Re-engineering of the PAM1 phage display monoclonal antibody to produce a soluble, versatile anti-homogalacturonan scFv

Antibody phage display is an increasingly important alternative method for the production of monoclonal antibodies (mAbs) and involves the expression of antibody fragments (scFvs) at the surface of bacteriophage particles. We have previously used this technique to generate a phage mAb (PAM1phage) with specificity for the un-esterified regions of the homogalacturonan backbone of pectic polymers. Although phage particles are essential during mAb selection and amplification, their large size results in phage mAbs being poor probes for immunocytochemistry. In order to overcome this and to extend the utility of the PAM1 mAb, we describe here the production of a phage-free, soluble scFv version of the PAM1 mAb (PAM1scFv). Using the new PAM1scFv probe, the occurrence of the HG epitope recognized can now be localized with high resolution within micro-domains of plant cell walls