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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:


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.

Key words: Confluent, Embryo, Hybridomas, Infectious laryngotracheitis, Isotype, Monoclonal antibodies, Purification, Screening, Tissue culture


ISSN 0253-8318 (Print)
ISSN 2074-7764 (Online)