Bioanalytical Assay Development and Validation for SARS-CoV-2 therapeutics

Today effective therapies and prophylactic vaccines are the need of time for severe acute respiratory syndrome coronavirus (2 SARS-CoV-2). Bioanalytical assays, hplc analysis, and bioanalytical methods must efficiently characterize the efficacy and safety of SARS-CoV-2 therapeutics. Nevertheless, repurposed therapies need to be tested with new assays specific for SARS-CoV-2.

 The current pandemic has increased the demand for human sample assays capable of detecting antibodies, biomarkers, and therapeutic responses in patients, and in vitro assays showing neutralization of the virus.

Notably, it is crucial to understand that the components of the assay and method validation of the assay must be tailored to the intended use of the assay. This current article discusses some vital aspects of Covid-19 bioanalytical assay development and validation.

 Viral detection/Viral load assays

 Measurement of viral load is required for enrolling positive patients of SARS-CoV-2. The detection further prevents and improves the rate of infection. Ideally, regulatory bodies recommend Quantitative polymerase chain reaction (qPCR) in the detection of SARS-CoV-2 RNA.

The FDA believes that a validated single viral target SARS-CoV-2 assay could provide acceptable performance. Primary considerations for SARS-CoV-2 viral load measurement include proper collection, storage, transport, and extraction of RNA.

 SARS-CoV-2 antibody assays

 Antibodies against SARS-CoV-2 are detected with immunoassays to qualitatively define active/past infection (immunized) which is necessary for patient selection. Antibodies also help to ascertain whether the vaccine or therapy produces antibodies against the virus.

However, antibody assays must not be used alone for diagnosis and patient enrollment. The time course of action determines the type of antibodies detected.

IgM and IgG antibodies can be detected usually four days after SARS-CoV-2 infection, followed by IgA antibodies. Generally, human serum from a minimum of 30 patients with past infection should be used in assay validation to determine its sensitivity and specificity.

 Neutralizing antibody assays

 The antibodies produced are tested for their functional efficacy to neutralize the target virus for both vaccines and therapeutics. Modern neutralizing assays use a 2-part method with ligand-binding assays (LBAs) that use human serum, as well as in vitro cell-based assays to shorten the time and increase the throughput needed for such assays.

For SARS-CoV-2 infection, ELISA assay is used to identify the positive samples. Cell-based assays with infectious viral particles are then employed to determine if positive serum neutralizes entry and replication of the virus.

 Vaccine antigen and antibody assays

 For vaccine trials, the viral component antigen in the vaccine must be measured after dosing as a measurement of PK. Customized assay design and validation are necessary for each vaccine as the antibodies or primers used in the assay should match the vaccine immunogen.

Initial potency measurements for vaccine trials include antibody titers against vaccine antigens and determination of antiviral neutralizing activity. 

 Cytokine biomarkers

 The release of cytokine biomarkers after the presentation of SARS-CoV-2 viral particles on antigen-presenting cells initiates a cytokine storm, likely responsible for the respiratory complications of the disease.

Alterations in serum IL-2, IL-6, IL-7, granulocyte-colony stimulating factor, IP-10, MCP-1, MIP1-α, and TNF-α levels, are positively correlated with a hyperinflammatory response associated with COVID-19 disease severity and fatality.


 With the urgent need for therapeutics, laboratories characterizing SARS-CoV-2 therapies should understand their responsibility in developing assays with implications for individual patients and the public.

The combination of a human sample (e.g., anti-CoV-2 antibodies) and in vitro assays (e.g., neutralizing antibodies) presented above may streamline the time and cost of bioanalytical testing to support therapeutic development. The urgent need for therapeutics will require a sustained capacity of many laboratories to perform SARS-CoV-2 bioanalytical assays.

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