Development of a highly sensitive icELISA to detect semicarbazide based on a monoclonal antibody

A highly sensitive and specific monoclonal antibody was prepared to detect semicarbazide (SEM). Hapten 4-{[(aminocarbonyl)hydrazono]methyl}benzoic acid (4-CPSEM) was synthesised through the condensation reactions of SEM and 4-carboxybenzaldehyde (4-CBA). The active ester method was employed to couple the hapten to bovine serum albumin, keyhole limpet hemocyanin and ovalbumin (OVA) to obtain conjugates of immunogens and coating antigens. A novel hapten 2-[(aminocarbonyl)hydrazono]acetic acid (SEM-A) was also synthesised from SEM and oxoacetic acid to prepare a heterologous coating antigen (SEM-A-OVA). The linear ranges of inhibition curves in the optimised indirect competitive enzyme-linked immunosorbent assay (icELISA) method were in the range of 0.0071–0.056 ng mL⁻¹ and 0.018–0.209 ng mL⁻¹ for coating antigens SEM-A-OVA and 4-CPSEM-OVA, respectively. The IC₅₀ was 0.019 ng mL⁻¹ for SEM (SEM in the form of 4-nitrobenzaldehyde semicarbazone) and 0.13 ng mL⁻¹ for 2-nitrobenzaldehyde semicarbazone, both of which were well below the minimum required performance limit of 1 µg·kg⁻¹ set by the European Commission.