Application of Enzyme-Linked Immunosorbent Assay for Measurement of Polychlorinated Biphenyls from Hydrophobic Solutions.

Metadata:
Identification_Information:
Citation:
Citation_Information:
Originator:
James L. Zajicek, Donald E. Tillitt, James N. Huckins, Jimmie D. Petty, Michael E. Potts
Originator:
U.S. Geological Survey, Biological Resources Division, Columbia Environmental Sciences Center (formerly known as Midwest Science Center)
Originator: David A. Nardone, Ohmicron Corporation
Publication_Date: 1996
Title:
Application of Enzyme-Linked Immunosorbent Assay for Measurement of Polychlorinated Biphenyls from Hydrophobic Solutions.
Publication_Information:
Publication_Place: Columbia, Missouri
Publisher:
U.S. Geological Survey, Biological Resources Division, Columbia Environmental Research Center
Description:
Abstract:
Determination of Polychlorinated Biphenyls (PCBs) in biological tissue extracts by enzyme-linked immunosorbent assays (ELISAs) can be problematic, since the hydrophobic solvents used for their extraction and isolation from interfering biochemicals have limited compatibility with the polar solvents (e.g. methanol/water) and the immunochemical reagents used in ELISA. Studies of these solvent effects indicate that significant errors can occur when microliter volumes of PCB containing extracts, in hydrophobic solvents, are diluted directly into methanol/water dilutents. Errors include low recovery and excess variability among subsamples taken from the same sample dilution. These errors are associated with inhomogeneity of the dilution, which is readily visualized by the use of a hydrophobic dye, Solvent Blue 35. Solvent Blue is also used to visualize the evaporative removal of hydrophobic solvent and the dissolution of the resulting PCB/dye residue by pure methanol and 50% (v/v) methanol/water, typical ELISA diluents. Evaporative removal of isooctane by an ambient temperature nitrogen purge with subsequent dissolution in 100% methanol gives near quantitative recovery of model PCB congeners. They also compared concentrations of total PCBs from ELISA (ePCB) to their corresponding concentrations determined from capillary gas chromatography (GC) in selected fish sample extracts and dialysates of semipermeable membrane device (SPMD) passive samplers using an optimized solvent exchange procedure. Based on Aroclor 1254 calibrations, ePCBs (ng/mL) determined in fish extracts are positively correlated with total PCB concentrations (ng/mL) determined by GC: ePCB = 1.16 * total-cPCB - 5.92. Measured ePCBs (ng/3 SPMDs) were also positively correlated (r2 = 0.999) with PCB totals (ng/3 SPMDs) measured by GC for dialysates of SPMDs: ePCB = 1.52 * total PCB - 212. Therefore, this ELISA system for PCBs can be a rapid alternative to traditional GC analyses of PCBs in extracts of biota or in SPMD dialysates.
Purpose:
The objectives of this work were 1) to study the recovery and homogenity of PCBs in solution prepared for ELISA by direct dilution of isooctane solutions; 2) to evaluate nitrogen evaporation as a quick and efficient means for solvent removal; 3) to examine the efficacy of methanol and aqueous/methanol mixtures for quantitative recovery of residual PCBs, following evaporation of the hydrophobic solvent; and 4) to compare concentrations of ePCBs with their corresponding concentrations determined by gas chromatography (GC) in selected fish extracts and in dialysates of semipermeable membrane devices (SPMDs) used to monitor laboratory air.
