Diagnostics of egg-based paintings in cultural heritage: an innovative combined dot-ELISA and UPLC-based amino acid analysis approach

In this work we want to highlight the capability of two different techniques, a modified Dot-Enzyme-Linked ImmunoSorbent Assay (Dot-ELISA) and an amino acid analysis performed by the new methodology AccQ•Tag Ultra UPLC (Ultra Performance Liquid Chromatography). The Dot-ELISA is a highly versatile solid-phase immunoassay for antibody or antigen detection. The technique is based on the amplification of the immunochemical reaction between an antigen, dotted onto solid support, and a primary antibody, using an enzyme-conjugated secondary antibody that binds to the primary one. The addition of a precipitable, chromogenic substrate causes the formation of a colored dot on the solid phase which is visually read. The determination of colour staining was performed by a visible light spectrophotometer on the solid support. The AccO-Tagm Ultra UPLC is a dedicated technology useful for amino acids analysis based on a pre-column derivatization method with 6-aminoquinolyI-N-hydroxysuccinimidyl carbamate (ADC). The techniques involves first an hydrolysis, a derivatization process and a separation/detection in UPLC. Moreover, the UPLC allows an increase in speed. operating ease, sensitivity. and resolution with respect to traditional HPLC of the oldest amino acid derivatization techniques. Different concentrations of ovalbumin, whole egg, and egg white samples and specially prepared specimens of various tempera paintings as model samples (not pretreated) were used in both the techniques. Both methods were demonstrated to be valid in detecting protein binder in analysed samples.