Immunoassays for Residue Analysis - American Chemical Society

Chapter 30

Immunoassay for Mercury in Seafood and Animal Tissues

Downloaded by GEORGETOWN UNIV on June 2, 2018 | https://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0621.ch030

Larry Carlson, Bart Holmquist, Roger Ladd, Mal Riddell, Fred Wagner, and Dwane Wylie BioNebraska, Inc., 3820 Northwest 46th Street, Lincoln, NE 68524

The measurement of mercury in swordfish, tuna, scallop, bass, shark, and alligator tissue by immunoassay correlated well with cold-vapor atomic absorption. Methylmercury, which bioaccumulates in fish and other animals, was extracted from the tissue with a procedure optimized for the immunoassay. Since methylmercury is extremely toxic, it is important to identify fish or animal tissues with mercury levels considered unsafe for human consumption. Current analytical methods for mercury are expensive and time-consuming, and they must be performed in a laboratory. The mercury immunoassay can be adapted for quick, on-site screening of large numbers of tissue samples. Mercury contamination of rivers, lakes, and oceans originates from both natural and human-related sources (7,2). Elemental mercury is distributed throughout the atmosphere and deposited as inorganic mercury in precipitation, thereby introducing mercury into remote bodies of water (3). Microbial methylation of inorganic mercury produces highly toxic methylmercury, which bioaccumulates in fish and other animals (1,4-6). Continued consumption of food containing methylmercury results in damage to the human central nervous system (7). Mercury in many marine and freshwater fish exceeds the 1 μg/g (ppm) action level established by the U.S. Food and Drug Administration. Given the extreme toxicity of methylmercury, it is important to identify fish or other animals with mercury levels above those considered fit for human consumption. Cold-vapor atomic absorption spectrophotometry (CVAAS) and cold-vapor atomic fluorescence are two methods in current use, but these methods are expensive and must be performed in a laboratory. A rapid and inexpensive mercury-specific immunoassay developed by BioNebraska has been used to detect mercury in water and soil in the field (7-9). This paper presents the results of experiments in which this immunoassay was used to measure mercury in animal tissue. Mercury was extracted

0097-6156/96/0621-0388$15.00/0 © 1996 American Chemical Society Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

30. CARLSON ET AL.

Immunoassay for Mercury in Seafood & Animal Tissues 389

from tissue samples using a procedure designed for use with CVAAS, but with modifications that make it amenable to the immunoassay. Material and Methods


Tissue Samples. Alligator and bass samples were obtained from the Florida Game and Fresh Water Fish Commission. Shark, swordfish, scallop, and tuna samples were obtained from the Environmental Research Institute, Storrs, CT. Cormorant liver samples were obtained from the Florida Keys Wild Bird Center, Tavernier, FL. Mercury was extractedfromthe tissues and analyzed by both cold-vapor atomic absorption and the BiMelyze mercury immunoassay.

Extraction Procedure. Tissue samples were kept frozen at -20 °C until used. Prior to extraction, they were thawed and cut into smal pieces (approximately 0.2 cm) using a clean razor blade, and 5 g were placed in acid-washed 125-mL glass bottles with plastic lids. Controls consisted of a reagent blank and Dorm-1 (National Research Council of Canada), a lyophilized certified reference material of dogfish muscle containing 0.798 μg/g total mercury. Tissue digestion procedures involving strong acids and oxidizing agents were performed in afiimehood by personnel wearing appropriate protective clothing. The procedure used to prepare samples for the immunoassay was modified from that described by Delany et al. (4). Concentrated nitric acid (5 mL) was added to 5 g of tissue. The bottles were capped, incubated for 2 h at room temperature with occasional agitation, then placed in a water bath at 95 °C for 2 h. After cooling to room temperature, 30% H 0 (7 mL) was added. The samples were heated again for 2 h at 95 °C. The extracts were alowed to cool, and the total volume of the extrac was measured. The Dorm-1 powder was treated similarly except that 20 mL of acid per gram was used to allow for complete mixing, and 14 mL of 30% H 0 was added This modified extraction procedure was compared with EPA Method 245.6, in which 4 mL of concentrated sulfuric acid and 1 mL of concentrated nitric acid were added to 0.25 g of tissue. After a 30 min incubation at 80 °C, the sample was cooled at 4 °C to prevent excessive bubbling when 15 mL of 5% potassium permanganate and 8 mL of 5% potassium persulfate were added. The sample was then heated for 90 min at 30 °C and diluted with 55 mL of deionized water. Excess permanganate was reduced by adding 6 mL of a solution containing 12% sodium chloride and 12% hydroxylamine sulfate. 3

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Cold-Vapor Atomic Absorption. Tissue extracts were assayed for mercury using an LDC Analytical Model 1255 Mercury Monitor equipped with a Linear Scientific Model 1201 chart recorder. Extracts were diluted 1:100 or 1:25 with 1.6 Μ HN0. Standards of 20, 10, 5, 2, and 1 ppb mercury were prepared by diluting the certified standard, SRM 3133 (National Institute of Standards and Technology, 10 mg Hg/mL in 10% HN0), in 1.6 Μ HN0. Ten milliliters of 5% tin(II) chloride dihydrate in 0.5 N HC1 was added to the reservoir of the bubbler assembly, and 0.7 mL of standard or diluted extract was injected. High purity grade nitrogen was used to carry the elemental mercury vapor to the detection cell. After the recorder pen returned to the 3