Time_Period_of_Content:
Time_Period_Information:
Single_Date/Time:
Calendar_Date: unknown
Currentness_Reference: ground condition
Status:
Progress: Complete
Maintenance_and_Update_Frequency: None planned
Keywords:
Theme:
Theme_Keyword_Thesaurus: None
Theme_Keyword: polychlorinated biphenyls
Theme_Keyword: PCBs
Taxonomy:
Taxonomic_Keywords: fish
Taxonomic_Coverage:
Specific_Taxonomic_Information:
Kingdom: Animal
Division-Phylum: Chordata
Class: Actinopterygii
Applicable_Common_Names: fish
Access_Constraints: None
Use_Constraints: None
Point_of_Contact:
Contact_Information:
Contact_Organization_Primary:
Contact_Organization:
U.S. Geological Survey, Biological Resources
Division, Columbia Environmental Research Center
Contact_Person: James L. Zajicek
Contact_Address:
Address_Type: 4200 New Haven Road
City: Columbia
State_or_Province: Missouri
Postal_Code: 65201
Contact_Voice_Telephone: (573) 876-1885
Contact_Facsimile_Telephone: (573) 876-1896
Contact_Electronic_Mail_Address: James_Zajicek@usgs.gov
Cross_Reference:
Citation_Information:
Originator:
James L. Zajicek, Donald E. Tillitt, James N. Huckins, Jimmie D. Petty, Michael E. Potts, and
Originator:
U.S. Geological Survey, Biological Resources Division, Columbia Environmental Science Center (formerly Midwest Science Center)
Originator: David A. Nardone, Ohmicron Corporation
Publication_Date: 1996
Title:
Application of Enzyme-Linked Immunosorbent Assay for Measurement of Polychlorinated Biphenyls from Hydrophobic Solutions.
Publication_Information:
Publication_Place: unknown
Publisher: American Chemical Society
Data_Quality_Information:
Attribute_Accuracy:
Attribute_Accuracy_Report: unknown
Logical_Consistency_Report: not applicable
Completeness_Report: unknown
Lineage:
Methodology:
Methodology_Type: Lab
Methodology_Identifier:
Methodology_Keyword_Thesaurus: None
Methodology_Keyword: solvent exchange
Methodology_Description:
Microliter aliquots of isooctane solutions of two carbon-14 radiolabeled PCB congeners were diluted directly into 100 mL and 5.00 mL of 50% (v/v) methanol/water (50% MeOH/H20) or Ohmicron diluent (Ohm-diluent, a 50% [v/v] methanol/buffered aqueous solution with stabilizers). In some direct dilution experiments, after mixing, the undissolved isooctane droplets were evaporated by gently purging with nitrogen.
Methodology_Type: Lab
Methodology_Identifier:
Methodology_Keyword_Thesaurus: None
Methodology_Keyword: radioactivity measurements
Methodology_Description:
Liquid scintillation counting (LSC) analyses used a Model LS 3801 scintillation counter (Beckman Instruments, Fullerton, CA). Microliter volumes of Ohm-diluent, 50% MeOH/H20, methanol, or isooctane solutions of 14 C-TePCB or 14C-HxPCB were mixed with 10 mL of Ecolume liquid scintillation cocktail (ICN Biomedicals, Costa Mesa, CA) and counted.
Methodology_Type: Lab
Methodology_Identifier:
Methodology_Keyword_Thesaurus: None
Methodology_Keyword: sample preparation
Methodology_Keyword: fish
Methodology_Description:
Fish samples were a subset (23 of 117 monitoring stations) of the U.S. Department of Interior's National Contaminant Biomonitoring Program (NCBP) fish collected in 1988. Extracts of whole fish composites were prepared as described (Steinbraeber et al, 1994) for mayflies (Hexagenia bilineata). Briefly, whole fish samples were ground and mixed with sodium sulfate, column extracted with methylene chloride, applied to multi-layer reactive chromatography columns (sodium sulfate, 40% sulfuric acid silica gel [SA/SG], potassium silicate [KS], silica gel [SG], and sodium sulfate), and extracted with methylene chloride. The partially purified extracts were concentrated, and applied to a final reactive chromatography column (sodium sulfate, SA/SG, SG, and sodium sulfate), and were eluted with 0.5% benzene/99.5% hexane (v/v). Together these chromatography steps removed > 99.5% of the co-extracted lipids from the tissue extracts. During the subsequent solvent reduction of the clean extracts were exchanged into isooctane, which is a preferred solvent for GC analysis and for sample storage. For separate ELISA determinations, small aliquots (40-200 microliters) of the concentrated fish extracts (in isooctane) were brought to dryness in 1.1-mL conical glass vials, and the PCB residues were redissolved in 200 microliters to 1000 microliters of methanol to give solutions referred to as the "Primary ELISA Samples". Aliquots of each "primary ELISA Sample" were subsequently diluted with Ohm-diluent to give concentrations in the appropriate range defined by the ELISA calibration standards (0.25 ng to 5.0 ng Aroclor 1254/mL).
