depth Information
Engineered antibody fragments, such as F(ab), have been employed
for immunodetection as they retain the specificity of antibodies but offer
improvements in production cost and substrate coverage densities, and
they can accommodate systems that require small-sized receptors.
13
Initial
approaches to generation of antibody fragments utilized chemical agents for
proteolytic cleavage of certain bonds. This method was used to create anti-
body fragments for a
Cryptosporidium
biosensor.
13
Alternatively, using genetic
modifications, recombinant antibodies can be generated. Conroy provides
an excellent introduction to recombinant antibody technology and the
application to biosensors.
1
At present, there have only been a few biosensors
reported using recombinant antibodies, but this area is predicted to grow
significantly due to the advantages offered by these antibodies
1
(
Fig. 7.4
).
Immunization
Rabbit
Mouse
Chicken
cDNA
Synthesis
Serum
analysis
Extraction of B-cells
(spleen/lymph nodes)
Isolation
of mRNA
Species-
specific
primers
Generation of
hybridoma
Monoclonal
Amplification of
V
H
and V
L
Recombinant
Development
of immunization
regime
Purification
from serum
scAb
Fab
scFv
Identification of
target
Screening
Robotics,
High-throughput
Expression in
E. coll
Polyclonal
Further engineering
Mutagenesis, Reformatting
Characterization
ELISA, Biacore,
Affinity, Stability
Key
Common to all Ab forms
Polyclonal Ab generation
Monoclonal Ab generation
Recombinant Ab generation
Platform assay testing
Inter/Intra day analysis, Sensitivity,
LOD, LOQ, Specificity
Figure 7.4
Overview of antibody generation, screening, and characterization. This flow
diagram illustrates the overall steps in the generation of polyclonal, monoclonal, and
recombinant antibodies. LOD, limit of detection; LOQ, limit of quantitation. Source:
Figure 4 from Ref.
1
. Reproduced with permission.
(For color version of this figure, the
reader is referred to the online version of this topic.)
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