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Extramural Research

Final Report: Quantitation of Heavy Metals by Immunoassay

EPA Grant Number: R824029
Title: Quantitation of Heavy Metals by Immunoassay
Investigators: Blake, Diane A.
Institution: Tulane University of Louisiana
EPA Project Officer: Levinson, Barbara
Project Period: September 1, 1995 through August 31, 1998
Project Amount: $381,920
RFA: Exploratory Research - Chemistry and Physics of Water (1995)
Research Category: Engineering and Environmental Chemistry



The contamination of the environment with heavy metals poses a continuing and increasing threat to plant and animal life. At least twenty metals are known to be toxic and fully half of these, including arsenic, cadmium, chromium, copper, lead, nickel, silver, selenium, and zinc are released into the environment in sufficient quantities to pose a risk to human health. Metals are classified as persistent environmental toxins because they cannot be metabolized to CO2 and H2O or easily rendered harmless by chemical or biological remediation processes. In the environment, heavy metals often persist for long periods bound to soils or sediment, where they are relatively non-toxic except to bottom-feeding animals. However, changes in weather, in the pH of the soil or water, or in other combinations of environmental factors can mobilize bound metals and greatly increase their toxicity. For this reason, sites contaminated with heavy metals must be monitored on a regular basis. In addition, sites undergoing attempted remediation must be monitored frequently to assess progress in heavy metals cleanup. Existing technologies to measure heavy metals require complex instrumentation (atomic absorption or inductively coupled plasma emission spectroscopy) in a centralized facility. These instruments measure the total amount of a specific metal in an environmental sample, but provide no information about metal oxidation state or speciation.

Immunoassays offer an alternate approach for metal ion detection, and they have significant advantages over more traditional metal ion detection methods. Immunoassays are quick, inexpensive, simple to perform, and reasonably portable; they can be highly sensitive, selective, and species-specific. Sample analysis is one of the major costs in the remediation of a contaminated site, and studies have shown that the use of immunoassays can reduce analysis costs by 50% or more. A limited number of immunoassays are now available that measure environmental contaminants, including industrial pollutants, pesticides, and herbicides. Although most commercial immunoassays are directed toward complex organic chemicals, this method is theoretically applicable to any pollutant for which a suitable antibody can be generated. During the course of this research, the Blake laboratory has demonstrated the feasibility of immunoassays for specific metal ions. Experiments with an anti-cadmium monoclonal antibody showed that soluble cadmium and ionic mercury could each be measured by immunoassay at concentrations comparable down to 5 ppb.

At present, there remains an urgent need for low cost, rapid biosensors that can also be used on-site by relatively untrained personnel. Future research by our laboratory will be directed toward using the antibodies developed during this project to construct automated antibody-based biosensors for metal ion contamination. Although the EPA has chosen not to renew this project, the Department of Energy has recently awarded a grant to our laboratory to support our efforts in biosensor development.

Description and Objectives of Research:

The specific aims of this project were as follows:

  1. To construct and optimize an immunoassay for cadmium with respect to sensitivity, precision, and interfering substances;

  2. To prepare new metal-specific antibodies directed towards EDTA complexes of copper, lead, or zinc;

  3. To prepare new metal-specific antibodies directed towards metals complexed with other chelating agents (DTPA or DOTA).

Summary/Accomplishments (Outputs/Outcomes):

  1. A new method was developed to determine antigen-antibody binding affinities (Blake, D.A. et. al, 1996, I997).

    The development of any immunoassay is greatly facilitated by a detailed knowledge of the binding characteristics of the antibody used in the assay. Assay sensitivity is directly related to the antibody's affinity for the antigen, assay selectivity is directly related to its binding specificity, and assay performance is directly related to the kinetic properties of the antibody binding reaction. For these reasons, we have developed a new technique for characterizing the monoclonal antibodies generated in our laboratory. A KinExA Automated Immunoassay Instrument (one of the 6 prototype instruments in the country when these experiments were initiated) was used to determine the binding affinity of the 2A81G5 (anti-cadmium) monoclonal antibody for 16 different metal-EDTA complexes. The ability of purified antibody to recognize 16 different metal-EDTA complexes was assessed by measuring equilibrium binding constants. The antibody bound to cadmium- and mercury-EDTA complexes with equilibrium dissociation constants of 21 and 26 nM, respectively. All other metal-EDTA complexes tested, including those of Mn(II), In(III), Ni(II), Zn(II), Co(II), Cu(II), Ag(I), Fe(III), Pb(II), Au(III), Tb(III), Ga(III), Mg(II), and Al(III) bound with affinities from 20-fold to 40,000-fold less than that determined for the cadmium-EDTA complex. With the exception of mercury and magnesium, the binding of divalent metal-chelate complexes was weli-correlated with the size of the metal ion. The amino acid sequences of the heavy and light chain variable regions were deduced from PCR amplified regions of the corresponding genes and subsequently used to construct molecular models of the antigen binding region. The key residue for cadmium binding in the model for 2A81G5 appeared to be histidine 96 in the heavy chain. These experiments were published in 1996 in the Journal of Biological Chemistry (Blake, D.A. et. al, 1996). We have subsequently used this instrument to characterize all the monoclonal antibodies generated from the activities of this project (Blake, D.A. et al., 1997).

