A Nanoparticle-based Lateral Flow Immunoassay for Detection of Anti-SARS-CoV-2 Neutralizing Antibodies

The rapid pandemic of a new coronavirus, namely severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to the coronavirus disease 2019 (COVID-19) pandemic that has claimed more than 6.4 million lives worldwide. caused Scientists have worked tirelessly to develop vaccines. Some of them have been approved for emergency use by global regulatory bodies such as the US Food and Drug Administration (FDA). Immunization programs were then initiated in most parts of the world.

Background

It is very important to assess the level of acquired immunity after COVID-19 vaccination. Efficacy of newly developed vaccines is usually determined by measuring anti-SARS-CoV-2 immunoglobulin M (IgM) and immunoglobulin G (IgG) levels after vaccination. However, only a small proportion of IgM and IgG can neutralize SARS-CoV-2, so estimating the concentrations of these immunoglobulins alone does not account for the acquired immunity generated against SARS-CoV-2. It cannot be represented exactly.

During the course of infection, the receptor binding domain (RBD) spike protein A portion of SARS-CoV-2 binds to the host angiotensin-converting enzyme 2 (ACE2) receptor. New outbreak after COVID-19 vaccination Neutralizing antibody (NAbs) bind to RBD and neutralize SARS-CoV-2. Therefore, the estimate of NAbs is efficacy of vaccination. Additionally, it can be used to determine the efficacy of new vaccination strategies in the management of SARS-CoV-2 variants.

The high cost of conventional virus neutralization tests and the need for specialists to conduct them make methods for mass detection of NAbs in whole vaccinated populations less practical. To overcome the limitations of conventional systems, scientists have recently developed enzyme-linked immunosorbent assays, lateral flow immunoassays (LFIA), digital microfluidic systems, surface plasmon resonance assays, etc. to detect SARS-CoV-2 NAbs. developed new technology.

Among the newly developed SARS-CoV-2 NAbs detection methods, LFIA has become the most popular point-of-care immunosensor due to its portability, low cost, and rapid evaluation process. However, some limitations of this method include poor sensitivity and limited colorimetric range.

Sandwich immunoassays show higher specificity and sensitivity, but cannot accurately detect SARS-CoV-2 NAbs. This is because the SARS-CoV-2 NAb consists of a complex range of antibodies for the neutralization process and in the NAb it is very difficult for him to find two different binding sites. In contrast, direct competitive immunoassay is a reliable and cost-effective process that can be used for high-volume detection of SARS-CoV-2 NAbs.

new research

Organoluminogens with aggregation-induced luminescence (AIEgens) exhibit bright fluorescence at high concentrations, but their nanocrystalline forms cannot bind antibodies and release from the pad. Therefore, their utilization in LIFA’s ultrabright AIEgen nanocrystals is limited. To overcome this limitation, a suitable format for introducing ultra-bright AIEgens into her LIFA is required.

recently biomaterial Researchers have developed an AIEgen-embedded polystyrene (PS) nanoparticle-based LFIA that can accurately detect anti-SARS-CoV-2 NAb in serum samples of vaccinated individuals.

It was hypothesized that the rigidity of the PS polymer, which contains steric phenyl rings and hydrophobic chains, severely inhibits intramolecular motion and causes the super-bright fluorescence of AIEgen embedded in PS nanoparticles.

Survey results

Interestingly, encapsulation of green-blue emitting AIE₄₉₀ Carboxyl-modified PS nanoparticles (AIE₄₉₀NP) enhanced the fluorescence signal more than 10-fold. The fluorescence intensity of AIE490-PS nanoparticles was much greater than that of quantum dot nanoparticles with intrinsic fluorescence ability.

AIE combination₄₉₀-PS nanoparticles and LFIA provided a reliable detection method for anti-SARS-CoV-2 NAb in human serum. AIE₄₉₀NP modified with ACE2 Fc chimera (ACE2-AIE₄₉₀NP) served as a fluorescent marker to sense anti-SARS-CoV-2 NAb. A nitrocellulose membrane coated with SARS-CoV-2 nucleocapsid S RBD fusions served as a test line.

The test line exhibited an ultra-bright fluorescent signal upon encountering a negative serum sample. That is, samples did not contain anti-SARS-CoV-2 NAb due to strong ACE2-RBD binding. However, in the presence of positive samples containing NAb, the test line exhibited mild fluorescence due to the high incidence of NAb-RBD binding.

AIE₄₉₀– The PS nanoparticles-based LFIA method for detecting anti-SARS-CoV-2 NAb was validated using 63 COVID-19 vaccination samples and 70 SARS-CoV-2 serum samples. All samples were positively identified by the newly developed method. AIE₄₉₀-PS nanoparticle-based LFIA can also be used to theoretically quantify NAb in test samples using available standard NAb samples. The estimated detection time for one sample was 20 minutes.

Conclusion

Taken together, the utilization of AIEgen as a fluorescent marker significantly improved the performance of LFIA. The main advantages of this technique are the portability and cost effectiveness of the LFIA strips. This is of great importance for its widespread application in the detection of SARS-CoV-2 antibodies. AIE₄₉₀-PS nanoparticle-based LFIA is a highly useful device for determining the efficacy of COVID-19 vaccines and assessing the duration of immune protection after vaccination. To increase detection sensitivity, researchers are currently working on developing multicolor marker-based LFIAs.