Environ. Sci. Technol. 2002, 36, 1042-1047
Lead Analysis by Anti-Chelate Fluorescence Polarization Immunoassay D A V I D K . J O H N S O N , * ,† SHERRY M. COMBS,‡ JOHN D. PARSEN,‡ AND MICHAEL E. JOLLEY§ BioMetalix, Inc., 330 East Main Street, P.O. Box 601, Twin Lakes, Wisconsin 53181-601, University of Wisconsin Soil & Plant Analysis Laboratory, 5711 Mineral Point Road, Madison, Wisconsin 53705, and Jolley Consulting & Research, Inc., 683 Center Street, Grayslake, Illinois 60030
Lead concentrations were determined by a fluorescence polarization immunoassay (FPIA) method that uses polyclonal antibodies raised against the lead(II) chelate of ethylenediamine-N,N,N′,N′-tetraacetic acid (EDTA). The technique is based on competition for a fixed concentration of antibody binding sites between Pb-EDTA, formed by treating the sample with excess EDTA, and a fixed concentration of a fluorescent analogue of the Pb-EDTA complex. The objective was to correlate results obtained by FPIA with those produced by conventional atomic spectroscopy analysis of soils, solid waste leachates (produced by the Toxicity Characteristic Leachate Procedure; TCLP), airborne dust, and drinking water. Linear regression analysis of FPIA results for 138 soil samples containing 0-3094 ppm Pb(II) by flame atomic absorption spectroscopy and 40 TCLP extracts containing 0-668 ppm Pb(II) by inductively coupled plasma atomic emission spectroscopy produced correlation coefficients (r2) of 0.96 and 0.93, respectively. Pilot studies of mineral acid extracts of airborne dust trapped on fiberglass filters and of two sources of drinking water demonstrated the feasibility of also measuring lead in these matrixes by FPIA. The limit of detection under conditions that minimized sample dilution was approximately 1 ppb, and cross reactivity with 15 nontarget metals was below 0.5% in all cases. The methods are simple to perform and are amenable to field testing and mobile laboratory use, allowing timely and cost-effective characterization of suspected sources of lead contamination.
Introduction Lead is ubiquitous in the environment. It occurs naturally but can also be present at elevated local concentrations and in nonnaturally occurring chemical forms as a result of its past widespread use in a range of products (e.g., leaded gasoline, lead-based paint) and industrial processes (e.g., smelting, battery manufacturing). Over the past several decades, epidemiological studies have led to a growing recognition of the chronic neurotoxicity of lead and of the * Corresponding author phone: (262)877-8945; fax: (262)877-2682; e-mail:
[email protected]. † BioMetalix, Inc. ‡ University of Wisconsin Soil & Plant Analysis Laboratory. § Jolley Consulting & Research, Inc. 1042
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 36, NO. 5, 2002
particular susceptibility of children to lead poisoning (1). The predominant routes by which lead enters the body are via inhalation of lead-containing airborne particulates and through the ingestion of lead-contaminated water and food and, in young children, through pica behavior involving paint chips, soil, and dust (2). Although the use of lead in paint and gasoline has been phased out, it is estimated that 75% of the U.S. housing stock contains some lead-based paint, and roadside dust and soils can still contain lead deposited during past combustion of leaded fuels. Lead emissions from industrial facilities have been brought under increasingly stringent control, but lead released during past operation of such facilities remains in the environment and can constitute another potential source of human exposure. As a result of these widespread past releases, lead is currently the singlemost prevalent environmental health concern for children in the U.S., and better methods for identifying and delineating areas of suspected lead contamination would be advantageous. Although X-ray fluorescence and anodic stripping voltametry techniques can sometimes be used for in situ measurements, lead concentrations in matrixes of environmental interest are generally determined by returning samples collected in the field to a central laboratory where they are analyzed by either atomic absorption spectroscopy (AAS) or inductively coupled plasma atomic emission spectroscopy (ICP-AES). For soils and solid wastes, two types of measurements are typically made. The total acid-extractable lead content of the sample is determined by digestion or extraction with strong mineral acid while the mobile, or leachable, fraction is measured by a milder procedure in which the sample is extracted with 0.1 M acetate buffer (the Toxicity Characteristic Leachate Procedure; TCLP) (3). Airborne particulates are collected on fiberglass filters from which the lead is eluted with mineral acid(s) while drinking water samples are analyzed directly, although generally after being acidified to convert heavy metals into the ionic form. The relatively high cost per result, limited throughput, and lengthy turnaround times typical of fixed base atomic spectroscopy testing currently limit the precision and timeliness with which sources of suspected lead contamination can be characterized. Immunoassay technology has the potential to provide relatively simple, inexpensive analytical methods that are suitable for use in field testing applications. A variety of immunoassays for common organic pollutants were introduced in the early 1990s (4), and attempts have been made to extend the application of such methods to heavy metal analysis. Prototype enzyme-linked immunosorbent assays (ELISA) have been described to date for indium(III) (5), mercury(II) (6), cadmium(II) (7, 8), and lead(II) (9), in all cases in a water matrix. The work described here was undertaken as part of a program to develop quantitative methods for heavy metal analysis based on an alternative homogeneous immunoassay format that uses a fluorescent label (10). The purpose of the present study was to correlate results obtained by such fluorescence polarization immunoassay (FPIA) methods with the corresponding atomic spectroscopy values for a number of matrixes that are typically sampled when monitoring lead concentrations in the environment.
Experimental Section Chemicals. EDTA‚Na2‚2H2O (ACS, 99+%), lead(II) nitrate (ACS, 99+%), iron(III) nitrate nonahydrate (ACS, 98+%), aluminum(III) chloride monohydrate, nontarget metals as 10.1021/es011114t CCC: $22.00
2002 American Chemical Society Published on Web 01/26/2002
TABLE 1. Cross-Reactivity of Nontarget Metal Ions in the FPIA for Lead(II)
metal ion
crossreactivity (%)
mercury(II) silver(I) copper(II) cadmium(II) bismuth(II) zinc(II) calcium(II) cobalt(II)
0.37 0.19 0.15 0.15 0.13 0.11 0.11 0.10
metal ion
crossreactivity (%)
chromium(III) gold(III) nickel(II) manganese(II) magnesium(II) aluminum(III) iron(III)
0.10 0.08 0.08
