Chapter 8 : Immunoenzymatic Techniques for Detection of Viral Antigens in Cells and Tissue

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Preview this chapter:
Zoom in

Immunoenzymatic Techniques for Detection of Viral Antigens in Cells and Tissue, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555815974/9781555814625_Chap08-1.gif /docserver/preview/fulltext/10.1128/9781555815974/9781555814625_Chap08-2.gif


Since their introduction, immunoenzymatic techniques for the detection of viral antigens have served as important tools to detect, confirm, and identify viruses from direct specimen, cell culture, and tissue. In this chapter, the author reviews the developments in immunohistochemistry (IHC) techniques, with particular emphasis on antibody preparations, pretreatment antigen retrieval (AR), and state-of-the-art enzymatic signal amplification methods. The use of labeled antibodies for the detection of infectious agents in tissue was first demonstrated in 1942, with identification of pneumococcal antigens by direct fluorescent antibody in the livers and spleens of experimentally infected mice. Proper fixation of tissue prior to examination is necessary to maintain tissue architecture, preserve antigenicity, and prevent degradation. Immunohistochemistry method is independent of production of cytopathic effect (CPE) or typical viral inclusions, generates permanent preparations that can be viewed with an ordinary light microscope, and allows simultaneous evaluation of stained and unstained cells in tissue sections. Nonspecific staining due to antibody trapping around defects or edges of culture or tissue sections and endogenous peroxidase activity (e.g., neutrophils and plasma cells) in inflamed tissues or specimens can be sources of false reactivity. Detection and confirmation of cytomegalovirus (CMV) infection in tissue remains one of the most common viral indications for performance of IHC stain. The performance of IHC for detection of herpes simplex virus (HSV) from tissue is similar to that of IHC for CMV.

Citation: Polage C, Petti C. 2009. Immunoenzymatic Techniques for Detection of Viral Antigens in Cells and Tissue, p 103-109. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch8
Highlighted Text: Show | Hide
Loading full text...

Full text loading...