  2. A competitive immunoassay for Cd(II) was constructed and validated for water samples (Khosraviani, M. etal., 1998, Blake, D.A. etal., l998).

    The 2A81G5 (anti-cadmium) monoclonal antibody was exploited to assemble a competitive immunoassay for Cd(II). An immunoassay was constructed that measured Cd(II) in aqueous samples at concentrations from approximately 7 to 500 ppb. The assay utilized a monoclonal antibody that bound tightly to a cadmium--ethylenediaminetetraacetic acid (EDTA) complex but not to metal-free EDTA. A inhibition immunoassay format was employed for this analysis; ionic cadmium was diluted into an excess of EDTA before being incubated with the antibody in the presence of an immobilized Cd(II)-EDTA conjugate. Ca(II), Na(I), and K(I), cations commonly encountered in ambient water samples, did not interfere with the cadmium immunoassay at concentrations approaching their solubility limit. The assay reliably measured Cd(II) in the presence of a 1 mM excess of Fe(III), Mg(II), and Pb(II). Zn(II) and Ni(II) had minimal effect on the assay at levels below 100 1uM, and the immunoassay was relatively insensitive to interferences by In(III) and Mn(II) at concentrations up to 10 uM. Hg(II had the ability to cause a false positive in the assay, but only at concentrations higher than 1 uM. The assay compared favorably with atomic absorption spectroscopy in its ability to measure cadmium in spiked water samples taken from a Louisiana bayou.

  3. A new monoclonal antibody (I SB4) has been purified that recognizes Zn- and Ni-chelate complexes.

    The 1 5B4 hybridoma cell line synthesizes and secretes a monoclonal antibody which recognizes diethylenetriamine pentaacetic acid (DTPA) complexes of cobalt with greatest affinity and nickel and zinc complexes with approximately equivalent affinity. The hybridoma was generated with an immunogen prepared using a p-isothiocyanatobenzyl derivative of DTPA obtained from Dr. Martin Brechbeil of the NIH. Like the previously characterized 2AS1G5 monoclonal antibody, 15B4 binds with low affinity to metal-free chelators, but recognizes DTPA and structurally related chelators loaded with Co(II), Zn(II), or Ni(II) with much greater affinity. The equilibrium dissociation constant for 9 different metals has been determined on the KinExA Immunoassay Instrument, and the relevant binding constants are shown in Table 1. Since Co(II) is unlikely to occur as a contaminant in most of the environmental samples we are likely to encounter, we feel that this antibody could be used to detect levels of Zn(II) and/or Ni(II) in water or soil samples.

    Table 1. Binding of chelated metals to 15B4 monoclonal antibody

    Constant (M)
    Constant (M)
    DTPA-Co(II) 5.2 + 0.3 x 10-8 pNB-DTPA-Co(II) 8.0 + 0.5 x 10-l0
    DTPA-Ni(II) 2.7 + 0.2 x 10-7 pNB-DTPA-Ni(II) 3.0 + 0.4 x 10-9
    DTPA-Zn(II) 2.5 + 0.3 x 10-7 pNB-DTPA-Zn(II) 2.5 + 0.2 x 10-9
    DTPA-Mn(II) 7.0 + 0.5 x 10-7 pNB-DTPA-Mn(II) nd
    DTPA-Cu(II) 1.4 + 0.07 x 10 6 pNB-DTPA-CulII) 9.8 + 1.8 x 10-9
    DTPA-Cd(II) 3.2 + 0.3 x 10-6 pNB-DTPA-Cd(II) nd
    DTPA-Ca(II) > 5 x 10-6 pNB-DTPA-Ca(II) nd
    Fe(III), Au(III) Fe(III), Au(III)

    1 pNB-DTPA, p-nitrobenzyl-DTPA, a chelator with structural similarities to the original immunogen.
    2 not determined.