Methodology_Type: Lab
Methodology_Identifier:
Methodology_Keyword_Thesaurus: None
Methodology_Keyword: sample preparation
Methodology_Keyword: semipermeable-membrane devices
Methodology_Keyword: SPMDs
Methodology_Description:
Semipermeable-membrane devices were prepared and exposed to room air as described by Petty et al, 1993. Briefly, SPMDs were exposed to laboratory air for varying lengths of time up to 28 days and then dialyzed with hexane. Concentrated dialysates were then subjected to gel permeation chromatography, Florisil adsorption chromatography, and exchanged into hexane. Separate aliquots of the clean hexane extracts were brought to dryness in 1.1 -mL conical glass vials, and the PCB residues were redissolved in 1000 microliter of pure methanol. These solutions were referred to as the "Primary ELISA Samples". Aliquots of each "Primary ELISA Sample" were subsequently diluted at least 100-fold with Ohm-diluent to give concentrations in the appropriate range defined by the ELISA calibration standards (0.25 ng/mL to 5.0 ng Arocolor 1254/mL). the less than 1% increase in the methanol content of the resulting sample dilutions was considered to be trivial with respect to ELISA interference.
Methodology_Citation:
Citation_Information:
Originator: Petty, J.D., Huckins, J.N. & Zajicek, J.L.
Publication_Date: 1993
Title:
Application of Semipermeable Membrane Devices (SPMDs) as passive air samplers.
Series_Information:
Series_Name: Chemosphere
Issue_Identification: 27
Other_Citation_Details: pp. 1069-1624
Process_Step:
Process_Description:
Aliquots of the concentrated fish extracts were analyzed by capillary gas chromatography with electron capture to determine the concentrations of 105 individual PCB congeners (cPCBs, Steingraeber et al, 1994). The sum of the individual congeners is referred to as total-cPCBs.
Process_Date: unknown
Process_Step:
Process_Description:
Aliquots of the concentrated dialysates were analyzed by a separate gas chromatography method to measure concentrations of total PCBs (Petty et al, 1993). This GC method used to screen for total PCBs in SPMDs required less time per sample injection (45 minutes versus 160 minutes), required less time for data reduction (minutes versus hours/sample), but measured only about one half the number of cPCBs measured by the GC method for fish extracts. Total PCBs measured by this GC screening method are generally similar to those obtained by the more rigorous GC method used for fish, but the relationship between the two methods has not been determined statistically.
Process_Date: unknown
Process_Step:
Process_Description:
Enzyme-Linked Immunosorbent Assay. ELISA. The PCB-competitive ELISA (Ohmicron PCB RaPID Assay), a tube format assay employing paramagnetic particles that are covalently coated with anti-PCB antibodies, was supplied by Ohmicron Corporation and used according to the manufacturer's recommendations. Briefly, individual samples (200 mciroliter aliquots) were analyzed singly or in duplicate together with four duplicate calibration standards: 0, 1.25, 1.0, 5.0 ng/mL. Up to thirty individual sample dilutions (or 15 duplicate) were analyzed with one Ohmicron positive control (Ohmicron-control) per 20 tubes in batches of 20 to 40 tubes. The immunochemical reagents, 250 microliters of PCB enzyme conjugate (a horseradish peroxidase-labeled PCB analog) and 500 microliter of the particle suspension, were added to aliquots of each sample, standard, and control in test tubes. The tubes were vortexed, and the competitive binding reaction was allowed to occur at room temperature for 15 minutes. The paramagnetic particles containing bound PCBs and/or PCB enzyme conjugate were isolated using the vendor supplied magnetic separation rack and two washes. The magnetic rack was separated from the tube rack, 500 microliters of substrate/chromogen solution (hydrogen peroxide/3,3', 5,5'-tetramethylbenzidine) was added to each tube, and the color was developed at room temperature. After 20 minutes the color reaction was stopped by adding 500 microliters of 2 M HcS04. It is important to note that although, some of the ELISA measurements were performed over one year after the reference GC measurements, it has been the investigators experience that repeat GC analysis of similar fish extracts stored in isooctane for comparable periods of time show no measurable losses of PCBs (J.L. Zajicek, unpublished data).