1. Arbustini, E.,, M. Grasso,, M. Diegoli,, E. Percivalle,, P. Grossi,, M. Bramerio,, C. Campana,, C. Goggi, and, A. Gavazzi. 1992. Histopathologic and molecular profile of human cytomegalovirus infections in patients with heart transplants. Am. J. Clin. Pathol. 98: 205213.
2. Arbustini, E.,, P. Morbini,, M. Grasso,, M. Diegoli,, R. Fasani,, E. Porcu,, N. Banchieri,, V. Perfetti,, C. Pederzolli,, P. Grossi,, D. Dalla Gasperina,, L. Martinelli,, M. Paulli,, M. Ernst,, B. Plachter,, M. Vigano, and, E. Solcia. 1996. Human cytomegalovirus early infection, acute rejection, and major histo-compatibility class II expression in transplanted lung. Molecular, immunocytochemical, and histopathologic investigations. Transplantation 61: 418427.
3. Banks, R. E.,, R. A. Craven,, P. A. Harnden, and, P. J. Selby. 2003. Use of a sensitive EnVision +-based detection system for Western blotting: avoidance of streptavidin binding to endogenous biotin and biotin-containing proteins in kidney and other tissues. Proteomics 3: 558561.
4. Benjamin, D. R. 1977. Use of immunoperoxidase for rapid diagnosis of mucocutaneous herpes simplex virus infection. J. Clin. Microbiol. 6: 571573.
5. Benjamin, D. R.,, and C. G. Ray. 1974. Use of immunoperoxidase for the rapid identification of human myxoviruses and paramyxoviruses in tissue culture. Appl. Microbiol. 28: 4751.
6. Burns, J. 1975. Background staining and sensitivity of the unlabelled antibody-enzyme (PAP) method. Comparison with the peroxidase labelled antibody sandwich method using formalin fixed paraffin embedded material. Histochemistry 43: 291294.
7. Cevenini, R.,, M. Donati,, A. Moroni,, L. Franchi, and, F. Rumpianesi. 1983. Rapid immunoperoxidase assay for detection of respiratory syncytial virus in nasopharyngeal secretions. J. Clin. Microbiol. 18: 947949.
8. Chemaly, R. F.,, B. Yen-Lieberman,, E. A. Castilla,, A. Reilly,, S. Arrigain,, C. Farver,, R. K. Avery,, S. M. Gordon, and, G. W. Procop. 2004. Correlation between viral loads of cytomegalovirus in blood and bronchoalveolar lavage specimens from lung transplant recipients determined by histology and immunohistochemistry. J. Clin. Microbiol. 42: 21682172.
9. Cioc, A. M.,, and G. J. Nuovo. 2002. Histologic and in situ viral findings in the myocardium in cases of sudden, unexpected death. Mod. Pathol. 15: 914922.
10. Colina, F.,, N. T. Juca,, E. Moreno,, C. Ballestin,, J. Farina,, M. Nevado,, C. Lumbreras, and, R. Gomez-Sanz. 1995. Histological diagnosis of cytomegalovirus hepatitis in liver allografts. J. Clin. Pathol. 48: 351357.
11. Coons, A. H.,, H. J. Creech,, R. N. Jones, and, E. Berliner. 1942. The demonstration of pneumococcal antigens in tissues by the use of the fluorescent antibody. J. Immunol. 45: 159170.
12. Coons, A. H.,, J. C. Snyder,, F. S. Cheever, and, E. S. Murray. 1950. Localization of antigen in tissue cells; antigens of rickettsiae and mumps virus. J. Exp. Med. 91: 3138.
13. Crist, G. A.,, J. M. Langer,, G. L. Woods,, M. Proctor, and, D. R. Hillyard. 2004. Evaluation of the ELVIS plate method for the detection and typing of herpes simplex virus in clinical specimens. Diagn. Microbiol. Infect. Dis. 49: 173177.
14. Curran, R. C.,, and J. Gregory. 1978. Demonstration of immunoglobulin in cryostat and paraffin sections of human tonsil by immunofluorescence and immunoperoxidase techniques. Effects of processing on immunohistochemical performance of tissues and on the use of proteolytic enzymes to unmask antigens in sections. J. Clin. Pathol. 31: 974983.
15. De Bartolomeis, C.,, A. Collini,, R. Barni,, G. Ruggieri,, M. Bernini, and, M. Carmellini. 2005. Cytomegalovirus infection with multiple colonic perforations in a renal transplant recipient. Transplant. Proc. 37: 25042506.
16. de Macedo, F. C.,, A. F. Nicol,, L. D. Cooper,, M. Yearsley,, A. R. Cordovil Pires, and, G. J. Nuovo. 2006. Histologic, viral, and molecular correlates of dengue fever infection of the liver using highly sensitive immunohistochemistry. Diagn. Mol. Pathol. 15: 223228.
17. Gardner, P. S.,, M. Grandien, and, J. McQuillin. 1978. Comparison of immunofluorescence and immunoperoxidase methods for viral diagnosis at a distance: a WHO collaborative study. Bull. W. H. O. 56: 105110.
18. Giorno, R. 1984. A comparison of two immunoperoxidase staining methods based on the avidin-biotin interaction. Diagn. Immunol. 2: 161166.
