Development and validation of two new sensitive ELISAs for Hesperetin and Naringenin in biological fluids

Carboxylic acid haptens were synthesised, for Hesperetin (Hesp) and Naringenin (Nar). They had a spacer arm (4 or 6 carbons long) on the C7 position. Haptens were coupled to bovine serum albumin. Polyclonal antibodies were generated. Enzyme-linked immunosorbent assays (ELISAs) were developed. Owing to sensitivity, one antibody for each flavanone was retained, namely anti-h4-Hesp and anti-h4-Nar. For Hesp and Nar, IC50 of the standard curves were 1.29 pmol/well and 0.72 pmol/well, respectively. Intra-assay and inter-assay variations were 10.24% and 10.53%, respectively for Hesp and 10.03% and 10.79%, respectively for Nar. Anti-h4-Hesp was highly specific when anti-h4-Nar showed cross-reactions with liquiritigenin and eriodictyol, which were significantly reduced in heterologous assay conditions. Assays were validated by comparisons with HPLC Coularray. Measurements were done on diet, plasma and urine samples from mice fed on diets enriched with Hesp or Nar and on a range of diluted mice urine samples.