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Analytics and virus production processes
Published in Amine Kamen, Laura Cervera, Bioprocessing of Viral Vaccines, 2023
The hemagglutination assay is still in use in many labs for different types of viruses. This includes the viral families of orthomyxoviridae, paramyxoviridae, togaviridae, reoviridae, adenoviridae with for example influenza, measles, or rubella viruses [21]. The hemagglutination assay detects the interaction between the virus and red blood cells. Virus suspensions are incubated with red blood cells (RBCs) to allow for attachment of viral antigens with RBC specific receptors. In highly concentrated viral suspension, RBC and virus will then form a network blocking the RBC sedimentation. When performed within a conical bottom well plate, it is thus easy to visually distinguish a condition where the network has been formed (no sedimentation) from a condition where not enough viruses were present to form a network (sedimentation observed with a red dot). Hemagglutination assay is highly dependent on the purity of the viral material tested and on the RBC quality and origin. Mostly RBCs are used from chicken blood, but guinea pigs and other types of poultry can also be used. RBCs are used fresh, ideally collected the day before the assay which renders the analysis process complex to plan. Donor-to-donor animal variability could strongly impact the results; therefore, standard reference samples are required. The assay sensitivity is also quite poor compared to further detailed immune-based assay. Nevertheless, this essay is simple to perform, rapid, easy to read by visual evaluation,and allows for the comparison of many conditions. Such assay has already been used to quantify total viral particles. Indeed, in 1954, Donald and Issacs established a quantification method of viral particles based on the hypothesis that there is approximately one influenza virus for each red blood cell at the end point of agglutination [22]. Viruses preparations were quantified by both electron microscopy and red blood cells assay to establish such correlation. Nevertheless, because of its high degree of variability and dependency on RBC origin or operator reading, hemagglutination assay should be used with care to compare production levels between conditions, and even more between laboratories.
Soluble expression, rapid purification, biological identification of chicken interferon-alpha using a thioredoxin fusion system in E. coli and its antiviral effects to H9N2 avian influenza virus
Published in Preparative Biochemistry and Biotechnology, 2019
Jun Zhao, Hai-Yang Yu, Yu Zhao, Feng-Hua Li, Wei Zhou, Bin-Bin Xia, Zhi-Yuan He, Jason Chen, Guo-Tuo Jiang, Ming-Li Wang
Each chicken embryo allantoic fluid sample containing influenza viral antigens was subjected to a hemagglutination assay (HA) to determine the number of hemagglutinating units (HAU) present or the amount of virus needed to agglutinate an equal volume of a standardized red blood cell (RBC) suspension. Briefly, The specimen of chicken embryo allantoic fluid was serially 2-fold diluted in PBS (pH 7.2) across a U-shaped well microtiter plate to yield a volume of 50 μl. PBS alone was added to two wells to be used as a blank control. After adding standardized RBCs (50 μl) to each well, the plate was agitated and incubated at room temperature for 30 min. Hemagglutination titers were read as the reciprocal dilution of the virus in the last well with complete hemagglutination.[28]