An antigen microarray immunoassay for multiplex screening of mouse monoclonal antibodies.
The mouse monoclonal antibody (mAb) technology still represents a key source of reagents for research and clinical diagnosis, although it is relatively inefficient and expensive and therefore unsuitable for high-throughput production against a vast repertoire of antigens. In this article, we describe a protocol that combines the immunization of individual mice with complex mixtures of influenza virus strains and a microarray-based immunoassay procedure to perform a parallel screening against the viral antigens. The protocol involves testing the supernatants of somatic cell hybrids against a capture substratum containing an array of different antigens. For each fusion experiment, we carried out more than 25,000 antigen-antibody reactivity tests in less than a week, a throughput that is two orders of magnitude higher than that of traditional antibody detection assays such as enzyme-linked immunosorbent assays and immunofluorescence. Using a limited number of mice, we can develop a vast repertoire of mAbs directed against nuclear and surface proteins of several human and avian influenza virus strains.
Microbiology Section, Department of Experimental Medicine, University of Perugia, Perugia, Italy.
Manlio
M
Di Cristina
Luisa
L
Nunziangeli
Maria Angela
MA
Giubilei
Barbara
B
Capuccini
Lorenzo
L
d'Episcopo
Giorgio
G
Mazzoleni
Francesca
F
Baldracchini
Roberta
R
Spaccapelo
Andrea
A
Crisanti
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification*; D000956:Antigens, Viral / Q000276:immunology*; D007114:Immunization / Q000379:methods; D007118:Immunoassay / Q000379:methods*; D051379:Mice; D046228:Microarray Analysis / Q000379:methods*; D009975:Orthomyxoviridae / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral