Immunoassays

by Niranjan Aiyagari & Timothy La Breche

IMMUNOASSAY IN FIELD USE

Table of Contents


Description of Immunoassays Example of Immunoassays
Animation of an Immunoassay Advantages and Disadvantages of Immunoassays
EPA Methods That Use Immunoassays

DESCRIPTION OF IMMUNOASSAYS



Immunoassays have been in use since the the early 1960's when Radioimmunoassays were used to quantify insulin in plasma samples. Since their introduction, immunoassays have been used to detect and quantify hundreds of types of molecules both native to living organisms, such as hormones, and foreign molecules, such as pharmaceuticals. The molecules detected by immunoassays vary widely in size, chemical and physical properties, and biological activity.

The crux of an immunoassay is the natural reponse of a living organism to a foreign agent. For example, the common cold is caused by a virus. A virus is a piece of RNA (ribonucleic acid) with a protein coat surrounding it. When this virus enters the body it triggers the immune system to produce antibodies that will attach themselves to the virus or "antigen". Once the antibody and antigen are bound together the antigen's activity is reduced or eliminated and the cluster is removed from the body. It is estimated that the individual animal can produce antibodies specific for 10,000,000 to 100,000,000 antigens. The ability of an antibody to discriminate between the millions of naturally occuring molecules in a living organism is critical to the use of immunoassays in environmental analysis.

While a virus is a very small physical object, it is larger than most macromolecules. Macro molecules such as proteins, polysaccharides, and nucleic acids (like RNA) are usually discernable by immunoassays. However, the molecules that environmental engineers are typically concerned with are much smaller than these molecules. Pesticides, fungicides, and herbicides are typically less than 1,000 atomic mass units and therefore do not usually provoke a response by a specific antibody unless they are bound to an antigenic macromolecule. A molecule that doesn't specifically incite an antibody but can attach to its specific immunogenic macromolecule is termed a hapten. Selection or syntheis of a hapten specific antibody permits the detection of these smaller molecules that don't ordinarily illicit an immune response.

The table shows the relative size of particles of interest in environmental engineering. Most insecticides and fungicides fit in the lower region of the molecular size range

PARTICLE SIZE COMPARISON

Frequently an antibody that recognizes one hapten will recognize other similar molecules, as is the case with the cyclodiene insecticide Aldrin and other organochlorine pesticides. Further separation techniques such as HPLC are required in cases like these to distinguish between specific molecules, like DDT, lindane etc.. While some immunoassays may be limited in distinguishing between small differences in molecular structure within a family of compounds, they do have the distinct advantage of being highly selective even in the the midst of much obscuring material, like humic acids, fulvic acids, petroleum spills etc. A sample that might require days of clean up in the lab, can in some cases be checked in the field by immunoassay in about an hour without excessive cost or bulky equipment.



EXAMPLE OF IMMUNOASSAYS



OHMICRON RaPID Assays (Rapid Pesticide Immuno Detection Assays)

ELISA's
(Enzyme linked immunosorbent assays) are based on selective antibodies attached to solid supports combined with enzyme reactions to produce systems capable of detecting low levels of chemicals. The Ohmicron RaPID assay uses magnetic particles as the solid support and means of separating test pesticides. Following this description is an animation that demonstrates the concepts of an Ohmicron immunoassay.



Step One: Immunological reaction
The water sample, the pesticide labeled with an enzyme, and magnetic particles with antibodies for the pesticide of interest attached are added to a test tube. The pesticide of interest in the water sample and the enzyme labeled pesticide compete for binding sites on the magnetic particles. The reaction proceeds for 5 to 10 minutes.

Step Two: Magnetic separation
A magnetic field is applied to the test tube and the magnetic particles along with their attached pesticides and enzyme labeled pesticides are drawn toward the field and held in place against the tube wall. The test tube is emptied and the particles washed once with water. The ratio of labeled and non labeled pesticides remaining in the tube are in direct proportion the original concentration in the tube.

Excluding the ISCO, the large tan cylinder, this is all it takes for field sampling with Immunoassays.


The compact, fast nature of Immunoassays makes them ideal for samling in remote locations like this:

Step Three: Color development
The presence of labeled pesticide is determined by adding the enzyme substrate, hydrogen peroxide, and the chromogen 3,3',5,5' - tetramethylbenzidine. These react to form a colored product that is directly proportional to the concentration of labeled pesticide and thus inversely proportional to the concentration of pesticide in the water sample. In other words the more intense the color generated the less pesticide that was in the sample. The color intensity may then be measured with a spectrophotometer and quantified from a standard curve that the user generates.