19. Hahon, N.,, J. Simpson, and, H. L. Eckert. 1975. Assessment of virus infectivity by the immunofluorescent and immunoperoxidase techniques. J. Clin. Microbiol. 1: 324329.
20. Hashizume, K.,, Y. Hatanaka,, Y. Kamihara, and, Y. Tani. 2001. Automated immunohistochemical staining of formalinfixed and paraffin-embedded tissues using a catalyzed signal amplification method. Appl. Immunohistochem. Mol. Morphol. 9: 5460.
21. Hendrix, R. M.,, M. Wagenaar,, R. L. Slobbe, and, C. A. Bruggeman. 1997. Widespread presence of cytomegalovirus DNA in tissues of healthy trauma victims. J. Clin. Pathol. 50: 5963.
22. Jang, H. J.,, S. C. Kim,, Y. P. Cho,, Y. H. Kim,, M. S. Han, and, D. J. Han. 2004. Cytomegalovirus infection of the graft duodenum and urinary bladder after simultaneous pancreas-kidney transplantation. Transplant. Proc. 36: 22002202.
23. Kaplan, M. E.,, A. H. Coons, and, H. W. Deane. 1950. Localization of antigen in tissue cells; cellular distribution of pneumococcal polysaccharides types II and III in the mouse. J. Exp. Med. 91: 1530.
24. Ketteler, M.,, L. Preuschof,, A. Mertz,, M. Stoffler-Meilicke,, H. Schafer,, A. Distler, and, G. Offerman. 2000. Fatal cytomegalovirus pneumonia after preemptive antiviral therapy in a renal transplant recipient. Clin. Nephrol. 54: 418424.
25. Ljungman, P.,, P. Griffiths, and, C. Paya. 2002. Definitions of cytomegalovirus infection and disease in transplant recipients. Clin. Infect. Dis. 34: 10941097.
26. Milde, P.,, J. Merke,, E. Ritz,, M. R. Haussler, and, E. W. Rauterberg. 1989. Immunohistochemical detection of 1,25-dihydroxyvitamin D3 receptors and estrogen receptors by monoclonal antibodies: comparison of four immunoperoxidase methods. J. Histochem. Cytochem. 37: 16091617.
27. Miller, M. J.,, and C. L. Howell. 1983. Rapid detection and identification of herpes simplex virus in cell culture by a direct immunoperoxidase staining procedure. J. Clin. Microbiol. 18: 550553.
28. Mokry, J. 1996. Versatility of immunohistochemical reactions: comprehensive survey of detection systems. Acta Medica (Hradec Kralove) 39: 129140.
29. Mori, T.,, S. Mori,, Y. Kanda,, K. Yakushiji,, S. Mineishi,, Y. Takaue,, H. Gondo,, M. Harada,, H. Sakamaki,, T. Yajima,, Y. Iwao,, T, Hibi, and, S. Okamoto. 2004. Clinical significance of cytomegalovirus (CMV) antigenemia in the prediction and diagnosis of CMV gastrointestinal disease after allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant. 33: 431434.
30. Moseley, R. C.,, L. Corey,, D. Benjamin,, C. Winter, and, M. L. Remington. 1981. Comparison of viral isolation, direct immunofluorescence, and indirect immunoperoxidase techniques for detection of genital herpes simplex virus infection. J. Clin. Microbiol. 13: 913918.
31. Mukai, K.,, and J. Rosai. 1981. Immunocytochemistry in diagnostic histopathology: its contributions and limitations. Basic Appl. Histochem. 25: 153158.
32. Nagle, R. B.,, V. A. Clark,, K. M. McDaniel, and, J. R. Davis. 1983. Immunohistochemical demonstration of keratins in human ovarian neoplasms. A comparison of methods. J. Histochem. Cytochem. 31: 10101014.
33. Nakane, P. K.,, and G. B. Pierce, Jr. 1966. Enzyme-labeled antibodies: preparation and application for the localization of antigens. J. Histochem. Cytochem. 14: 929931.
34. Nakane, P. K.,, and G. B. Pierce, Jr. 1967. Enzyme-labeled antibodies for the light and electron microscopic localization of tissue antigens. J. Cell Biol. 33: 307318.
35. NCCLS. 1999. Quality Assurance Guide for Immunocytochemistry; Approved Guideline. Document MM4-A19. NCCLS, Wayne, PA.
36. Nuovo, G. J. 2006a. The surgical and cytopathology of viral infections: utility of immunohistochemistry, in situ hybridization, and in situ polymerase chain reaction amplification. Ann. Diagn. Pathol. 10: 117131.
37. Nuovo, G. J. 2006b. The utility of immunohistochemistry and in situ hybridization in placental pathology. Arch. Pathol. Lab. Med. 130: 979983.
38. Nuovo, G. J.,, and M. L. Alfieri. 1996. AIDS dementia is associated with massive, activated HIV-1 infection and concomitant expression of several cytokines. Mol. Med. 2: 358366.
39. Nuovo, G. J.,, D. L. Defaria,, J. G. Chanona-Vilchi, and, Y. Zhang. 2005. Molecular detection of rabies encephalitis and correlation with cytokine expression. Mod. Pathol. 18: 6267.
