How Immunoassays Work


Antibody-based Enzyme-Linked ImmunoSorbent Assay (ELISA) methods have been widely used in medical applications since the early-70's. Countless studies have demonstrated the correlation between ELISA results and traditional methodologies such as gas chromatography (GC) and high performance liquid chromatography (HPLC).

Immunoassays are based on the principles that specific antigens will stimulate very specific (unique) immune responses and that the proteins produced by the immune response, called antibodies, can be used to signal the presence of a target compound in a sample.


Plate and tube format assays:
Most EnviroLogix 96-well format (Plate) kits and tube kits (including all the pesticide ELISA assays) are competitive assays.

In many cases, working with customers or anticipating alternate uses, EnviroLogix has developed methods for extracting analytes from other matrices so that matrix effects are eliminated or can be factored into interpretation of results. See Contacting EnviroLogix for further information.


Extending the Assay Range:
Each product insert specifies the range of analyte concentrations over which the assay is most effective. Customers may wish to decrease the sensitivity of an assay (testing samples with 10 to 100 parts per million (ppm) rather than the 1 to 10 parts per billion around which the assay may have been designed). In these cases, EnviroLogix may have prepared a written protocol for dilution of the samples, or may help through a call for technical support. In cases where the customer prefers the assay to detect an analyte at even lower concentrations than the limit of detection, it may be possible through concentration of the analyte. EnviroLogix technical support would be happy to share their experiences in this regard. See Contacting EnviroLogix for further information.


In these tests, pesticide residues in the sample compete with known amounts of pesticide analogue that has been enzyme labeled (typically with horseradish peroxidase) for a limited number of antibody binding sites on the inside surface of the test wells.

After a simple wash step, the outcome of the competition is visualized with a color development step.

As with all competitive immunoassays, sample concentration is inversely proportional to color development:

Darker color = Lower concentration of the suspected analyte
Lighter color = Higher concentration of the suspected analyte


Note: Certain non-pesticide tests, such as the Bt Endotoxin assay, use a different format for displaying immune response called a "sandwich" assay. In such assays, darker color means higher concentration of the analyte. Be careful to read the product insert that accompanies each test to be certain of the proper interpretation.

Interpreting immunoassays:

EnviroLogix plate and tube kits are supplied with Calibrators (known concentrations of the target analyte in solution) and a negative Control (known to be free of the target analyte) for both visual and instrumented interpretation of the test results. These Standards (Calibrators and Control) exhibit distinctly different shades of blue color at the different concentrations provided (for example: zero, 0.5 ppb (parts per billion), 1 ppb, 5 ppb, 10 ppb). By comparing the color of the sample against the Standards, the person testing may visually determine the concentration range of the sample, for example, "between 1 and 5 ppb". This interpretation is semi-quantitative.

Quantitative interpretation can be performed by inserting the microwells in a "plate reader" which precisely measures the optical density of all samples and all Standards at the same time. Using software provided with the reader the user then calculates the sample concentration from the Standard. EnviroLogix does not distribute plate readers, but will assist customers in making an appropriate selection.

For field use, a "strip reader" or "mini-photometer" can be used to process one eight-well or twelve-well strip at a time. This type of device is much more portable than a plate reader, and less costly, but it does require more manual manipulation. EnviroLogix carries one line of mini-photometers as a convenience to customers. See Accessories for further information.


QuickStix and QuickTox

EnviroLogix' QuickStix and QuickTox lateral flow test devices employ the same immunoassay principles as the plate and tube formats, but coat the antibodies and other reagents on a nitrocellulose membrane rather than on the inside of test wells or tubes. The sample is added at one end of the device and travels by capillary action to the other end. In passing along the membrane the sample is exposed to zones of antibody reactive to the target analyte. The QuickStix/QuickTox assay is designed to develop a "control line" near the far end of the device.

Interpretation of results with QuickStix:
If no "control line", the test has not run properly and should be repeated. As with the plate and tube formats, certain tests are "competitive assays" and others are so-called "sandwich assays". For example, EnviroLogix' pesticide assays, whether plate, tube or QuickStix, are all competitive assays, while the Bt endotoxin assays are all sandwich assays.

In competitive QuickStix/QuickTox assays, a positive result is indicated by the absence of a line in the test result zone.
In sandwich assays, a positive result is indicated by the presence of a line in the test result zone.




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