Eric Senning


Although the basics to immunoassaying were discovered around fifty years ago, it wasn't until the sixties before the immunoassay became a serious analytical technique. On returning to the US, it was noted that many Vietnam war veterans were addicted to opiate based drugs. Needless to say that these drug habits were acquired to alleviate the harrowing effects of war, the US government was now confronted with the problem of  monitoring the drug usage of these veterans, and this was not an inexpensive matter. The available chromatographic detection of morphine, a principle component of poppy plant extract, was costly and required a large amount of sample form the patient. Demand for  more efficient heroin screening escalated drastically because of this, and it was only a short time before the I-125 radioimmunoassay became the analytical technique of choice to detect heroin usage in human patients.

The sudden interest in immunoassays was based on several factors. As previously mentioned, the immunoassay is cheaper than most chromatographic techniques since it can be selectively manufactured to be sensitive to only one analyte. In conjunction with this,  only small quantites of analyte need be present for detection; this brought an end to the requirement for large serum samples. The third factor I want to mention is the convenience in time it takes to perform an immunoassay. As it was necessitated by the US government after the Vietnam War, immunoassays could be run on a mass scale to monitor veterans' health.

The mechanism of the immunoassay is modeled after the behavior of the humoral immune system. Immunoglobulins, which are quaternary proteins also called antibodies, that have a high affinity for the intended analyte are produced in large numbers and covalently anchored to a surface such as the inside of a platic test-tube. The physical nature of antibodies used in an immunoassay are generally ìYî shaped with the base being anchored to the test-tube and the forked end having the affinity for the analyte (antigen). A simplified model of an Ig G antibody is located at the beginning of the first paragraph. When the test-tube is accordingly filled with a sample, the analyte in solution comes in contact with an antibody and binds to it as follows:

Antibody + Analyte (Antigen) <---> Antibody-Analyte(complex)

This reaction mechanism also has an affinity constant that in the case of immunoassays is usually a high value near 10^10:

K = Antibody-Analyte(complex)

Thusly, most of the Analyte in solution will interact with the anchored Antibodies and bind to them. At this point a third reagent can be introduced to the test-tube. By various ways, depending on the choice of immunoassay, the amount of analyte can then be determined by the interaction of the added reagent(s) with the antigen( analyte ) bound antibodies. The interaction can be quantified with an analytical instrument depending, again, on the implemented immunoassay . This is a fairly generic example of an immunoassay. In comparison to the previous description, modern immunoassays can be very comples and may involve numerous steps.


 Student Laboratories from the J. of Chem. Education

1. Yalow, Rosalyn S. Development and Proliferation of Radioimmunoassay Technology J. Chem. Educ. 1999 76 767. (June 1999)

2. Ekins, Roger P. Immunoassay, DNA Analysis, and Other Ligand Binding Assay Techniques: From Electropherograms to Multiplexed, Ultrasensitive Microarrays on a Chip J. Chem. Educ. 1999 76 769. (June 1999)

3. Ullman, Edwin F. Homogeneous Immunoassays: Historical Perspective and Future Promise J. Chem. Educ. 1999 76 781. (June 1999)

4. Straus, Eugene Radioimmunoassay of Gastrointestinal Hormones J. Chem. Educ. 1999 76 788. (June1999)

5. Russo, Salvatore F.; Dahlberg, Judy Utter. An enzyme immunoassay for human transferrin. J. Chem. Educ. 1990 67 175.

6. Angstadt, Carol N.; Barbieri, Edward J.; Chase, Grafton D. A new application of radioimmunoassay: measurement of thermodynamic constants. J. Chem. Educ. 1983 60 513.

7. Juaristi, Eusebio. Immunoassay in therapeutic drug monitoring. J. Chem. Educ. 1983 60 721.

8. Kohl, Herbert H.; Wheatley, W. B. Laboratory introduction to competitive protein binding and radioimmunoassay. J. Chem. Educ. 1978 55 612.


Helpful Links:


To get a better grasp on comprehending the mechanisms of an immunoassay, the following web-site provides animated sequences of several immunoassays. These techniques are very up-to-date and modern, but they should not be overwhelmingly complicated:

The following site discusses a broad range of important factors in the immunoassay:

Environmental studies also use the immunoassay:

The ELISA is illustrated on the following web-site:

Review Articles on Immunoassays

1. INVITED REVIEW - Immunoassay and Other Ligand Assays: From Isotopes to Luminescence Source: Journal of clinical ligand assay : official publication of the Clinical Ligand Assay Society and the European Ligand Assay Society. 22, no. 1, (1999): 61 (17 pages).

2. Retrospective review of dot enzyme immunoassay test for typhoid fever in an endemic area Source: The Southeast Asian journal of tropical medicine and public health. 26, no. 4, (1995): 625

3.  A Review of Enzyme-Immunoassay and a Description of a Competitive Enzyme-Linked Immunosorbent Assay for the Detection of Immunoglobulin Concentrations. Source: Biochemical education : a quarterly publication of the International Union of Biochemistry. 18, no. 3, (July 1990): 136

4.   Particle-labeled immunoassays: a review Source:  J. Chromatogr. 376 (1986): 175-89

5.  Impact of emerging technologies on immunochemical methods for environmental analysis Source: ACS Symp. Ser. 586, no. Immunoanalysis of Agrochemicals (1995): 1-19

6.  Biosensors. Their role in detection systems for cargo  inspection  Source: Proc. SPIE-Int. Soc. Opt. Eng. 2276, no. Cargo Inspection Technologies (1994): 112-19

7.   Immunoassays for Pesticides Source: Environ. Sci. Technol. 29, no. 3 (1995): 553-61