Production and characterization of monoclonal antibodies against binary ethylenimine inactivated Nipah virus.
Nipah virus, a zoonotic paramyxovirus which emerged recently was chemically inactivated using binary ethylenimine (BEI). The inactivated virus was concentrated and purified by sucrose gradient centrifugation. The gradient fractions were examined by electron microscopy and Western immunoblot, and gradient fraction containing mainly Nipah matrix (M) and nucleocapsid (N) proteins was used for immunizing BALB/c mice to generate hybridomas. Screening of the resultant hybridoma clones identified five strongly positive clones producing IgG monoclonal antibodies (mAbs) reactive to the Nipah virus antigen. The protein specificity of these mAbs was determined by Western immunoblot using Nipah virus and recombinant Nipah virus proteins expressed in mammalian cells. Four mAbs reacted with Nipah N protein and one reacted with Nipah M protein. None of the mAbs neutralized Nipah virus infectivity in vitro. However, all mAbs recognized Nipah virus in ELISA and immunofluorescence assay. F45G2 mAb was most suitable for immunohistochemistry on long term formalin-fixed Nipah virus infected swine tissues. Three of the anti-nucleocapsid mAbs (F45G2, F45G3 and F45G6) showed cross-reactivity with closely related Hendra virus N protein in both immunofluorescence and Western Immunoblot assays. Two of the mAbs were specific for the Nipah virus only, F45G4 (anti-N) and F45G5 (anti-M), and could be used in the primary identification of Nipah virus. The use of these immunoreagents to develop new diagnostic assays is discussed.
Berhane
Yohannes
Y
National Centre for Foreign Animal Disease, Canadian Food Inspection Agency, Canadian Science Center for Human and Animal Health, 1015 Arlington Street, Winnipeg, MB, Canada R3E 3M4. berhaney@inspection.gc.ca
Berry
Jody D
JD
Ranadheera
Charlene
C
Marszal
Peter
P
Nicolas
Brigitte
B
Yuan
Xin
X
Czub
Markus
M
Weingartl
Hana
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000914:Antibodies, Viral / Q000096:biosynthesis / Q000276:immunology*; D001388:Aziridines / Q000494:pharmacology*; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D003429:Cross Reactions; D004202:Disinfectants / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D019085:Fluorescent Antibody Technique, Direct; D045404:Hendra Virus / Q000276:immunology; D045464:Henipavirus Infections / Q000175:diagnosis; D006825:Hybridomas; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D045405:Nipah Virus / Q000187:drug effects / Q000276:immunology* / Q000302:isolation & purification; D019590:Nucleocapsid Proteins / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D014763:Viral Matrix Proteins / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification; D038301:Virus Inactivation
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D001388:Aziridines; D004202:Disinfectants; D019590:Nucleocapsid Proteins; D014763:Viral Matrix Proteins; C033132:aziridine