Detection of α-, β- and γ-interferon-induced antiviral activity
The cells used in the assay are mouse L 929 fibroblasts, which have a high sensitivity to interferon and V S V . The cells should be derived from laboratories that have established this assay and not from commercial sources. Continuous and consistent cell passaging is beneficial to the credibility of the test results. However, after 4 to 6 months of passaging at a frequency of one passaging per week, the time to reach 100 % CPE is prolonged and the endpoints may become ambiguous. When this occurs, the cell line should be replaced with a new one. Author: J.E. Colligan et al, Translated by Xuitao Cao et al. This experiment is from the "Compendium of Immunology Experiments Guide".
Operation method
Detection of α-, β- and γ-interferon-induced antiviral activity Move Detection of interferon-induced antiviral activity in basic program mice Materials Interferon-sensitive L 929 fibroblasts E M E M Medium (Eagle's Minimum Required Medium) Tryptic digest Reference standards for mouse interferon α-interferon, β-interferon, γ-interferon, and αβ-interferon (obtained from Dr. Laughlin, National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA) Vesicular stomatitis virus (VSV; Indiana strain) EBSS (Earles Balanced Salt Solution) at 4°C 5 % (W V ) formalin 20 % ethanol containing 0.05 % (m/V) crystalline violet 100 % methanol (optional) 75c m 2 straight neck tissue culture flasks (Falcon or Costar) 8-way micropipettes (fixed or adjusted to 50M1 and IOOiLd; Costar) 9 6-well flat-bottomed microplate (Falcon or Costar) 8-well pipette (Drummond; Thomas Scientific) Inverted microscope Fixed-rail shaking table (optional) Microtiter Plate Reader (ELISA) (optional) 1 . Suspend approximately I X l O6 Interferon-sensitive L929 adult fibroblasts in 25 ml of EMEM medium and inoculate into 75 cm2 tissue culture flasks. Incubate at 37°C in a 6% (:02) incubator for approximately 1 week (or until the flasks are full). 2 . Add 3 m l of tryptic digest to the cell-covered culture flasks and collect the cells after they detach from the bottom of the flasks. 6 . Determination of the neutralizing titer of the antibody product, i.e. the reciprocal of the highest dilution of the antibody that neutralizes 50 % of the 5 U/m l interferon activity. 7 . Optional: The neutralizing activity of the antibody is expressed in U/mL. The potency is multiplied by 5 (adjusted to neutralize 5U/ml), then by 4 (adjusted to a 4-fold dilution of the antibody relative to the sample and cells added), and then by 5 (since the assay volume is 0.2 ml). For more product details, please visit Aladdin Scientific website.
Test Sample (interferon active serum or culture supernatant)
Supplementary program 2 Antibody neutralization assay ![抗体介导的干扰素诱导的抗病毒活性的逆转可以用来区分样品中发挥抗病毒活性的 干扰素的种类，或者用来检测特定种类干扰素的特定的抗体制品。 1 . 用 E M E M 培养基将待检测抗体进行2 倍系列倍比稀释，终体积为IOOm I/孔 。 2 . 基本方案中所测定的相应干扰素样品的浓度调整为20U /ml。每孔加入50/xl。 此 实 验 中 必 须 测 定 干 扰 素 的 浓 度 [在 对 照 抗 体 和 （或） E M E M 存在情况下]， 以确定其对V S V 感染的细胞的1 0 0 % 的保护效应。而且，也应设立病毒对照孔，以 确定可发生1 0 0 % 的 C P E 。 3 . 每孔加人50M1 L 929成纤维细 胞 悬 混 液 （干扰素终浓度5U /ml)。用手轻轻悬转培养 板使细胞均勻分散。 37°C ， 6 % C 0 2培养箱中培养24h 。 4 . 吸出孔中液体，感 染 V S V (见基本方案，步 骤 9)。 5 . 感 染 约 24h 后 ，显微镜下观察病毒对照孔，收集 ，洗涤，固 定 并 染 色 细 胞 （见基本 方案，步 骤 10〜11)。分 光 光 度 计 读 板 （步 骤 13b)](http://img.dxycdn.com/trademd/upload/userfiles/image/2016/07/B14695988003553958iyzud4png_small.jpg)
