Production of Monoclonal Antibodies
against the Challenge Strain of Infectious Laryngotracheitis Virus
of Chickens and Their Use in an Indirect Immunofluorescent
Diagnostic Test
Ferhat Abbas*, James Andreasen1, Rockey Becker1,
Masroor Ahmed, M Arif Awan, Abdul Wadood and Anita Sonn1
Center for Advanced Studies in Vaccinology and Biotechnology,
University of Balochistan, Quetta, Pakistan; 1Oregon State University,
USA. *Corresponding author: ferhatcasvab@yahoo.com
Abstract
The objective of the present research was to produce monoclonal antibodies
(MCAs) against the USDA challenge strain of infectious laryngotracheitis virus
and to perform an initial investigation of their use in an indirect
immunofluorescence diagnostic test. Fourteen-day old chicken embryo liver cells
were grown in tissue culture plates. Confluent monolayers were obtained after 48
hours. Monolayers were infected with the USDA challenge strain of infectious
laryngotracheitis virus (ILTV). Cytopethic effect of the virus in the form of
syncytial formation and clumping of cells was observed after 24 hours. The virus
from the tissue culture flasks was collected and purified using discontinuous
sucrose gradient. A clear band of the virus from sucrose gradient was obtained.
The refractory index and the density measured were 1.410 and 1.20 g/cm3,respectively. Spectrophotometry of the purified virus showed 68.117 ug/ml
of protein and 9.8948 ug/ml of nucleic acid concentration. Spleen cells from
immunized mice with pure virus were fused with myeloma cells and hybridomas were
obtained after 10 days. Screening was performed using indirect
immunofluorescence antibody test (IFAT) using rabbit anti-mouse immunoglobulins
as secondary antibodies. Three hybridomas, 2D1D8, 2E11G2 and 2C6C7 were found
producing antibodies against ILTV. All monoclonal antibodies were of isotype IgM
and reacted with different strains of ILTV (ILTV USDA, S 88 00224, 86-1169) in
IFAT. None of the monoclonals reacted with Parrot herpesvirus and avian
adenovirus 301 in IFAT.