RT Journal Article
T1 Optimization of fixed-permeabilized cell monolayers for high throughput micro-neutralizing antibody assays: application to the zebrafish/viral hemorrhagic septicemia virus (vhsv) model
A1 Chinchilla Rodríguez, Blanca
A1 Encinas, Paloma
A1 Estepa, Amparo
A1 Coll, Julio
A1 Gómez Casado, Eduardo
AB A new high throughput centrifugation-free method to estimate viral neutralizing antibody levels in low volumes and large numbers of plasma blood samples is described. Cell monolayers were, (i) plated on poly-D-Lys coated 96-wells, (ii) infected with viruses previously incubated with fish plasma containing antibodies, (iii) fixed with formaldehyde to increase cell recovery and avoid centrifugation steps, (iv) permeabilized with Saponin, (v) immunostained in the presence of Saponin by using a monoclonal antibody (MAb) to viral protein, (vi) digested with trypsin to detach cells from the monolayer, in the absence of Saponin to reduce damage of intracellular MAb-antigen complexes, and (vii) gated by flow cytometry using automatic 96-well batch analysis. The method was applied to the determination of plasma neutralizing antibodies from zebrafish (Danio rerio) surviving infections with viral hemorrhagic septicemia virus (VHSV) (an important rhabdovirus of salmonids). This semi-automatic, rapid and practical assay detected anti-VHSV neutralizing antibodies in the plasma (∼3 μl per fish) of 95.1% of the zebrafish surviving VHSV infections. The fixed-permeabilized monolayer (FIXPERM) micro-neutralization method might help to analyze sera/plasma from small fish under standarized high throughput conditions.
PB Elsevier
SN 0166-0934
YR 2013
FD 2013
LK https://hdl.handle.net/20.500.14352/97352
UL https://hdl.handle.net/20.500.14352/97352
LA eng
NO Chinchilla, B., Encinas, P., Estepa, A., Coll, J., & Gomez-Casado, E. (2013). Optimization of fixed-permeabilized cell monolayers for high throughput micro-neutralizing antibody assays: application to the zebrafish/viral hemorrhagic septicemia virus (vhsv) model. Journal of virological methods, 193(2), 627–632. https://doi.org/10.1016/j.jviromet.2013.07.049
NO Blanca Chinchilla holds a FPI doctoral fellowship (2012) from the Ministry of Economy and Competitiveness (MINECO) of Spain
NO Ministerio de Economía y Competitividad (España)
NO Comisión Interministerial de Ciencia y Tecnología (España)
DS Docta Complutense
RD 12 abr 2025