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Chapter 9 : Neutralization

Author: David Schnurr
Content Type: Reference
Publication Year: 2009

Category: Clinical Microbiology; Viruses and Viral Pathogenesis

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Abstract:

The neutralization test has been used in virology longer than any other serologic procedure. To test for virus neutralization, virus and serum are mixed together, incubated under appropriate conditions, and then inoculated into a susceptible living host for detection of nonneutralized virus. Nonneutralized virus is detected by looking for viral growth, using indicators such as cytopathic effect (CPE), plaque formation, or metabolic inhibition. The use of standardized components is critical to performing neutralization assays. For viral identification, well-characterized pretitered antiserum or well-standardized immune serum pools are used. Similarly, to measure antibody response to a virus, a well-characterized, pretitered virus is required. Finally, to monitor for viral inactivation (neutralization), a living host system is required. The antiserum must be standardized for use in the neutralization test by titration against both its homologous virus and heterologous virus(es). Standardization of immune serum requires that the serum be tested in the neutralization assay against the virus used to immunize the animal and closely related viruses. The neutralization capacity of antiviral monoclonal antibodies (MAb) is determined by titration against the virus of interest. Neutralization is used for typing viruses and for diagnosing infection based on the host’s immune response. It is also a research tool for dissecting how antibodies protect the host from infection and probing viral function.

Citation: Schnurr D. 2009. Neutralization, p 110-118. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch9
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Neutralization
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Citation: Schnurr D. 2009. Neutralization, p 110-118. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch9
/content/10.1128/9781555815974.ch09.ch09fig01
FIGURE 1
This virus titration demonstrates that the endpoint titer is the highest dilution of virus that infects 50% of the inoculated cell cultures.
10.1128/9781555815974/ch09fig1_thmb.gif
10.1128/9781555815974/ch09fig1.gif
Image of FIGURE 1
FIGURE 1

This virus titration demonstrates that the endpoint titer is the highest dilution of virus that infects 50% of the inoculated cell cultures.

Citation: Schnurr D. 2009. Neutralization, p 110-118. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch9
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/content/10.1128/9781555815974.ch09.ch09fig02
FIGURE 2
This serum titration demonstrates that the antibody titer is the highest dilution of antiserum that protects against the standardized virus.
10.1128/9781555815974/ch09fig2_thmb.gif
10.1128/9781555815974/ch09fig2.gif
Image of FIGURE 2
FIGURE 2

This serum titration demonstrates that the antibody titer is the highest dilution of antiserum that protects against the standardized virus.

Citation: Schnurr D. 2009. Neutralization, p 110-118. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch9
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/content/10.1128/9781555815974.ch09.ch09fig04
FIGURE 4
Intersecting pool scheme for identification of adenoviruses.
10.1128/9781555815974/ch09fig4_thmb.gif
10.1128/9781555815974/ch09fig4.gif
Image of FIGURE 4
FIGURE 4

Intersecting pool scheme for identification of adenoviruses.

Citation: Schnurr D. 2009. Neutralization, p 110-118. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch9
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References

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1. Bartosch, B.,, J. Bukh,, J. Meunier,, C. Granier,, R. E. Engle,, W. C. Blackwelder,, S. U. Emerson,, F. Cosset, and, R. H. Purcell. 2003. In vitro assay for neutralizing antibody to hepatitis C virus: evidence for broadly conserved neutralization epitopes. Proc. Natl. Acad. Sci. USA 100: 14199– 14204.
2. Braun, W.,, and C. Schacherer. 1988. Rapid (24h) neutralization assay for the detection of antibodies to human cytomegalovirus using a monoclonal antibody to an HCMV early nuclear protein. J. Virol. Methods 22: 1– 40.
3. Crawford-Miksza, L. K.,, R. N. Nang, and, D. P. Schnurr. 1999. Strain variation in adenovirus serotypes 4 and 7a causing acute respiratory disease. J. Clin. Microbiol. 37: 1107– 1112.
4. Crawford-Miksza, L. K.,, and D. P. Schnurr. 1994. Quantitative colorimetric microneutralization assay for characterization of adenoviruses. J. Clin. Microbiol. 32: 2331– 2334.
5. Hang, D. P.,, G. H. Kim,, Y. B. Dim,, L. L. M. Poon, and, M. W. Cho. 2004. Development of a safe neutralization assay for SARS-CoV and characterization of S-glycoprotein. Virology 326: 140– 141.
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7. Khawplod, P.,, K. Inoue,, Y. Shoji,, H. Wilde,, S. Ubol,, A. Nishizono,, I. Kurane, and, K. Morimoto. 2005. A novel rapid fluorescent focus inhibition test for rabies virus using a recombinant rabies virus visualizing a green fluorescent protein. J. Virol. Methods 12: 35– 40.
8. Kolokoltsov, A. A.,, W. Wang,, T. M. Colpitts,, S. C. Weaver, and, R. A. Davey. 2006. Pseudotype viruses permit rapid detection of neutralizing antibodies in human and equine serum against Venezuelan equine encephalitis virus. Am. J. Trop. Med. Hyg. 75: 702– 709.
9. Lim, K. A.,, and M. Benyesh-Melnick. 1960. Typing of viruses by combination of antiserum pools. Application of typing of enteroviruses (coxsackie and ECHO). J. Immunol. 84: 309– 317.
10. Lu, X.,, and D. D. Erdman. 2006. Molecular typing of human adenoviruses by PCR and sequencing of a partial region of the hexon gene. Arch. Virol. 151: 1587– 1602.
11. Malasig, J. D.,, P. R. Goswami,, L. K. Crawford-Miksza,, D. P. Schnurr, and, G. C. Gray. 2001. Simplified microneutralization test for serotyping adenovirus isolates. J. Clin. Microbiol. 39: 2984– 2986.
12. Oberste, S. M.,, K. Maher,, D. R. Kilpatrick,, M. R. Flemister,, B. A. Brown, and, M. A. Pallansch. 1999. Typing of human enteroviruses by partial sequencing of VP1. J. Clin. Microbiol. 37: 1288– 1293.
13. Schmidt, N. J. 1979. Cell culture techniques for diagnostic virology, p. 100–111. In E. H. Lennette and, N. J. Schmidt (ed.), Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections, 5th ed. American Public Health Association, Washington, DC.
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18. Zielinska, E.,, D. Liu,, H. Y. Wu,, J. Quiroz,, R. Rappaport, and, D. P. Yang. 2005. Development of an improved microneutralization assay for respiratory syncytial virus by automated plaque counting using imaging analysis. Virol. J. 2: 84.

Tables

Neutralization
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Citation: Schnurr D. 2009. Neutralization, p 110-118. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch9
/content/10.1128/9781555815974.ch09.ch09tab01
TABLE 1
Calculation of 50% mortality (virus titration) with sample data a
Generic image for table
TABLE 1

Calculation of 50% mortality (virus titration) with sample data a

Citation: Schnurr D. 2009. Neutralization, p 110-118. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch9
/content/10.1128/9781555815974.ch09.ch09fig03a
Untitled
Generic image for table
Untitled

Citation: Schnurr D. 2009. Neutralization, p 110-118. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch9

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