Development of a double sandwich ELISA able to discriminate between free PAP (pokeweed antiviral protein) and complexed PAP in leaf extracts

Pokeweed antiviral protein (PAP) is a soluble leaf protein produced by the plant Phytolacca americana. It is able to specifically remove an adenine re sidue from the 28 S ribosomal RNA of eukaryotic ribosomes, thus blocking pr otein synthesis. Although PAP seems to be synthesized constitutively at ver y high- concentrations during plant development, the mechanism of its synth esis remains unclear since the protein is toxic to pokeweed ribosomes. Rece ntly, our group identified an inactive complexed form of PAP (PAPi) which c ould play a significant role in the plant protection mechanism during PAP s ynthesis. In order to estimate the concentration of PAP in plant extracts, a double sandwich ELISA was developed. This test enabled the detection and the quantification of PAP in a crude leaf extract at concentrations ranging from 2-1000 ng ml(-1). It was specifically developed to discriminate betwe en free PAP and PAPi. This report shows that the use of a monoclonal antibo dy specific to the C-terminal extremity of mature PAP, combined with an ant i-PAP polyclonal antibody did not detect PAPi, unless it was denaturated pr ior to ELISA. Thus, the specificity of this test was restricted to free PAP and the test proved to be a reliable and unique tool to estimate the integ rity of PAPi. (C) 1999 Elsevier Science B.V. All rights reserved.