The rapid global outbreak of a novel coronavirus, namely, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), led to the coronavirus disease 2019 (COVID-19) pandemic, which has claimed more of 6.4 million lives worldwide. Scientists have worked tirelessly to develop vaccines, many of which have received emergency use authorization from global regulatory bodies such as the US Food and Drug Administration (FDA). Subsequently, vaccination programs were initiated in most regions of the world.
Study: ultrabright nanoparticle-labeled lateral flow immunoassay for the detection of anti-SARS-CoV-2 neutralizing antibodies in human serum. Image credit: Design_Cells/Shutterstock
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After vaccination against COVID-19, assessing the level of acquired immunity is extremely important. Typically, the effectiveness of a newly developed vaccine has been determined by measuring anti-SARS-CoV-2 immunoglobulin M (IgM) and immunoglobulin G (IgG) levels after vaccination. However, only estimating the concentration of these immunoglobulins does not correctly represent the acquired immunity generated against SARS-CoV-2, since only a small part of IgM and IgG can neutralize SARS-CoV-2.
During the course of infection, the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein binds to the host’s angiotensin-converting enzyme 2 (ACE2) receptor. After vaccination against COVID-19, next-generation neutralizing antibodies (NAb) bind to the RBD and neutralize SARS-CoV-2. Therefore, the estimation of NAbs could provide more accurate information about vaccine efficacy. In addition, it could also be used to determine the effectiveness of new vaccination strategies in the management of SARS-CoV-2 variants.
The high costs of conventional virus neutralization tests and the requirement for experts to perform them make it a less practical approach for mass screening of NAbs in the vaccinated population. To overcome the limitations of the conventional system, scientists have recently developed new techniques such as enzyme-linked immunosorbent assay, lateral flow immunoassay (LFIA), digital microfluidic systems, and surface plasmon resonance assay to detect NAbs SARS-CoV-2.
Among newly developed SARS-CoV-2 NAb 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 insufficient sensitivity and limited colorimetric range.
Although the sandwich immunoassay exhibits higher specificity and sensitivity, it cannot accurately detect SARS-CoV-2 NAbs. This is because the SARS-CoV-2 NAb is composed of a complex array of antibodies for the neutralization process, and it is extremely difficult to find two different binding sites in the NAb. In contrast, direct competitive immunoassay is a reliable and cost-effective process that can be used for mass detection of SARS-CoV-2 NAbs.
A new study
Although aggregation-induced emission organic luminogens (AIEgens) exhibit bright fluorescence at high concentration, their nanocrystal form cannot be conjugated to antibodies and cannot be released from the pad. Therefore, its use in ultrabright AIEgen nanocrystals in LIFA is restricted. A suitable format for introducing ultrabright AIEgens into LIFA is required to overcome this restriction.
In a recent study in Biomaterials, researchers developed an LFIA based on AIEgen-embedded polystyrene (PS) nanoparticles that can accurately detect anti-SARS-CoV-2 NAb in serum samples from vaccinated individuals.
It was hypothesized that the rigidity of the PS polymer containing steric phenyl rings and hydrophobic chains would significantly inhibit intramolecular motions and trigger the ultrabright fluorescence of the AIEgen embedded in the PS nanoparticles.
Results of the study
Interestingly, encapsulation of a blue-green emissive AIE490 in the carboxyl-modified PS nanoparticles (AIE490NP) enhanced the fluorescence signal more than tenfold. The fluorescence intensity in the AIE490-PS nanoparticle was much higher than in the quantum dot nanoparticles, which have an inherent fluorescence capability.
The combination of AIE490-PS and LFIA nanoparticles provided a reliable detection method for anti-SARS-CoV-2 NAb in human serum. ACE2 Fc chimera-modified AIE490NP (ACE2-AIE490NP) acted as a fluorescence marker to detect anti-SARS-CoV-2 NAb. Nitrocellulose membrane coated with SARS-CoV-2 nucleocapsid S RBD fusion served as test lines.
The test line showed an ultra-bright fluorescence signal upon encountering a negative serum sample, meaning the samples did not contain anti-SARS-CoV-2 NAb due to strong ACE2-RBD binding. However, in the presence of a positive sample containing NAbs, the test line flowed slightly due to the high incidence of NAb-RBD binding.
The AIE490-PS nanoparticle-based LFIA method for the detection of anti-SARS-CoV-2 NAb was validated using sixty-three COVID-19 vaccinated samples and seventy pre-SARS-CoV-2 serum samples. All samples were positively identified by the newly developed method. The AIE490-PS nanoparticle-based LFIA can also theoretically quantify NAbs in test samples using the available standard NAb sample. The estimated detection time for one sample was 20 minutes.
Conclusions
Overall, the use of AIEgen as a fluorescent marker has markedly improved the performance of LFIA. The main advantages of this technique are the portability and cost-effectiveness of the LFIA strip, which are extremely important for its widespread application in SARS-CoV-2 antibody detection. The AIE490-PS nanoparticle-based LFIA is an extremely useful device for determining the efficacy of vaccines against COVID-19 and assessing the duration of immune protection after vaccination. To improve detection sensitivity, researchers are currently working on the development of multicolor marker-based LFIA.