  4. A new hybridoma cell line (2C12) has been isolated that synthesizes alld secretes a monoclonal antibody which recognizes chelatedforms of Ph(II).

    A monoclonal antibody 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 using a KinExA? immunoassay instrument. The monoclonal antibody bound to metal-free cyclohexyldiethylenetriamine pentaacetic acid (CHXDTPA) with an equilibrium dissociation constant of 2.3 x 10-7 M. Addition of Pb(II) to the complex increased the affinity of the antibody for the complex by approximately 25-fold; Pb(II) was the only metal cation (of 15 different di-, tri-' and hexavelent 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 10,000-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 complexing agent, addition of Pb(II) increased the affinity of the antibody for the complex by 200-fold, while Ca(II) increased the affinity by 750-fold. Prototype immunoassays for soluble Pb(II) were constructed using an antigen inhibition format. The sensitivity of the immunoassay 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.

    Unfortunately, the 2C12 antibody bound to Pb-DTPA complexes with only ~M affinity, and prototype immunoassays constructed using the 2C 12 antibody and DTPA as the chelator detected soluble Pb(II) only at the low pm level. We need an antibody with nM affinity to construct an immunoassay that will detect Pb(II) at the low ppb levels. We have recently received funding from the Department of Energy that will support experiments to modify the antibody and/or assay format to increase assay sensitivity.

  5. A new hybridoma cell line has been developed that synthesizes and secretes an antibody with specificity for Cu(II)-EDTA complexes.

    This hybridoma was isolated in the final year of this funded grant. Because the EPA did not renew this project, we do not have any detailed information about the affinity or specificity of this antibody.


Although immunoassays for heavy metals are still in their infancy, very promising results have been obtained. There are clearly many avenues for further improvement and extension of current assay reagents. Since the sensitivity and specificity of any immunoassay is critically dependent upon the binding properties of the antibody in question, the greatest assay improvements will probably come from the production of new antibodies with improved binding characteristics.

Two types of immunoassays can be envisioned. One involves the generation and exploitation of a family of highly specific antibodies, each of which binds exclusively to a single metal-chelate complex (the one antibody, one metal ion scenario). A second scenario involves a biosensor array comprised of a family of antibodies, each with a broad, overlapping specificity for a variety of metal-chelate complexes. In this latter case, identification and quantification of metal ions in complex mixtures would then be deduced from the response obtained in the multifunctional binding array. Further funding is being sought to advance both of these concepts.

The immunoassays under development are not meant to supplant or replace existing technologies; rather, portable immunoassays for metal ions will serve as useful adjuncts to those workers who need a real-time/on-site assays for metal ion contamination. Although some assay improvements have been attained by careful manipulation of assay format, more significant gains will be achieved by the isolation of antibodies with optimized binding properties.

Journal Articles on this Report : 3 Displayed | Download in RIS Format

Other project views: All 11 publications 5 publications in selected types All 3 journal articles

Type Citation Project Document Sources
Journal Article Blake DA, Chakrabarti P, Khosraviani M, Hatcher FM, Westhoff CM, Goebel P, Wylie BE, Blake II RC. Metal binding properties of a monoclonal antibody directed toward metal-chelate complexes. Journal of Biological Chemistry 1996;271(44):27677-27685. R824029 (Final)
not available
Journal Article Blake DA, Blake RC, Khosraviani M, Pavlov AR. Immunoassays for metal ions. Analytica Chimica Acta, December 1998;376(1):13-19. R824029 (1998)
R824029 (Final)
not available
Journal Article Khosraviani M, Pavlov AR, Flowers GC, Blake DA. Detection of heavy metals by immunoassay: optimization and validation of a rapid, portable assay for ionic cadmium. Environmental Science & Technology 1998;32(1):137-142. R824029 (1998)
R824029 (Final)
not available
Supplemental Keywords:

Scientific Discipline, Toxics, Water, Hydrology, National Recommended Water Quality, Physics, Environmental Chemistry, Chemistry, Engineering, Chemistry, & Physics, Mercury, immunoassay, metal-chelate complexes, lead, ambient emissions, metal speciation, metal contaminated sediment, metal ions, bifunctional derivatives, monoclonal antibodies, remediation, Zinc, soil contaminants, copper, immunochemical-based detection, cadmium

Progress and Final Reports:
Original Abstract
1998 Progress Report

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The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.

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