Production and Evaluation Criteria of Specific Monoclonal Antibodies to the Hemagglutinin of the H7N2 Subtype of Avian Influenza Virus

Abstract

To enhance the rapidity in diagnosing the spread of avian influenza virus (AIV) in chicken layer flocks, studies were initiated to develop more sensitive and specific immunological and molecular methods for the detection of AIV. In this study, the purification of the hemagglutinin protein (H) from field isolates of H7N2, the production of monoclonal antibodies (MAbs), and their evaluation as diagnostic reagents are reported. Hybridomas were generated by fusion of SP2/0-Ag14 myelomas and spleen cells from immunized mice. Hybridomas secreting antibodies specific for the H protein were assayed by an ELISA and cloned using limiting dilution. The MAbs produced were characterized by hemagglutination inhibition (HI), immunohistochemistry (IHC), indirect fluorescent antibody assay (IFA), Western blots, and IFA flow cytometry using various AIV subtypes (i.e., H4N2, H5N3, H7N2). Of the various MAbs assayed, 6 had consistent and reproducible results in each of the assays used. The results obtained in this investigation enhanced the usage of the MAbs to viral H protein in the surveillance of AIV in chickens.

References

Castro

Stoffregen

Brigman

Hillard

: 1997, A method to detect bovine viral diarrhea virus contamination in cell cultures using immunoperoxidase staining. J Vet Diagn Invest 9:427–431.

Easterday

Hinshaw

: 1991, Influenza. In: Diseases of poultry, 9th ed., eds., Calnek

Barnes

Beard

Reid

Yoder

Jr. , pp. 532–551. Iowa State University, Ames, Iowa.

Kawaoka

Yamnikowa

Chambers

: 1990, Molecular characterization of a new hemagglutinin, subtype H14, of influenza A virus. Virology 179:759–767.

Longobardi Givan

: 1992, Instrumentation: beyond the black box. In: Flow cytometry, first principles, pp. 15–40. Wiley-Liss, New York, NY.

Perdue

Latimer

Greene

Holt

: 1994, Consistent occurrence of hemagglutinin variants among avian influenza virus isolates of the H7 subtype. Virus Res 34:15–29.

Qiu

Tannock

Barry

Jackson

: 1992, Western blot analysis of antibody responses to influenza virion proteins. Immunol Cell Biol 70(Pt 3):181–191.

Rohm

Horimoto

Kawaoka

: 1995, Do hemagglutinin genes of highly pathogenic avian influenza viruses constitute unique phylogenetic lineages? Virology 209:664–670.

Rohm

Zhou

Suss

: 1996, Characterization of a novel influenza hemagglutinin H15: criteria for determination of influenza A subtypes. Virology 217:508–516.

Selleck

Hooper

Grix

Morrow

: 1994, The characterisation of an influenza A virus isolated from Victorian ducks. Aust Vet J 71:222–223.

10.

Wiley

Skehel

: 1987, The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. Ann Rev Biochem 56:365–394.

11.

Wiley

Wilson

Skehel

: 1981, Structural identification of the antibody-binding sites of Hong Kong influenza hemagglutinin and their involvement in antigenic variation. Nature 289:373–378.

12.

Yamagishi

Takekuma

Ide

: 1984, Indirect immunofluorescent technique for measurement of influenza virus antibody. Kitasato Arch Exp Med 57:273–277.

13.

Yokoyama

. 1994, Production of monoclonal antibodies. In: Protocols in immunology, ed. Coico

, pp. 1–17. John Wiley and Sons, New York, NY.

14.

Zhou

Chan

McIsaac

Heckert

: 1998, Evaluation of antibody responses of emu (Dromaius novaehollandiae) to avian influenza virus infection. Avian Dis 42:757–761.