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Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

390

IMMUNOASSAYS FOR RESIDUE ANALYSIS


baseline position, the trap of the bubbler assembly was emptied, 10 mL of fresh tin chloride solution was added, and the next sample was injected. The concentration of mercury in the samples was calculated on a wet-weight basis. Immunoassay Procedure. Digests also were analyzed with the BiMelyze Mercury Assay Plate Kit following a 1:1,000 dilution with 0.1 M HEPES, pH 7. A standard curve of 8, 2, 0.5, 0.25, and 0.125 ppb mercury in 0.1 M HEPES, pH 7, was made from S R M 3133. For the analysis of cormorant liver samples, for which 0.5 g samples were used, the standard curve was prepared by adding mercury to a 1:1,000 dilution of a low-mercury liver digest in 0.1 M HEPES, pH 7. Mercury Assay Plates were prepared by coating each well of a 96-well polystyrene plate with a glutathionebovine serum albumin conjugate (GSH-BSA) (8) in phosphate buffered saline. Samples and standards (100 μL) were added to each of two wells of the Mercury Assay Plate and incubated for 15 min at room temperature. The plate was rinsed three times with deionized water, then 100 of a solution containing a mercuryspecific monoclonal antibody was added to each well and incubated for 10 min at room temperature. The plate was washed three times with a buffered rinse solution and three times with water, then 100 μL of a solution containing goat anti-mouse IgG horseradish peroxidase conjugate was added to each well. Following a 10 min incubation at room temperature, excess reagent was washed away as before, and 100 \iL of ABTS substrate solution (2,2'-azinobis[3-ethylbenzothiazoline]-6 sulfonic acid diammonium salt) was added to each well. Color development was stopped after 10 min by the addition of 100 \\L of 1% sodium dodecyl sulfate to each well. Absorbencies measured at 405 nm using a BioTek EL311 microplate autoreader were proportional to the log of the mercury concentration. Detection Limit Determination. The lower detection limit (LDL) of the immunoassay for mercury in tissue was determined using a scallop tissue extract which did not contain detectable mercury. The extract was diluted 1:1000 in 0.1 M HEPES, pH 7, and then mercury from S R M 3133 was added to final concentrations of 0.5, 0.25, 0.125, 0.062, 0.031, 0.016, and 0.008 ppb. The mercury dilutions and diluted extract without added mercury were then analyzed with the immunoassay. Results and Discussion 2+

The basic procedure used to measure H g in solution is diagrammed in Figure 1. After tissue digestion, samples were diluted in pH 7 buffer and added to plates which were coated with glutathione-bovine serum albumin (GSH-BSA). After mercury was bound to the GSH-BSA, the mercury-specific antibody was added, and it binds to the immobilized mercury. Horseradish peroxidase (HRP)-conjugated antibody was added next, followed by the addition of ABTS substrate. Cleavage of substrate resulted in color formation. The HNO3/H2O2 digestion procedure converts methylmercury to Hg *, the form recognized by the mercury-specific antibody. This procedure was modified slightly so that analyses by C V A A S and immunoassay could be completed on the same day. In the original procedure, samples were digested with nitric acid for 2 h, heated at 95 °C 2


30.

CARLSON ET AL.


for 4 h, and heated at 95 °C for an additional 2 h following addition of hydrogen peroxide. In the modified procedure, the 4-h heating step was shortened to 2 h, and this procedure adequately extracted mercury from the tissue as determined by C V A A S (data not shown). These extracts were then analyzed by the immunoassay. The results from these initial experiments showed that mercury concentrations determined by the immunoassay were lower than those determined by C V A A S . Experiments were performed to determine optimal H N 0 and H 0 concentrations for sample digestion and mercury detection with the immunoassay. The effect of H 0 concentration was examined with five different tissue samples digested with 70% H N 0 and 5, 9, or 13% H 0 (Table I). Mercury concentrations determined by immunoassay (IA) were closest to 100% of C V A A S values for samples digested with 13% H 0 , so this concentration was used in subsequent extractions. 3

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Table I. Effect of Hydrogen Peroxide on Detection of Mercury in Tissue H 0 (%) 5 9 13 2

Sample 1

2

(IA / C V A A S ) χ 100 (%) 47 67 88

2

5 9 13

40 45 90

3

5 9 13

50 62 75

4

5 9 13

60 80 100

5

5 9 13

50 60 114

Many tissue digestion procedures are available, but the high acid concentration and strong oxidizing agents used are not amenable to the immunoassay. An important issue, therefore, is the development of an efficient extraction procedure which is also compatible with the immunoassay. The procedure utilizing 70% H N 0 and 13% H 0 was compared to EPA Method 245.6 which employs chemicals that interfere with the immunoassay. Tissue samples were digested with both procedures and analyzed by C V A A S . The HN03/H 0 procedure recovered 64, 75, 68, and 69% of the mercury from shark, swordfish, tuna, and alligator tissue compared to the EPA 3

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392


Figure 1. Mercury immunoassay procedure.

