(de Haard et al. 1999). Phage-display antibody libraries have been constructed by

using blood samples from immunized/vaccinated human subjects or those suffering

from infections or other diseases. In addition, naive human antibody gene

repertoires, which are close to the human germline with low risk of immunogenicity,

have also been developed. Further, synthetic human antibody phage-display libraries

have also been constructed. From the phage-display library, antigen-specific binders

are selected by “panning” using antigen-coated solid surface such as ELISA plates.

Antigen-nonbinding phages are removed by stringent washing. Subsequently, the

bound specific antibody-expressing phages are eluted and re-amplified by transfec-

tion of E. coli and packaging with helper phage, which are again used for next round

of panning under higher stringent conditions of washing. Usually, two to three

rounds of panning are required to enrich specific antibody-expressing phage. The

selected antibodies can be either expressed as soluble proteins in the bacterial

expression system or the specific antibody variable regions can be excised and

cloned into whole human antibody expression vector and antibody can be expressed

using a mammalian expression system. Figure 22.2 schematically depicts the outline

of the procedure to make human antibody by phage-display library. Adalimumab

Phage-display scFv library

Binding

Antigen coated ELISA plate

Washing

Elute

Unbound phages

Amplify

Repeat 2-3 rounds

(Biopaning)

Periplasmic

fraction

Supernatant

scFV

Full length

antibody

Fig. 22.2 Outline of the procedure to generate human antibodies by using phage-display library:

The phage-display scFv library is incubated with antigen-coated microtitration plate, followed by

washing to remove the unbound phages. The antigen-binding phages are eluted, followed by their

amplification, and this process called biopanning is repeated two to three times using increasing

stringent conditions for washing to enrich phages expressing antigen-specific antibodies. The

specific antibody-expressing phages are amplified and the target antibody could be expressed either

in E. coli or the specific antibody variable regions excised, cloned into whole antibody expression

vectors and antibody expressed in mammalian cells

22

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