Polyvinylpolypyrrolidone Reduces Cross-reactions between Antibodies and Phenolic Compounds in Enzyme-linked Immunosorbent Assay for the Detection of Ochratoxin A

Introduction: Ochratoxin A (OTA) occurs in a variety of foods worldwide, and has been shown to be nephrotoxic and carcinogenic in animals.  Enzyme-linked immunosorbent assay (ELISA) has emerged as a rapid test with its high sensitivity and low cost.  However, presence of phenolic compounds, which have structural similarities to OTA, in foods may result in false positive or erroneous quantitative measurements due to the interference or cross-reaction between OTA and antibody in ELISA.

Purpose: Polyvinylpolypyrrolidone (PVPP), a fining reagent in wine, can reduce anthocyanin and other phenolic compounds. The objective of this project was to test the efficacy of PVPP in reducing cross-reaction between OTA and phenolic compounds in quantification of OTA.

Methods: The PVPP powder was mixed with 5 mg/ml of gallic acid and 100 ng/ml of OTA in  50% methanol; 1 g of pistachio skins and different PVPP amount were mixed with 4 ml of 50% methanol. The mixed sample was agitated with a wrist action shaker at high speed for 15 min, followed by centrifugation. The concentration of OTA and gallic acid were assayed using high performance liquid chromatography (HPLC), and cross-reactivity was assayed using commercial ELISA kits.

Results: HPLC analysis results of OTA and gallic acid concentrations demonstrated that 100 mg/ml PVPP could remove 95% of 5 mg/mL gallic acid while keeping OTA levels constant. Ten mg/ml, 25 mg/ml, and 50 mg/ml of PVPP reduced the cross-reactivity of pistachio skins (4.6 mg of total phenolic compounds/ml of extract) by 14%, 37%, and 80%, respectively. One hundred fifty mg/ml of PVPP was shown to completely remove the cross-reactivity of pistachio skins.

Significance: The results demonstrated the usefulness of PVPP addition extract method for decreasing cross-reactivity in OTA ELISA kits by binding with phenolic compounds.