Background We identified five Cryptomeria japonica trees producing Cry j 1 isoforms that cannot be detected in a sandwich ELISA using two monoclonal antibodies, J1B01 and J1B07, suggesting that the binding affinity of these isoforms for both monoclonal antibodies is low.
Objectives The binding properties of the Cry j 1 isoforms produced by five trees to J1B07 and J1B01 were examined. The complementary DNA (cDNA) sequences of the Cry j 1 isoforms were also determined.
Methods To clarify the binding properties of these Cry j 1 isoforms to J1B01 and J1B07, Cry j 1 was quantified in pollen samples collected from each of the five trees, by sandwich ELISAs using polyclonal antibodies and either J1B01 or J1B07. The cDNA sequences of isoforms with different binding properties were determined. To test the assumption that amino acid substitutions affect the binding affinities of Cry j 1 isoforms for monoclonal antibodies, cleaved amplified polymorphic sequences (CAPS) markers representing the putative polymorphisms were used to analyse additional trees.
Results Four of the five trees produced Cry j 1 isoforms with extremely low binding affinity for J1B07, whereas the other tree produced two different isoforms with low binding affinity for either J1B01 or J1B07. Cry j 1-encoding cDNA sequences for one of the four trees and for the exceptional fifth tree indicate that amino acid substitutions at positions 55 and 352 in mature Cry j 1 affect its binding to J1B01 and J1B07, respectively. This was supported by the results of CAPS analysis.
Conclusion The existence of Cry j 1 isoforms with low binding affinity for either J1B01 or J1B07 was established. Furthermore, a single amino acid substitution is involved in this difference in binding affinity for each monoclonal antibody.