Optimization of fixed-permeabilized cell monolayers for high throughput micro-neutralizing antibody assays: application to the zebrafish/viral hemorrhagic septicemia virus (vhsv) model.
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.
Instituto Nacional Investigaciones Agrarias (INIA), Dpto. Biotecnología. Crt. La Coruña, Km. 7, 28040 Madrid, Spain. Electronic address: blanca.chinchilla@inia.es.
Blanca
B
Chinchilla
Paloma
P
Encinas
Amparo
A
Estepa
Julio
J
Coll
Eduardo
E
Gomez-Casado
D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood*; D018929:Cell Culture Techniques / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods; D009500:Neutralization Tests / Q000379:methods*; D029141:Novirhabdovirus / Q000276:immunology*; D013048:Specimen Handling / Q000379:methods; D015027:Zebrafish
D057134:Antibodies, Neutralizing
Assay; Fixed-permeabilized monolayers; Highthroughput; Neutralizing antibodies; VHSV; Zebrafish