Applying recombinant antibodies for quality control of mixed-culture peanut bradyrhizobial inoculant

Bradyrhizobium is a nitrogen-fixing bacterium that crucially supports peanut production as a biofertilizer. Using mixed-culture inoculant proves beneficial for field application. This study explores the application of recombinant human single-chain fragment variable (scFv) antibodies for examine and monitor the quality of a mixed-culture inoculant. Bradyrhizobium sp. DASA03028 is one of the five elite strains recommended by the DOA for peanut production in Thailand. Using phage display technology, a specific scFv antibody, yi028-F11, was developed against strain DASA03028 without reacting with other tested bradyrhizobia. At a concentration of 20 µg/mL, the scFv antibody yi028-F11 detects bacteria as low as 10⁴ cells. The binding of the antibody depends on the colony's age, where the colonies that are older or younger than 7 days show a reduced enzyme-linked immunosorbent assay signal. Additionally, the study showcased the application of recombinant scFv antibodies through the fluorescent antibody technique to evaluate viable cell counts of Bradyrhizobium strains in liquid and peat-based mixed-culture inoculants. Interestingly, yi028-F11 could distinguish between the two strains only in bacteroid form inside mung bean nodules, but not in peanut cultivars, representing an initial observation of host-associated differences in bacteroid detection. Dot blot analysis indicated differences in the binding properties of the two antibodies. While the yi028-F11 scFv antibody binds to the exopolysaccharide (EPS) on the surface of strain DASA03028 cells, the yiN92-e10 scFv binds to a protein on strain SUTN9-2. Despite the diagnostic limitations, the findings reaffirm the uniqueness of individual plant hosts' nodule formation and bacteroid differentiation, impacting the binding of scFv antibodies. This study demonstrates the application of recombinant scFv antibodies as a quality control tool for single- and mixed-culture peanut bradyrhizobial inoculants.