40. Patel, R. M.,, J. R. Goldblum, and, E. D. Hsi. 2004. Immunohistochemical detection of human herpes virus-8 latent nuclear antigen-1 is useful in the diagnosis of Kaposi sarcoma. Mod. Pathol. 17: 456460.
41. Pitts, A.,, and E. E. Williams. 1974. Destruction of endogenous peroxidase activity in order to locate cellular antigens by peroxidase-labeled antibodies. J. Histochem. Cytochem. 22: 5154. (Letter.)
42. Pucci, A.,, V. Ghisetti,, E. Donegani,, A. Barbui,, E. David,, M. Fortunato,, C. Papandrea,, S. Pansini,, G. Zattera, and, M. di Summa. 1994. Histologic and molecular diagnosis of myocardial human cytomegalovirus infection after heart transplantation. J. Heart Lung Transplant. 13: 10721080.
43. Ramos-Vara, J. A.,, and M. A. Miller. 2006. Comparison of two polymer-based immunohistochemical detection systems: ENVISION+ and ImmPRESS. J. Microsc. 224: 135139.
44. Roberts, T. C.,, D. C. Brennan,, R. S. Buller,, M. Gaudreault-Keener,, M. A. Schnitzler,, K. E. Sternhell,, K. A. Garlock,, G. G. Singer, and, G. A. Storch. 1998. Quantitative polymerase chain reaction to predict occurrence of symptomatic cytomegalovirus infection and assess response to ganciclovir therapy in renal transplant recipients. J. Infect. Dis. 178: 626635.
45. Schmidt, N. J.,, J. Dennis,, V. Devlin,, D. Gallo, and, J. Mills. 1983. Comparison of direct immunofluorescence and direct immunoperoxidase procedures for detection of herpes simplex virus antigen in lesion specimens. J. Clin. Microbiol. 18: 445448.
46. Shi, S. R.,, R. J. Cote, and, C. R. Taylor. 2001. Antigen retrieval techniques: current perspectives. J. Histochem. Cytochem. 49: 931937.
47. Shi, Z. R.,, S. H. Itzkowitz, and, Y. S. Kim. 1988. A comparison of three immunoperoxidase techniques for antigen detection in colorectal carcinoma tissues. J. Histochem. Cytochem. 36: 317322.
48. Speel, E. J.,, A. H. Hopman, and, P. Komminoth. 1999. Amplification methods to increase the sensitivity of in situ hybridization: play CARD(S). J. Histochem. Cytochem. 47: 281288.
49. Sternberger, L. A.,, P. H. Hardy, Jr.,, J. C. Cuculis, and, H. G. Meyer. 1970. The unlabeled antibody enzyme method of immunohistochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorse-radish peroxidase) and its use in identification of spirochetes. J. Histochem. Cytochem. 18: 315333.
50. Straus, W. 1971. Inhibition of peroxidase by methanol and by methanol-nitroferricyanide for use in immunoperoxidase procedures. J. Histochem. Cytochem. 19: 682688.
51. Tamm, M.,, P. Traenkle,, B. Grille,, M. Soler,, C. T. Bolliger,, P. Dalquen, and, G. Cathomas. 2001. Pulmonary cytomegalovirus infection in immunocompromised patients. Chest 119: 838843.
52. Taylor, C. R. 2000. The total test approach to standardization of immunohistochemistry. Arch. Pathol. Lab. Med. 124: 945951.
53. van Burik, J. A.,, E. J. Lawatsch,, T. E. DeFor, and, D. J. Weisdorf. 2001. Cytomegalovirus enteritis among hematopoietic stem cell transplant recipients. Biol. Blood Marrow Transplant. 7: 674679.
54. Weller, T. H.,, and A. H. Coons 1954. Fluorescent antibody studies with agents of varicella and herpes zoster propagated in vitro. Proc. Soc. Exp. Biol. Med. 86: 789794.
55. Wood, G. S.,, and R. Warnke. 1981. Suppression of endogenous avidin-binding activity in tissues and its relevance to biotin-avidin detection systems. J. Histochem. Cytochem. 29: 11961204.
56. Zedtwitz-Liebenstein, K.,, P. Jaksch,, C. Bauer,, T. Popow,, W. Klepetko,, H. Hofmann, and, E. Puchhammer-Stockl. 2004. Association of cytomegalovirus DNA concentration in epithelial lining fluid and symptomatic cytomegalovirus infection in lung transplant recipients. Transplantation 77: 18971899.
57. Zhang, H.,, Y. Li,, T. Peng,, M. Aasa,, L. Zhang,, Y. Yang, and, L. C. Archard. 2000. Localization of enteroviral antigen in myocardium and other tissues from patients with heart muscle disease by an improved immunohistochemical technique. J. Histochem. Cytochem. 48: 579584.


Generic image for table

Strengths and weaknesses of immunoenzymatic methods for antigen detection

Citation: Polage C, Petti C. 2009. Immunoenzymatic Techniques for Detection of Viral Antigens in Cells and Tissue, p 103-109. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch8

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error