294
JOHANN PETER HOSSLE
• Antibody choice:
Both monoclonal and polyclonal antiborlies have been used successfully.
Ideally, the use of several monoclonal antibodies, which recognize distinct epitopes, would be most advantageous. Because expressed fusion
proteins may contain only portians (preferentially C-terminal) of the
desired protein, utilization of a single monoclonal antibody carries
the risk of missing positive clones. In practice polyclonal antisera rather
than a mix of monoclonal antibodies are often used. In any case it is of
paramount importance to test beforehand, whether partially denatured
antigen bound onto ftlter can be recognized by the antibody that will be
used.
• Antigen detection:
Sensitivity is critically dependant on the quality (titer, binding characteristics and preparation) of the antibody used. Antibody dilutions must
be optimized individually. A dilution 1/100 of the primary antibody is
useful to start with. In most cases, non-radioactively labeled secondary
antiborlies ( e.g. peroxidase or phosphatase-conjugated) work as well as
radiolabeled antibodies. The amount of antigen bound to the filter replica after plaque lift-off is in the range of 50 to several hundred picograms, depending on expression and stability of the fusion protein in the
host cells (Mierendorf et al. 1987).
• Preadsorbtion of antibodies:
Since IgG often binds non-specifically to E. coli proteins it may be necessary to preadsorb the primary as well as secondary antibody using
immobilized host cell extracts ( e.g. bound to a cyanogen bromide activated matrix).
• Plaque density:
affects the ability to detect specific recombinants with antibody. Plaque
density should not exceed 3 to 4 x 10 4 plaques per 90 mm petri dish.
Screening based on function
In addition to immunological screening other approaches can be used to
screen cDNA expression libraries. Possibly the most appealing are screening strategies that are based on the biological function of the protein of interest. For example, expression of receptors can be detected by screening
with a radiolabeled ligand (Sikela and Hahn 1987). Screening by antigen
binding permits identification of monoclonal Fab fragments in combina-
JOHANN PETER HOSSLE
• Antibody choice:
Both monoclonal and polyclonal antiborlies have been used successfully.
Ideally, the use of several monoclonal antibodies, which recognize distinct epitopes, would be most advantageous. Because expressed fusion
proteins may contain only portians (preferentially C-terminal) of the
desired protein, utilization of a single monoclonal antibody carries
the risk of missing positive clones. In practice polyclonal antisera rather
than a mix of monoclonal antibodies are often used. In any case it is of
paramount importance to test beforehand, whether partially denatured
antigen bound onto ftlter can be recognized by the antibody that will be
used.
• Antigen detection:
Sensitivity is critically dependant on the quality (titer, binding characteristics and preparation) of the antibody used. Antibody dilutions must
be optimized individually. A dilution 1/100 of the primary antibody is
useful to start with. In most cases, non-radioactively labeled secondary
antiborlies ( e.g. peroxidase or phosphatase-conjugated) work as well as
radiolabeled antibodies. The amount of antigen bound to the filter replica after plaque lift-off is in the range of 50 to several hundred picograms, depending on expression and stability of the fusion protein in the
host cells (Mierendorf et al. 1987).
• Preadsorbtion of antibodies:
Since IgG often binds non-specifically to E. coli proteins it may be necessary to preadsorb the primary as well as secondary antibody using
immobilized host cell extracts ( e.g. bound to a cyanogen bromide activated matrix).
• Plaque density:
affects the ability to detect specific recombinants with antibody. Plaque
density should not exceed 3 to 4 x 10 4 plaques per 90 mm petri dish.
Screening based on function
In addition to immunological screening other approaches can be used to
screen cDNA expression libraries. Possibly the most appealing are screening strategies that are based on the biological function of the protein of interest. For example, expression of receptors can be detected by screening
with a radiolabeled ligand (Sikela and Hahn 1987). Screening by antigen
binding permits identification of monoclonal Fab fragments in combina-