Abstract
Histamine (HA) is an important food contaminant generated during food fermentation or spoilage. However, an immunoassay for direct (derivatization free) determination of HA has rarely been reported due to its small size to induce the desired antibodies by its current hapten-protein conjugates. In this work, despite violating the classical hapten design criteria which recommend introducing a linear aliphatic (phenyl free) linker into the immunizing hapten, a novel haptens, HA-245 designed and synthesized with a phenyl-contained linker, exhibited significantly enhanced immunological properties. Thus, a quality-improved monoclonal antibody (Mab) against HA was elicited by its hapten-carrier conjugates. Then, as the linear aliphatic linker contained haptens, Hapten B was used as linker-heterologous coating haptens to eliminate the recognition of linker antibodies. Indirect competitive ELISA (ic-ELISA) was developed with a 50% inhibition concentration (IC50) of 0.21 mg/L and a limit of detection (LOD) of 0.06 mg/L in buffer solution. The average recoveries of HA from spiked food samples for this ic-ELISA ranged from 84.1% and 108.5%, and the analysis results agreed well with those of referenced LC-MS/MS. This investigation not only realized derivatization-free immunoassay for HA, but also provided a valuable guidance for hapten design and development of immunoassay for small molecules.
Highlights
Histamine (HA) is one of several biogenic amines closely associated with allergies and food poisonings
Hapten HA-245 designed with a phenyl-contained linker was synthesized by condensation of HA with 4-formyl-benzoic acid, followed by reduction of schiff base (C-N double bond) to secondary
The obtained anti-HA monoclonal antibody (Mab) was further used to develop an ic-ELISA for HA
Summary
Histamine (HA) is one of several biogenic amines closely associated with allergies and food poisonings. The anti-NPHA antibody shows no binding ability to Hapten B-OVA conjugate with the observed absorbance being at a blank level, suggesting the detrimental effect of modification of the amide (in Hapten C, Scheme 1B) to a secondary amine (in Hapten B, Scheme 1B). These results meant the incorporation of phenyl into immunizing hapten could significantly improve immunological properties of resultant anti-hapten immunogen.
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