Functional Viral and Bacterial
Nexelis has a strong track record and extensive expertise in viral and bacterial functional assay development/transfer, qualification, validation and clinical testing. We also possess multiple years of Pharma experience in developing viral and bacterial manual and automated functional assays. Our team of scientists has been working for many years with several assays used for GCLP clinical testing of vaccines from early to late-stage clinical development. Our experts work in close collaboration with clients to adapt, develop, qualify and validate specific tests to evaluate the level of neutralizing antibodies generated in vaccine trials.
- Fit-for-purpose assay development, qualification and method validation aligned with regulatory guidelines
- Preparation and characterization of Master and Working banks for viral and bacterial functional assays
- Transfer of developed (R&D), qualified and validated methods
- State-of-the-art high-throughput instrumentation
Viral Functional Assays
Plaque Reduction Neutralization Test (PRNT) and Micro-PRNT
Human serum samples are serially diluted and mixed with a standard concentration of virus in 96-well plates. Following incubation, serum-virus complexes are then transferred onto plates previously seeded with eukaryotic cells and incubated again. Following incubation, cells are fixed and stained when needed. The staining allows the detection of plaque forming units (PFUs) or focus forming units (FFUs) which correspond to infected cells. Images from each well are acquired and counted using an automated image analysis system. The number of resulting PFUs in the wells is inversely proportional to the level of neutralizing antibodies present in the serum, which is directly proportional to the immunological response of the subject.
Multiple Detection Methods Are Available:
Neutralization and Micro-Neutralization
The principle of the neutralization assay is the same as the PRNT. However, the readout of the test is different as it is indirect and PFUs are not counted as an endpoint. Depending on the approach, different readouts can be used and the level of infectivity of the virus can be correlated to the survival of the cells for instance.
Pseudo-Particle Neutralization Assay
In some cases, pseudo-particles (replication incompetent viruses) harboring a reporter (luciferase or fluorescent protein) can be used instead of a live virus. In such cases, the infectivity of the pseudo-particles is correlated to the quantification of the reporter. Several technologies are available at nexelis to quantify the different reporters. The pseudo-particle-based neutralization assay represents a much safer approach, which can be performed in a BSL2 environment, regardless of the viral strain being studied .
Assessment of Functional Antibody Induction by Hemagglutination Inhibition Assay (HAI)
Hemagglutination refers to the agglutination of red blood cells. As many viruses have the capacity to induce hemagglutination, this property can be used as a readout in different types of assays. In HAI, biological samples, which are serially diluted in a 96-well assay plate, and the virus are pre-incubated and then mixed with erythrocytes (red blood cells [RBCs]). Following a short period of incubation, the presence of virus, reflecting an insufficient quantity of anti-target antigen antibodies (anti-HA antibodies for instance, in the case of the influenza virus), is read visually by the hemagglutination of the RBCs. The antibody titer of a serum corresponds to the reciprocal of the last dilution of the serum samples which is completely inhibiting hemagglutination.
Bacterial Functional Assays
Serum Bactericidal Assays (SBA)
Serum samples are serially diluted in a 96-well assay plate and bacteria and complement (human or rabbit) are added to the sera in the assay plate. Following incubation, the bacteria-specific antibodies in the sera bind to the bacterial cell-surface via specific proteins or carbohydrate moieties. The complement binds to the Fc portion of the antibodies bound to the cell-surface and activates the classical complement pathway, which ultimately results in the lysis (death) of the target bacterial cells. The surviving bacteria form colonies which are captured and counted using an automated image analysis system. The bactericidal titer for each serum sample corresponds to the reciprocal serum dilution giving at least 50% killing.
Opsonophagocytic Assays (OPA)
Serum samples are serially diluted in a 96-well assay plate and bacteria are added to the sera in the assay plate. Meanwhile, phagocytic cells (such as differentiated HL60, RAW or fresh macrophages/neutrophils) are mixed to complement. Following incubation, the mixture composed of phagocytic cells and complement is added to the assay plate. Complement binds to the Fc portion of antibodies bound to the cell-surface which triggers phagocytosis of the target bacterial cells. Surviving bacteria are then plated on an appropriate solid medium and form colonies which are captured and counted using an automated image analysis system. The bactericidal titer for each serum sample corresponds to the reciprocal serum dilution giving at least 50% killing.
A similar assay can be performed with fluorescent bacteria and be acquired by flow cytometry. Proportion of phagocytic cells which engulf target bacteria will then be considered as a readout.
Qualification of Human Serum Complement
Collection, screening, production, and qualification of mini/large pools is performed .
Raw data from SBA & OPA assays are acquired using a High-Throughput Bacterial Image Analysis system: