Immunoassay for toxic congeners of polychlorinated biphenyls

Details Immunoassay for toxic congeners of polychlorinated biphenyls Immunoassay for toxic congeners of polychlorinated biphenyls

1995-03-16

2001-03-20

Housel, James C. (Department: 1641)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S007930, C436S518000, C436S815000, C436S822000

Reexamination Certificate

active

06204005

ABSTRACT:

TECHNICAL FIELD
This invention relates to methods for detecting or quantifying toxic congeners of polychlorinated biphenyls in a test sample by immunoassay, and to hapten and competitor reagents for use in such an analysis.
BACKGROUND OF THE INVENTION
Polychlorinated biphenyls (PCBs) are significant environmental pollutants that generally require complex and expensive methods for their analyses. Polychlorinated biphenyls are a class of discrete chemical compounds, called congeners, having the general formula C
12
H
x
Cl
y
, where x=0-9 and y=10-x. PCBs are produced by chlorinating the biphenyl compound at one through ten of the available positions to form a mono to decachlorobiphenyl.
PCBs were commercially produced as complex mixtures for a variety of uses, including dielectric fluids in capacitors and transformers. A major producer, Monsanto Corporation, marketed PCBs under the trade name Aroclor® from 1930 to 1977. Aroclor® PCBs were marketed for use in transformers, capacitors, and many other applications. Their chemical and physical stability, and their electrical insulating properties, led to the commercial utility of the PCBs.
The chemical and physical stability of PCBs is also the primary reason PCBs pose such a significant environmental contamination problem. Because PCBs do not readily degrade in the environment after disposal or dissemination, and are lipophilic, they are persistent and tend to accumulate in living organisms, such as humans and animals. In 1966, PCBs were found in eagles, herring and other Swedish environmental samples. S. Jensen, et al.,
Nature,
224, 247-250 (1969). Since then, PCBs have been shown to be nearly ubiquitous environmental pollutants, occurring in most human and animal adipose samples, milk, sediment and numerous other matrices.
As early as 1936, occupational exposure was reported to cause toxic effects, leading to establishment of workplace threshold limit values. Animal studies with both commercial mixtures and individual congeners have shown a variety of chronic toxic effects. National Research Counsel, “Polychlorinated Biphenyls,” National Academy of Science, Washington, D.C. (1979). PCB contaminated cooking oil caused a total of over 1200 “Yusho” patients in 1968 in western Japan. The clinical manifestations include various somatic complaints, low birth weights, chloracne and pigmentation. K. Higuchi,
PCB Poisoning and Pollution,
Academic Press, New York (1978). The discovery of widespread environmental occurrence, increased general environmental concern, and an apparent link to carcinogenesis culminated in the regulation of PCBs under the Toxic Substances Control Act in the United States.
Several EPA rules governing the use of PCBs are of concern to analytical chemists, as they require determination of PCBs in various matrices. Restrictions on the use of PCBs in the United States and other countries has made disposal of PCBs a major concern. Large quantities of PCB containing products, such as transformer and capacitor oils, are being removed from service and must be disposed of properly. For example, in the United States, the allowed methods of disposal are keyed both to concentration and to the matrix. If the PCB concentration in a transformer oil is 500 ppm or greater, disposal in a high efficiency incinerator is required. If the concentration is between 50 and 500 ppm other methods of disposal may be used.
While PCB regulations and disposal requirements differ from country to country, there is a common analytical interest in determining the presence and amount of PCBs in the environment, and in materials that are potential sources of PCBs to the environment. Regardless of the laws and rules, the analytical needs are similar: reliable, practical, and sensitive methods that can determine PCBs in a variety of matrices.
The present methods for detecting PCBs include mass spectrophotometry, x-ray fluorescence spectroscopy, gas chromatography, and high-performance liquid chromatography. One commonly employed analytical technique involves gas chromatography with ion-capture or mass spectrometer detection. Instrumental methods are relatively accurate, but expensive and time consuming. In addition, they require sophisticated analytical instrumentation and skilled operators.
A qualitative method for detecting PCBs in soil and oil based on dehalogenation followed by detection of the liberated chloride, is commercially available from the Dexsil Corporation. This method only measures total organic chlorine, and is prone to a wide variety of interferences.
Two papers on the development of PCB immunoassays have been published. M. I. Luster, et al.,
Toxicol. Appl. Pharmacol.,
50, 147-155 (1979); W. H. Newsome and J. B. Shields,
Intern. J. Environ. Anal. Chem.,
10, 295-304 (1981). Both papers describe the development of a radioimmunoassay (RIA) for specific PCB congeners which gave fair to poor specificity and sensitivity for the broad range of significant PCB congeners and congener mixtures. Also, U.S. Pat. No. 4,456,691, issued to S. Stark, teaches the preparation of polyclonal antibodies to PCBs using Aroclor® 1254 which has been aminated, diazotized and coupled to Bovine Serum Albumin (BSA). The antisera was evaluated by an RIA. M. Franek, et al.,
J. Agric. Food Chem.,
40, 1559-1565 (1992) teach a radioimmunoassay method for the detection of PCBs.
In European Patent Application No. 0 455 058 A2 and U.S. Pat. No. 5,145,790 Mattingly et al. (the '790 patent) describe an immunoassay method for detecting the presence or amount of polychlorinated biphenyls in a test sample. This assay is not congener specific. It is desirable to have an immunoassay method with greater sensitivity than disclosed by Applicants in the European application and in the '790 patent.
The widespread occurrence of polychlorinated biphenyls (PCBs) in the environment requires a comprehensive assessment of their environmental impact. This assessment is made more complicated by the recent realization that different forms of PCB exhibit different toxicity. There are many ways to distribute chlorines on a biphenyl ring system. These different structural forms, isomers, or congeners of PCB vary in toxicity. This is particularly complicated for PCBs since there are 209 congeners of PCB, which vary dramatically in their toxicity. The awareness of the varied toxicity of PCB congeners is recent and the analytical methods used to analyze the toxic PCB congeners is cumbersome and expensive.
The present methods for the congener specific analysis of PCBs typically use high-resolution gas chromatography with ion capture or mass spectrometer detection systems. D. E. Wells, et al., Intern.
J. Environ. Anal. Chem.,
47, 75-97 (1992). Although these methods are relatively accurate, they are also very expensive and time consuming because of their reliance on sophisticated analytical instrumentation and skilled operators. There is growing recognition that specific analysis of the most toxic PCB congeners in the environment is required for an objective evaluation of risk and environmental impact. However, the time, effort and expense associated with the toxic congener specific analysis places substantial constraints on the scope of risk assessment and site evaluation studies. C. S. Creaser, et al.,
Chemosphere,
25, 1981-2008 (1992). Immunoassay based analytical methods have demonstrated value for economical and specific, high throughput screening and for quantitiative analyses of many environmental analytes. Moreover, immunoassay can be accomplished with minimal sample preparation and instrumentation.
Accordingly, there is a need for a reliable and sensitive immunoassay for detecting or quantifying toxic PCB congeners. In particular there is need for an immunoassay that can offer significant advantages in cost, personnel training, and equipment requirements over present immunoassays and that provides substantially improved assay performance over known methods for detecting or quantifying toxic PCB congeners.


REFERENCES:
patent: 4456691 (1984-06-01), Sta

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