Kits for Neutralization Assays

The detection of neutralizing antibodies (NAbs) in the blood is an essential analysis to evaluate the efficiency of vaccines throughout their development, as well as to measure the duration of the immunity conferred by vaccination.

Unfortunately, the current detection technologies available on the market don’t adequately address the needs of the bioanalytical and biopharmaceutical industries. On one hand, cell-based technologies (involving viruses) are incompatible with high-throughput screening, time-consuming, unsafe, and laborious for the lab staff. On the other hand, the other technologies, based on the ELISA assay detect only one type of NAbs in an unrealistic biological environment leading to skewed data.

To solve this problem, at IVANO Bioscience, we are developing a new generation of neutralizing antibody detection bioassays, the AbVenger kit. First of its kind, it was inspired by the immunological process and designed to meet your needs at multiple levels.

Neutralizing antibodies inhibit viral infection by binding to viral particles. This leads to the blockade of the interaction with receptors or prevents the envelope glycoproteins conformational changes required for the fusion step.

Neutralization assays determine the ability of an antibody to inhibit a virus infection of cultured cells and the resulting cytopathic effects of viral replication, which are important for drug discovery and vaccine development. Virus neutralization is a specialized type of immunoassay since it detects antibodies that can block virus replication. Neutralizing antibodies generally recognize proteins or glycoproteins on the virus surface. Those antibodies have several mechanisms, including blocking the virus attachment to the host cell, inhibiting its penetration into the host cell membrane, or interfering with uncoating of the virus within the cell.

Understand the NAbs immune responses of infected people.

In some recent studies, the rapid decline of NAbs within a few weeks has been a concern, which is obvious in individuals with asymptomatic infection or mild symptoms (Seow J et al. 2020; Long QX et al. 2020). There have also been reports of some cases of re-infection, increasing concerns about susceptibility to re-infection (An J et al. 2020; Lee JS et al. 2020). However, in general, antibody levels will always decrease after the acute infection period. Most of the responses of effector B cells induced by infection are short-lived, while the antibody level needs to be maintained by longer-lived plasma cells and memory B cells (Stephens DS et al. 2020; Ripperger TJ et al. 2020).In the early stage, the decline of NAbs level should not cause concern. Neutralization assays are useful tools to determine the level of antibody produced as well as the duration of the antibody titer stability after a natural infection or a vaccination.

Evaluation of convalescent plasma (CP) therapy, mAbs and vaccines

Before effective drugs and vaccines are developed, convalescent plasma could represent an immediate stopgap treatment against viruses. Passive immunity driven by convalescent plasma can provide NAbs that inhibit infection. Other antibody-mediated pathways, such as complement activation, antibody-dependent cytotoxicity and/or phagocytosis, may also promote the therapeutic effect of convalescent plasma (Zhou G et al. 2020). Patients with high NAbs titers who have recovered from a virus may be a valuable source of donors for convalescent plasma. However, the titer of NAbs in the donor’s plasma, the efficiency of the plasma therapy, and the capacity of the recipient to produce efficient specific NAbs after the plasma administration are part of the data that need to be evaluated.
Passive antibody protection conferred by convalescent plasma can provide rapid but short-term immunity for susceptible individuals, while vaccines induce long-lasting protective antiviral immunity (Bloch EM et al. 2020). In many vaccine trials, the titer of NAbs is usually regarded as an important evaluation endpoint (Sahin U et al. 2020; Zhu FC et al. 2020). There are still some key questions to be answered in the development of vaccines, such as which antigen (epitope) will trigger powerful NAbs in most people.

In addition, neutralization assays can be used to do some valuable research on controlling infectious diseases. Studies could, for example, be performed to describe the NAbs response of recovered patients or normal individuals after immunization, including the average titers of asymptomatic, mild and severe patients and the titers of different age groups; to study the kinetics and lifespan of NAbs; to determine the titers of protective NAbs and the level of NAbs required to observe an antiviral effects.

Most sVNTs are based on the principle of blocking the interaction between the receptor binding site (RBD) and the cellular receptors (Abe KT et al. 2020; Tan CW et al. 2020). The selected specific antigen, which can be biotinylated, is coated on a plate and incubated with test serum. Then the soluble cellular receptor conjugated with HRP is added in the plate, followed by its colorimetric substrate 3,3′,5,5′-Tetramethylbenzidine. Or the plate is coated with the cellular receptor, and soluble antigen is used to compete with antibodies. The antibody that blocks the receptor-antigen interaction can be detected by the reduction of the HRP luminescence signal or surface plasmon resonance. Unfortunately, this detection method still has some limitations: it can only detect designated antibodies, such as blocking antibodies, but not all NAbs (Liu L et al. 2020). Therefore, the synergy between NAbs is difficult to assess. The sensitivity and specificity of sVNTs are lower than cell-based neutralization assays (Sholukh AM et al. 2020). These shortcomings make sVNTs currently only moderately used as supplementary tests for cell-based neutralization assays.

The live virus neutralization assay is an effective method to evaluate the level of NAbs (Wang K et al. 2021). Although the live virus neutralization assay can measure the level of NAbs blocking viral infection, it is expensive and requires well-trained professionals to work with highly pathogenic viruses in a biosafety level-3 or -4 (BSL-3, BSL-4) laboratory. It is time-consuming, labor-intensive, and usually takes 4-10 days to complete. Therefore, this method is not suitable for large-scale and high throughput serological diagnosis and vaccine evaluation (Xi Y. 2020).