Binding Properties of a Monoclonal Antibody Directed toward Lead−Chelate Complexes

Abstract

A monoclonal antibody (2C12) that recognizes a Pb(II)-cyclohexyldiethylenetriamine pentaacetic acid complex was produced by the injection of BALB/c mice with a Pb(II)-chelate complex covalently coupled to a carrier protein. The ability of purified antibody to interact with a variety of metal-free chelators and metal-chelate complexes was assessed by measuring equilibrium dissociation constants. The antibody bound to metal-free trans-cyclohexyldiethylenetriamine pentaacetic acid (CHXDTPA) with an equilibrium dissociation constant of 2.3 x 10(-)(7) M. Addition of Pb(II) increased the affinity of the antibody for the complex by 25-fold; Pb(II) was the only metal cation (of 15 different di-, tri-, and hexavalent metals tested) that increased the affinity of the antibody for CHXDTPA. The increased affinity was due primarily to an increase in the association rate constant. The antibody also had the ability to interact with ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), and structurally related derivatives, but with affinities from 50- to 10000-fold less than that determined for CHXDTPA. Addition of metals to EDTA-based chelators reduced the affinity of the antibody for these ligands. However, when DTPA was used as the chelator, addition of Pb(II) increased the affinity of the antibody for the complex by 200-fold. The sensitivity of prototype immunoassays for Pb(II) could be modulated by changing the structure of the immobilized metal-chelate complex and/or the soluble chelator used to complex Pb(II) in the test solution.

Development of Direct Competitive Enzyme-Linked Immunosorbent Assay for the Determination Cadmium Residue in Farm Produce

“…The cross-reactivity for several metal ions including Cd 2+ , Pb 2+ , Hg 2+ , Zn 2+ , Na + , Ca 2+ , Fe 3+ , Mg 2+ , Mn 2+ , Cu 2+ , Al 3+ , Co 2+ , Cr 2+ , Ni 2+ , Sn 2+ , or K + was tested using the DC-ELISA [15,16]. Metal ions with different concentrations which ranged from 0.01 to 10 5 μg/L were mixed with 5 mM EDTA overnight to form hapten metal chelate.…”

Development of ELISA for Detection of Mercury Based on Specific Monoclonal Antibodies Against Mercury-Chelate

“…The plates were incubated at 37°C in an atmosphere with 5% CO 2 for 7 days. The HAT medium was changed every other day and switched to HT medium when most of the non-fused cells were eliminated [13].…”

“…By contrast, immunoassays using specific monoclonal antibodies are rapid, inexpensive, simple, and highly sensitive [12]. Because metal ions are too small to elicit an immune response, they are usually chelated with isothiocyanate derivatives of bifunctional chelating agents, and conjugated to a carrier protein via the isothiocyanate [13][14][15][16]. This process is usually conducted by adjusting the pH to 9.0, but at this pH mercury would precipitation.…”

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

“…Blake et al [12,13,16] performed systemic research into synthesizing monoclonal antibodies to heavy metals by immunizing BALB/c mice with relevant antigens, antibody-based sensors for metal ions detection and relevant heavy metal immunoassays during the following years. To date, immunoassays based on specific antibodies for lead, indium, mercury, uranium and cadmium have been reported [23][24][25][26][27][28][29].…”

“…It has been shown that a suitable hapten design determines the features of the synthesized antibodies, which in turn determines the specificity and selectivity of the immunoassay [30]. The traditional design concept for metal ion haptens is through utilizing metal ions covalently conjugated to chelators (such as EDTA and DTPA) but in this approach, the monoclonal antibodies induced by the metal-chelateprotein complex preferably bind to the cage-like metal-chelate structure, rather than certain metal ions [28,31]. Wylie et al [17] proposed a new hapten design method by covalently coupling mercuric ion to the -SH group in glutathione.…”