ADVANTAGES AND DISADVANTAGES OF IMMUNOASSAYS COMPARED TO CONVENTIONAL METHODS :

ADVANTAGES


1) Immunochemical methods provide a rapid, sensitive, and cost effective analyses for a variety of environmental contaminants. These methods are highly sensitive to specific antibodies, and provide analytical systems capable of detecting very low levels of chemicals. This high sensitivity is due to the powerful catalytic ability of enzymes. These methods are also highly selective due to the extraordinary discriminatory capabilities of antibodies. The lowest concentrations(ppb) at which some pesticides can be detected using immunoassays are given in the following table:

COMPOUND LEAST DETECTABLE DOSE (ppb)
Dieldrin 0.55
Aldrin0.29
Heptachlor0.66
Endrin0.68
Endosulfan1.16
Chlorpyrifos0.10
Diazinon0.12
Lindane10.6

2) Immunoassay methods are fast and relatively easy compared to conventional GC/MS and HPLC. Whereas a conventional method may require 1 hour to analyze one sample, immunoassays can analyze about 30 samples per hour. This is because immunoassays require fewer steps and less training to operate. The Immunoassay instrumentation kit is portable and can be used for testing right at the sampling site.

3) Immunoassays are inexpensive when compared to traditional GC/MS and liquid chromatography. One can establish an enzyme immunoassay laboratory for as low as $1000, and the per-sample cost is only about $10. Setting up a laboratory to provide conventional analytical techniques can cost $150,000 or more, and the per-sample cost is about $200. Immunoassays also have the advantage of not requiring a multi-step cleanup process as do many traditional methods. While the cost is low for a given sample one should bear in mind that each chemical of interest requires a separate immunoassay. Therefore there is a point of diminishing returns in terms of cost and time when compared to GC/MS systems that are capable of discerning 70 or more chemicals from a single sample run.

4) Immunoassays are a very efficient screening tool. Detection limits are suitably low for environmental work, and sample preprocessing is not required with its abundant losses. Recovery of analyte from spiked sample can be better with immunoassays than with conventional methods.


DISADVANTAGES



Inspite of its numerous advantages in helping make significant progress in research, enzyme immunoassays cannot replace conventional methods altogether, and are best used in combination with them.

1) Some immunoassays are not highly selective, and they may respond to families of pesticides such as cyclodienes, rather than to individual compounds. As a result, more than one compound in a family shows up in the environmental sample In such cases, we will have to use the conventional GC/MS or HPLC methods to identify the analytes.

2) Because of the design of the immunoassay, sample contaminants that might interfere with the antigen-antibody reaction can produce positive readings when samples are indeed negative (called false positive results). This design eliminates the possibility of obtaining false negatives due to interfering materials. Conventional methods, can then have be used as a backup test for positive samples to be sure sample contamination is not skewing your results.

3) Lower molecular weight molecules often lack specific antigens, and sometimes there are crosslinking problems.

EPA METHODS USING IMMUNOASSAYS

The EPA has compiled a list of methods which are specific for immunoassays. The following are the method numbers and titles:

Method 4000Immunoassay
Method 4010Screening for Pentachlorophenol by Immunoassay
Method 4015Screening for 2,4-Dichlorophenoxyacetic Biphenyls by Immunoassay
Method 4020Screening for Polychlorinated Biphenyls by Immunoassay
Method 4030Soil screening for Petroleum Hydrocarbons by Immunoassay
Method 4035Soil screening for Polynuclear Aromatic Hydrocarbons (PAHs) by immunoassay
Method 4040Soil screening for Toxaphene by immunoassay
Method 4041Soil screening for Chlordane by immunoassay
Method 4042Soil screening DDT by immunoassay
Method 4050TNT Explosives in Water and soils by immunoassay
Method 4051Hexahydro -1,3,5 - trinitro - 1,3,5 -triazine(RDX) in Soil and water by Immunoassay


REFERENCES


  1. EPA Documents: SWA 846 Chapter 4 EPA Homepage
  2. Environmental Lab, December/January 1991/92, Edward J. Baum, Ph.D.
  3. Immunochemical Methods for Environmental Analysis, ed. Van Emon, Jeanette M. and Mumma, Ralph O., ACS Symposium Series 442, 1990
  4. Product Information Publications, Ohmicron Environmental Diagnostics, Inc., 375 Pheasant Run, Newtown, PA 18940 U.S.A. telephone: 215/860-5115


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Student Authors:
Timothy La Breche, tlabrech@vt.edu
Niranjan Aiyagari, naiyagar@vt.edu
Faculty Advisor: Andrea Dietrich, andread@vt.edu
Copyright © 1997 Daniel Gallagher
Last Modified: 12-22-1997