Synthesis and Characterization of Artificial Antigens for Copper and Application for Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay

Abstract

Copper was conjugated to two carrier proteins using penicillin G sodium salt to synthesize artificial antigens for copper. The antigens were identified by ultraviolet spectrometry, circular dichroism, and inductively coupled plasma–optical emission spectrometry. The results of ultraviolet spectrometry showed characteristic absorption peak shifts between haptens and carrier proteins. Circular dichroism showed that the secondary structure of the conjugates was an α-helix. These results suggested that the artificial antigens for copper were synthesized successfully. The polyclonal antibodies of copper were produced by immunizing New Zealand white rabbits with copper antigens. An indirect competitive enzyme-linked immunosorbent assay based on the polyclonal antibodies was developed. The concentration of copper ion was quantified based on the ability of its ethylenediaminetetraacetic acid complexes to inhibit the binding of the goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase and, subsequently, color formation in the assay. The assay showed an IC50 value of 0.475 microgram per milliliter with a detection limit of 0.007 microgram per milliliter. The antibody showed high affinity with copper and low cross-reactivity with other metals. The recovery of copper from fortified water was between 78.0 and 122 percent. The results indicate that the assay is a convenient supplemental analytical tool for monitoring copper ions in aquatic environments.