[Hg] by Immunoassay (ppm)

Figure 2. Detection of mercury in tissue by immunoassay and C V A A S . alligator (•), bass (Δ), scallop (Q), shark (φ), swordfish (•), tuna (•)


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CARLSON ET AL.

from tissue, it was incompatible with the immunoassay because the small sample size used in this procedure and the dilution required for neutralization resulted in low sensitivity. Subsequent immunoassays in this study were performed on tissue samples digested with H N 0 and H 0 . The F D A has established an action level of 1 ppm for mercury in fish (10), and some states have issued consumption advisories for fish containing greater than 0.5 ppm mercury. The immunoassay must be able to measure mercury at or below these concentrations in order to be useful as a screening test. The immunoassay's lower detection limit (LDL) was determined by analyzing a diluted scallop extract spiked with different concentrations of mercury. The L D L was considered to be the concentration of mercury which gave an absorbance equal to the absorbance of the negative control plus 3 times the standard deviation. An L D L of 0.03 ppb was obtained, which corresponds to 0.1 ppm of scallop tissue after the dilution of the extract is taken into consideration. With a detection limit of 0.1 ppm, this test possesses adequate sensitivity for the identification of samples which contain mercury at levels which are considered unsafe. The immunoassay's ability to measure mercury from various tissue samples was assessed. Swordfish, tuna, scallop, bass, shark, and alligator tissue was digested with the modified procedure and analyzed by both C V A A S and immunoassay (Figure 2). Linear regression analysis gave a slope of 1.02 and an r value of 0.97. The quantitation limit for mercury in these tissue samples was approximately 0.1 ppm for C V A A S and approximately 0.4 ppm for immunoassay. The immunoassay quantitation limit of 0.125 ppb in 0.1 M HEPES, pH 7, corresponds to a quantitation limit of 0.4 ppm in tissue after the mercury is extracted and diluted 1:1000. In addition, preliminary results indicate that the immunoassay can measure mercury in cormorant liver tissue (data not shown). Immunoassay reproducibility was assessed by performing five separate immunoassays of shark, swordfish and tuna tissue. C V ' s of 11.4, 5.7, and 9.2% were obtained for these samples, respectively. With this limited number of samples, the immunoassay reproducibly measured mercury in a variety of tissues. Mercury in food is a serious threat to human health, but the complexity of current analytical methods makes widespread screening difficult. The mercuryspecific immunoassay can be adapted for quick, portable analysis of mercury in tissue. A good correlation was obtained between the immunoassay and C V A A S for tissue samples digested with 70% H N 0 and 13% H 0 . Since this extraction procedure is lengthy and somewhat inefficient, the development of a quick, efficient, and safe extraction procedure which also is amenable to the immunoassay is a goal of future research. Once such a procedure is developed, the immunoassay can be used for quick, on-site determination of mercury in tissue. 3



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Literature Cited 1. 2. 3.

Environmental Health Criteria 101. Methylmercury; World Health Organization: Geneva, Switzerland, 1990. Environmental Health Criteria 86. Mercury-Environmental Aspects; World Health Organization: Geneva, Switzerland 1989. Fitzgerald, W. F. The World & I 1993, 8(10), 192-199.


394 4. 5. 6. 7. 8.


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Delaney, M. F.; Bell, J. U.; Sundlof S. F. J. Wildlife Dis. 1988, 24(1), 62-66. Grieb, T. M.; Driscoll, C. T.; Gloss, S. P.; Schofield, C. L.; Bowie, G. L.; Porcella, D. B. Environ. Toxicol. Chem. 1990, 9, 919-930. Driscoll, C. T.; Yan, C.; Schofield, C. L.; Munson, R.; Holsapple, J. Environ. Sci. Technol. 1994, 28(3), 136A-143A. Wylie, D. E.; Carlson, L. D.; Carlson, R.; Wagner, F. W.; Schuster, S. M. Anal. Biochem. 1991, 194, 381-387. Wylie, D. E.; Lu, D.; Carlson, L. D.; Carlson, R.; Funda Babacan, K.; Schuster, S. M.; Wagner, F. W. Proc. Nat. Acad. Sci. U.S.A. 1992, 89, 4104-4108. U.S. Department of Energy Methods for Evaluating Environmental and Waste Management Samples, Rev. 2; Goheen, S. C.; McCulloch, M.; Thomas, B. L.; Riley, R. G.; Sklarew, D. S.; Mong, G. M.; Fadeff, S. K., eds.; Pacific Northwest Laboratory: Richland, Washington, 1994, MB100. U.S. Food and Drug Administration. Federal Register 1979, 44, 4012.

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Immunoassays for Residue Analysis - American Chemical Society

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