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Immunocytochemistry Methodology

By : Biomeda Corporation, Copyright © 1996. All Rights Reserved.

The immunocytochemical methodology has evolved from the early works of Nakane and Pierce (l) and Avrameas (2) on antibody-enzyme conjugation tech-niques. The unlabeled Peroxidase Anti Peroxidase (PAP) system developed by Sternberger and coworkers (3) improved the sensitivity of preceding immunocyto-chemical methods. In 1974, Heitzman and Richards (4) introduced a new concept in cytochemistry, the Avidin-Biotin Affinity Cytochemistry. This system had been pioneered and used earlier by Becker and Wilchek (5) in an immunoassay application. The system relies on the extraordinarily high affinity that avidin has for biotin, approximately one million times higher than the affinity of most antibodies for their corresponding anti-gens. The covalent attachment of activated forms of biotin to biologically active proteins, particularly to antibodies, permits the detection of those labeled pro-teins following their binding to cells or tissues by using a variety of tracing probes linked to avidin. A substan-tial expansion and refinement of the Avidin-Biotin sys-tem was achieved by Bayer, Wilchek and Skutelsky (6-8). An excellent review on the use of the Avidin-Biotin Complex in bioanalytical applications has been pub-lished by Wilchek and Bayer (9). Due to the intrinsic flexibility of this system, it was uti-lized for developing a variety of valuable immunocyto-chemistry applications. The work of Warnke and Levy (10) on the differentiation of B and T cell antigens using monoclonal antibodies enhanced the use of the Avidin-Biotin Complex (ABC) in histology and cyto-chemistry. A variation of the Avidin-Biotin approach was introduced in early 1981 by Hsu, Raine and Fanger (11, 12). It was followed by a methodological modification developed by Tubbs and coworkers (13), O'Brien and associates (14), and Marty et al. (15). While undergoing continuous metamorphosis, the ABC technology has gained considerable recognition as a potent biochemical tool in histochemistry and in other areas of biology (8, 9). Immunocytochemical techniques are becoming a major tool for surgical pathologists and immunopathologists in the detection of specific antigens in tissue sections. These techniques have facilitated the assessment of a myriad of complex problems and have decisively assist-ed in the elucidation of cell lineage of many malignant proliferations. Lymph node biopsy specimens are currently being criti-cally analyzed for specific cell markers. The demonstra-tion of monotypic single light chain immunoglobulins is of greatest importance in the recognition of B cell lym-phomas, the sorting of histocytic lymphomas from non-lymphoid neoplasms, as well as the differentiation of reactive lymphoid infiltrates from neoplasms. Due to the use of these techniques, significant information on the subclassification of malignant lymphomas has been accumulated. The study of tissue-associated markers, whether of an embryonic, normal, tissue specific, path-ogenic or infectious origin, has become a major focus of the clinical laboratory in recent years. Immunohistological techniques such as immunofluores-cence and immunoperoxidase (a.k.a. horseradish perox-idase, HRP) have been demonstrated to be excellent tools in the search for many native or acquired cellular antigens in tissue sections (16-18). These techniques have been subjected to continuous modifications and improvements which have considerably expanded and enhanced their role in the immunodiagnostic and bio-medical research fields (9). Streptavidin, an avidin analog produced by a bacteria, has in some cases successfully substituted avidin and is generally considered to produce cleaner signals. In a recent study, immuno-researchers have reported that systems using streptavidin-peroxidase conjugates may be significantly more sensitive than those using the variation of the ABC approach of Hsu et al. (12) that utilizes the biotinylated peroxidase and avidin to form a complex prior to its use. The AutoProbe III, AutoProbe II, LAB/Probe, HistoScan and BioStain Super ABC kits are now formulated using this trepta-vidin-based technology. Biomeda's LymphoScan and HistoScan product series are immunohistochemical kits for the detection of human antigens, hormones and infectious agents such as hepatitis B virus surface antigens, herpes virus, and cytomegalovirus in fixed paraffin-embedded or acetone-fixed frozen tissue sections. These kits are based on the Streptavidin-Biotin technology and are available using three different detection systems: horseradish peroxi-dase (HRP), alkaline phosphatase (AP), or phycoery-thrin (PE). Universal kits containing the basic detec-tion system and a labeled second antibody directed against the specific animal species are also available in this series. The availability of the LymphoScan and HistoScan series of immunohistochemical staining kits provides the sensitivity and precision of the most advanced tech-nology in immunocytochemistry. These kits have been developed with dual objectives in mind: simplicity and quality. The superior performance of the Streptavidin-Biotin Complex methodology made the simplification of procedures feasible without undue sacrifice of the staining quality. The system employed in these kits provides a high signal to noise ratio, which translates into exceptionally low non-specific background stain-ing. Due to the extraordinarily high effciency of this method, shorter incubation times are required. Also included in the kits are instruction booklets containing detailed information about the staining procedure as well as a general description of the technology used in the kit. Most kits have a shelf life of one year when stored at 4-8C.

