Immunoassays are based on the ability of immunoglobulin proteins (antibodies) to distinguish three-dimensional structure at the molecular level. Antibodies are produced by the immune system of higher animals in response to and for the elimination of foreign materials. This ability to discriminate and bind strongly to unique structures in complex mixtures provides the means to measure those structures in controlled reaction conditions.
The radio-immunoassay (RIA) technique utilizing radioactive isotopes as label was first exploited by Yalow and Berson to quantitate insulin for which a Nobel prize was awarded in 1977. A second generation immunoassay technology replaced radio-isotopes with enzymes used with chromogenic substrates (EIA) eliminating handling and disposal issues. More recently, the use of polymeric membranes and colored particles have led to the development of field screening assays with minimal manipulations.
The immunoassay technique is widely used in clinical laboratories as the basis for decisions on diagnosis and therapy. The current world-wide market for clinical immunoassays is greater than 60 billion dollars. Since the late 1980s immunoassay technology has been applied to analytes of environmental and food safety concern. While these applications currently represent a small fraction of the clinical testing market, they are rapidly becoming more widely accepted and utilized due to the obvious advantages in cost, speed and decentralized testing possibilities.
Immunoassays can be divided into two major categories: competitive and non-competitive or sandwich assays. Competitive assays utilize a single specific antibody type immobilized to a solid surface and a corresponding analogue of the analyte to carry the label. In the assay, analyte in the sample competes with the labeled analogue for binding to the antibody. After separation of unbound analogue, the amount of label remaining is measured and is inversely proportional to the analyte concentration in the sample.
Non-competitive assays utilize two specific antibodies to sandwich the analyte. One antibody (capture antibody) is immobilized to a solid surface and the second antibody carries the label. In the assay, analyte is bound simultaneously by both the capture and label antibodies. After separation of unbound label antibody, the remaining label is measured and is directly proportional to analyte concentration in the sample. Sandwich assays are limited to those analytes of sufficient size to be able to bind two antibodies simultaneously, typically proteins and microorganisms. Competitive assays are compatible with a wide range of analytes and are used for the majority of low molecular weight organic analytes of environmental and food safety concern. However, even with competitive assays there is a lower limit of molecular weight (or structural uniqueness) to which the technique can be applied.
The underlying principle in all immunoassays is that assay reaction conditions do not vary except for the concentration of analyte in the calibrator or sample.
Considerations for Use
The applicability of immunoassay testing to analytical needs is determined by a number of factors including sample type, sample throughput, qualitative or quantitative and specificity. Immunoassay testing is probably best suited to large scale or routine screening programs where the nature of the analytes being tested is understood. The specificity of antibodies, or their ability to distinguish between closely related chemical structures varies widely. For instance, the antibody used in the Beacon s-Metolachlor kit can distinguish between the r and s isomers of Metolachlor as well as between Metolachlor and other chloro-acetanilides. On the other hand, the antibody used in the Beacon 2,4-D kit can not distinguish 2,4-D acid from 2,4-D esters or other closely related chlorophenoxy compounds. It the case of 2,4-D the immunoassay response will be the sum total of the concentrations of the reactive species without the ability to quantify the species individually. On the other hand, a negative result with an assay with wide specificity can yield information on the absence of a range of compounds. Immunoassays are, therefore, not typically well suited for survey situations where the specific contaminant needs to be identified from a wide range of possibilities.
Immunoassays utilize biologically-derived reagents and therefore function optimally in conditions which approximate physiologic. The ideal sample is either water or an aqueous sample extract. The sensitivity of immunoassays to organic solvents can also vary widely. For instance, the Beacon Aflatoxin kit achieves ppb sensitivity in 35% methanol. The important consideration is that the reaction conditions for samples are as similar as possible as conditions for calibrators.
Immunoassays can yield sub-ppb sensitivity in less than 15 minutes at a cost of less than $10 per sample at remote locations as well as centralized laboratories. Some typical examples of their use might be testing in-coming grain samples for mycotoxin contamination or testing of raw and finished drinking water for atrazine content.
The staff at Beacon is committed to working closely with customers to ensure that immunoassay technology is utilized in the most accurate and efficient manner to meet their testing needs. Please contact us for prompt response to inquiries.