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P&S Journal

P&S Journal: Winter 1997, Vol.17, No.1
Research Reports
Improving Diagnostic Tests

A new procedure that amplifies immunohistochemical tests used to detect cancer and infectious diseases markedly enhances the sensitivity of such tests while significantly reducing their cost. The amplification strategy, for which a patent application has been filed, was developed jointly by Drs. Bernard F. Erlanger, professor emeritus of microbiology; Bi-Xing Chen, staff associate in microbiology; and Matthias J. Szabolcs, assistant professor of clinical pathology. Their collaborative study appeared in the Journal of Histochemistry and Cytochemistry.

Drs. Erlanger and Szabolcs added a new twist to standard immunohistochemical procedures, which use marker-specific antibodies to identify proteins in cancerous cells or infectious agents. Those antibodies are known as primary antibodies. In order to detect whether the primary antibodies are bound to the targeted antigens, the primary antibodies are linked to either an enzyme or secondary antibodies that are linked to enzymes. Horseradish peroxidase is one commonly used enzyme.

In a typical immunohistochemical procedure, the primary antibody is added first, followed by the secondary antibody conjugated to the enzyme. Unbound secondary antibody is removed by washing. The presence of antibody-enzyme complex is then visualized by adding an enzyme substrate (for instance 3,3'-diaminobenzidine, or DAB, for peroxidase) which is converted into a pigmented insoluble product (DAB polymer), which is visible under the microscope. The brown DAB polymer is a signal to the pathologist that cancerous cells or infectious agents are present in the sample.

The innovation developed by the researchers increases the amount of pigment produced while also reducing the amount of primary antibodies that must be used in the test. Primary antibodies are expensive, costing as much as $400 per milliliter.

The researchers add another step to the procedure: They add antibodies specifically prepared to react with DAB polymer. Because one enzyme can "turn over" huge amounts of substrate, this additional step amplifies the results. For example, one molecule of enzyme might produce 1,000 molecules of end product (DAB polymer), which is then recognized by the anti-end-product antibody.

In trial procedures, Dr. Szabolcs has obtained as much as a 50-fold amplification, making possible a corresponding reduction in the amounts of primary antibodies used. This means that in some cases, pathologists will be able to reduce the amount of primary antibodies by up to 95 percent.

In addition, the amplification procedure is more sensitive to the presence of markers of infectious diseases or neoplastic cells and, for example, is able to pick out a few cancerous cells in a sample that, in the absence of amplification, would be missed. The procedure produces few false positive results and is easily used in automated testing systems found in most pathology labs.

Drs. Erlanger, Chen, and Szabolcs are now working on a similar system for tests to detect gene markers on chromosomes. In this case, antibodies have been raised to the product of the action of alkaline phosphatase on tetrazolium substrate.

copyright ©, Columbia-Presbyterian Medical Center

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