Automated Immunocytochemistry
The design and execution of automated immunocyto-chemistry by Brigati et al. (19) has provided improved efficiency to analytical immunopathology. Biomeda's implementation in automated immunocytochemistry is based on Brigati's heterologus formulation of enzyme labeled antibodies directed against a variety of animal species immunoglobulins. These systems, presently known as the AutoProbe and the UltraProbe Immunosystems, offer several advantages over conven-tional approaches currently used. The AutoProbe sys-tem is based on the use of peroxidase (HRP) as the tracer, while the UltraProbe utilizes alkaline phos-phatase (AP).

The AutoProbe II, III and Ultra Probe Immunosystems:
*Use a simple and reliable 3-step protocol
*Are compatible with primary antibodies from NINE different animal hosts
*Utilize a universal blocking reagent
*Eliminate possible sources or errors
*Support automated and manual procedures

AutoProbe II and the UltraProbe kits recognize multi-ple primary antibodies made in mouse, rat, guinea pig, rabbit, goat, sheep, cow, donkey and horse. Both kit series are based on the Avidin-Biotin Complex in a 3-step protocol. The reagents are color and number coded and provided in plastic dropper bottles. All the AutoProbe as well as the UltraProbe kits are available in two sizes: 150 tests and 500 tests. The 150 series is designed for both manual and volume auto-mated immunocytochemistry . The 500 series is dedicat-ed to automation and is designed to stain 500 tissue sections when used at the rate of O.l ml per slide. In addition, the ultra sensitive UltraProbe system is also available in a 60 test size. To complement these AutoProbe and UltraProbe immunodetection kits, Biomeda offers an extensive line of auxiliary reagents for automated immunocytochem-istry, including ready-to-use monoclonal and polyclonal primary antibodies, concentrated primary antibodies, aqueous hematoxylin, antibody diluting buffer, rinsing buffers, stabilized enzyme solution and other miscella-neous reagents.

References
1. Nakane, P.K. and Pièrce, G.B., Jr. J. Histochem. Cytochem. 14:929, 1966.
2. Avrameas, S. Immunocytochem. 6:43, 1969.
3. Sternberger, L.A. et al. J. Histochem. Cytochem. 18:S15, 1970.
4. Heitzmann, H. and Richards, F. Proc. Nat. Acad. Sci. US 71:$697, 1974.
5. Becker, J.M. and Wilchek , M. Biochim. Biophys. Acta 2â4:lô5, 1972.
6. Bayer, E.A. et al. J. Histochem. Cytochem, 24:933, 1976.
7. Bayer, E . et al. FEBS Letters 68:240, 1976.
8. Bayer, E.A. and Wilchek, M. Methods in Biochem. Anal. 26:1, 1980 (John Wiley & Sons, New York),
9. Wilchek, M. and Bayer, E.A. Analytical Biochemistry. 171:1, 1988.
10. Warnke, R. and Levy, R. J. Histochem. Cytochem. 28:771, 1980.
11. Hsu, S.M. et al. Am. J. Clin. Pathol. 75:734, 1981.
12. Hsu, S.M. et al. J. Histochem. Cytochem. 29:577, 1981.
13. Tubbs, R. et al. Lab Invest. 46:84A, 1982.
14. O'Brien, M. et al. Lab Invest. 46:62A, 1982.
15. Marty, J. et al. J. Histotechnol. 5:61, 1982.
16. Sternberger, L.A. Immunocytochemistry, 2nd ed., 1979 (John Wiley & Sons, New York).
17. Fenoglio, C. and Wolf, M. eds. Progress in Surgical Pathology, 1980 (Masson Publishing, New York).
18. DeLellis, R. ed. Diagnostic Immunohistochemistry, 1981 (Masson Publishing, New York).
19. Brigati, D. et al. J. Histotechnol. 11:165, 1988.
20. Shi, Z. R. et al. J. Histochem. Cytochem. 36:317, 1988.



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