Instituto Nacional Investigaciones Agrarias (INIA), Dpto. Biotecnología. Crt. La Coruña, Km. 7, 28040 Madrid, Spain.
J Virol Methods. 2013 Nov;193(2):627-32. doi: 10.1016/j.jviromet.2013.07.049. Epub 2013 Aug 6.
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.
一种新的高通量、无需离心的方法，用于在小体积和大量血浆样本中估计病毒中和抗体水平。该方法描述如下：（i）将细胞单层接种在涂有多聚-D-赖氨酸的 96 孔板上；（ii）用与含有抗体的鱼血浆孵育过的病毒感染细胞单层；（iii）用甲醛固定以增加细胞回收并避免离心步骤；（iv）用皂素通透化；（v）在皂素存在下通过使用针对病毒蛋白的单克隆抗体（MAb）进行免疫染色；（vi）在没有皂素的情况下用胰蛋白酶消化，以从单层中分离细胞，从而减少细胞内 MAb-抗原复合物的损伤；（vii）通过自动 96 孔批处理分析进行流式细胞术门控。该方法应用于检测感染病毒性出血性败血症病毒（VHSV）后幸存的斑马鱼（Danio rerio）的血浆中和抗体（每条鱼约 3 μl 血浆）。这种半自动、快速且实用的测定法可检测到 VHSV 感染后斑马鱼血浆中（∼3 μl 每条鱼）约 95.1%的抗 VHSV 中和抗体。固定-通透化单层（FIXPERM）微量中和法可能有助于在标准化高通量条件下分析小鱼的血清/血浆。