Process_Date: unknown
Process_Step:
Process_Description:
ELISA Reader. ELISA absorbances at 450 nm were measured with a Model RPA-1 spectrophotometer (Ohmicron Corp. Newtown, PA). Measured absorbance data (B) were related to that of the zero standard (B0), and concentrations were calculated from the relation: Logit B = Slope * Ln [PCB] + Intercept where Logit B = Ln ([B/B0]/[1-B/B0]). Concentration calculations were either performed automatically using the RPA-1 or by using Microsoft Excel (Microsoft Corporation, Redmond, WA).
Process_Date: unknown
Process_Step:
Process_Description:
Parallelism. Selected samples (one enriched extract each from fish and SPMDs) were serially diluted and analyzed by ELISA to compare the resulting dose-response curves of PCBs in these samples to similar curves resulting from dilutions of the Aroclor 1254 calibration standards. Lack of parallelism between standard and sample curves gives and indication of matrix effects associated with the sample, solvents, or the reagents.
Process_Date: unknown
Entity_and_Attribute_Information:
Overview_Description:
Entity_and_Attribute_Overview:
Entity - Biological tissue extracts; Associated attributes - polychlorinated biphenyls (PCBs)
Entity_and_Attribute_Detail_Citation: unknown
Distribution_Information:
Distributor:
Contact_Information:
Contact_Organization_Primary:
Contact_Organization:
U.S. Geological Survey, Biological Resources
Division, Columbia Environmental Research Center
Contact_Person: Christopher Henke
Contact_Position: Webmaster
Contact_Address:
Address_Type: mailing and physical address
Address: 4200 New Haven Rd
City: Columbia
State_or_Province: MO
Postal_Code: 65201
Contact_Voice_Telephone: 573-875-5399
Contact_Facsimile_Telephone: 573-876-1896
Contact_Electronic_Mail_Address: chris_henke@usgs.gov
Distribution_Liability:
Although these data have been processed successfully on a computer system at the U.S. Geological Survey, no warranty expressed or implied is made regarding the accuracy or utility of the data on any other system or for general or scientific purposes, nor shall the act of distribution constitute any such warranty. This disclaimer applies both to individual use of the data and aggregate use with other data. It is strongly recommended that these data are directly acquired from a U.S. Geological Survey server, and not indirectly through other sources which may have changed the data in some way. It is also strongly recommended that careful attention be paid to the contents of the metadata file associated with these data. The U.S. Geological Survey shall not be held liable for improper or incorrect use of the data described and/or contained herein.
Custom_Order_Process: Please contact distributor.
Metadata_Reference_Information:
Metadata_Date: 200004
Metadata_Contact:
Contact_Information:
Contact_Organization_Primary:
Contact_Organization:
Raytheon Information Technology and Scientific
Services (ITSS)
Contact_Person: Cheryl Solomon
Contact_Position: Ecosystem Coordinator
Contact_Address:
Address_Type: Mailing and Physical Address
Address: 4500 Forbes Boulevard
City: Lanham
State_or_Province: MD
Postal_Code: 20706
Country: USA
Contact_Voice_Telephone: 301 794-3049
Contact_Facsimile_Telephone: 301 794-3164
Contact_Electronic_Mail_Address: solomon@gcmd.nasa.gov
Metadata_Standard_Name:
NBII Content Standard for National Biological Information Infrastructure Metadata
Metadata_Standard_Version: December 1995
Metadata_Access_Constraints: None
Metadata_Use_Constraints: None
Generated by mp version 2.5.6 on Mon Jul 31 09:12:54 2000