Synthesis and characteristics of a novel artificial hapten using the copper mercaptide of penicillenic acid from penicillin G for immunoassay of heavy metal ions

Abstract

In this paper, we describe the synthesis of a novel copper ion hapten using the copper mercaptide of penicillenic acid (CMPA) derived from penicillin. Results from tests with immune rabbits indicate that: (i) A new antigen synthesized with CMPA has good stability and is safe for immunizing animals with no toxic phenomena being found in animal experiments; (ii) the immunogenic antigen (CMPA-BSA) can stimulate the immune system to produce specific antibodies with high titrations, up to 150000; and (iii) antibodies in antisera showed higher affinity to OVA-GSH-CuCl than OVA-GSH, which indicates that the antibodies have specific affinity towards copper ions. These results confirm that the novel hapten and relevant antigen for copper ion have been successfully synthesized, giving progress towards an immunoassay for copper ions in environmental and food samples. Heavy metals are a class of persistent toxic contaminants and residues found at various levels in the environment and in agricultural and industrial products [1]. Unlike carbon-based contaminants that can be completely degraded to relatively harmless products, metal ions can be transformed in only a limited number of ways through chemical or biological remediation processes [2]. What is worse, through biomagnifications, heavy metal ions existing in the environment can invade the human body and remain there for a long time, posing a serious threat to human health [3].Copper is an essential element of some metalloenzymes necessary for the normal course of biochemical processes but, like other heavy metals, it is toxic at high concentration [4,5]. All of these factors pose potential risks for the environment and human health [6] and therefore copper and other heavy metals must be monitored and identified in the environment. The traditional technologies for detecting heavy metals are instrument-based measurements such as atomic absorption spectrometry [7], inductively coupled plasma spectroscopy (ICP-AES and ICP-MS) [8] and stripping voltammetry [9]. Although these conventional instruments accurately detect the amount of metal in an individual sample, they all require expensive laboratory equipment in a centralized facility and sample turnaround is slow. In addition, instrumental analyses provide no information about the metal oxidation state.In this case, immunoassays based on the affinity and specificity of an antigen-antibody reaction offer an alternative approach in the field of metal ion measurement. Com-

Antibody developments for metal ions and their applications

“…An artificial antigen for copper was synthesized by utilizing penicillin G sodium salt as a chelating agent (Xi et al, 2015;Xu et al, 2011), similar as the concept of mercuric ion coupled to the sulfhydryl group (-SH) in glutathione in the pioneer work of Wylie et al in 1992(Wylie et al, 1992, which induced antibodies against the certain metal ions, rather than the traditional concept that antibodies preferably bound to the cagelike metal-chelate structure (we will discuss in the chapter below). Copper ions coupled to penicillin G sodium salt through the -SH group, and formed a stable copper mercaptide of penicillenic acid (CMPA).…”

“…Antibody-based assays exhibit great advantages of high efficiency, reliability, rapidness, and low cost, when compared to instrument-intensive methods (e.g. atomic absorption spectroscopy and inductively coupled plasma mass spectrometry), for metal ion detection (Date et al, 2012;Khosraviani et al, 1998;Xu et al, 2011). This technique is theoretically applicable to any hazard target, in which suitable antibodies can be generated.…”