The rapid global outbreak of a novel coronavirus, namely severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), caused the coronavirus disease 2019 (COVID-19) pandemic, which claimed more than 6.4 million lives worldwide. Scientists have worked tirelessly to develop vaccines, several of which have been approved for emergency use by global regulatory bodies such as the US Food and Drug Administration (FDA). Subsequently, vaccination programs began in most regions of the world.
Study: Ultrabright Nanoparticle-Labeled Lateral Flow Immunoassay for Detection of Anti-SARS-CoV-2 Neutralizing Antibodies in Human Serum. Image credit: Design_Cells/Shutterstock
Background
After vaccination against COVID-19, assessment of the level of acquired immunity is extremely important. Typically, the effectiveness of a newly developed vaccine is determined by measuring the levels of anti-SARS-CoV-2 immunoglobulin M (IgM) and immunoglobulin G (IgG) after vaccination. However, only assessing the concentration of these immunoglobulins does not correctly represent the acquired immunity generated against SARS-CoV-2, because only a small fraction of IgM and IgG can neutralize SARS-CoV-2.
During infection, the receptor-binding domain (RBD) in the spike protein of SARS-CoV-2 binds to the host’s angiotensin-converting enzyme 2 (ACE2) receptor. After vaccination against COVID-19, newly generated neutralizing antibodies (NAbs) bind to the RBD and neutralize SARS-CoV-2. Therefore, assessment of NAbs may provide more accurate information on vaccine efficacy. In addition, it can also be used to determine the effectiveness of new vaccination strategies in managing SARS-CoV-2 variants.
The high cost of conventional virus neutralization tests and the requirement for experts to perform them make it a less practical approach for mass detection 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 analysis to detect SARS-CoV-2 NAbs.
Among newly developed methods for the detection of SARS-CoV-2 NAbs, LFIA has become the most popular in-situ 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 showed higher specificity and sensitivity, it could not accurately detect SARS-CoV-2 NAbs. This is because SARS-CoV-2 NAbs are composed of a complex array of antibodies for the neutralization process, and it is extremely difficult to find two different NAb binding sites. In contrast, the 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 organic luminogens with aggregation-induced emission (AIEgens) show bright fluorescence at high concentration, their nanocrystalline form cannot be conjugated to antibodies and cannot be released from the support. Therefore, their use in ultrabright AIEgen nanocrystals in LIFA is limited. A suitable format for introducing ultrabright AIEgens into LIFA is needed to overcome this limitation.
In a recent Biomaterials study, researchers developed AIEgen-based polystyrene (PS) LFIA nanoparticles that can accurately detect anti-SARS-CoV-2 NAb in serum samples of vaccinated individuals.
It is predicted that the rigidity of the PS polymer containing steric phenyl rings and hydrophobic chains will significantly inhibit the intramolecular motions and induce ultrabright fluorescence of the incorporated AIEgen in PS nanoparticles.
Survey results
Interestingly, the encapsulation of green blue-emitting AIE490 in the carboxyl-modified PS nanoparticles (AIE490NP) increased the fluorescence signal more than tenfold. The fluorescence intensity in the AIE490-PS nanoparticles is much greater than in the quantum dot nanoparticles, which have intrinsic fluorescence ability.
The combination of AIE490-PS and LFIA nanoparticles provided a reliable method for the detection of anti-SARS-CoV-2 NAb in human serum. AIE490NP modified with ACE2 Fc chimera (ACE2-AIE490NP) acts as a fluorescent marker for anti-SARS-CoV-2 NAb sensing. Nitrocellulose membrane coated with SARS-CoV-2 nucleocapsid S RBD fusion served as test lines.
The test line shows an ultra-bright fluorescent signal when encountered with a negative serum sample, i.e. 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 fluoresces slightly due to the high binding frequency of NAb-RBD.
The AIE490-PS nanoparticle-based LFIA method for anti-SARS-CoV-2 NAb detection 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 NAbs standard sample. The estimated time to detect one sample is 20 minutes.
Conclusions
Taken together, the use 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 strip, which are extremely important for their widespread application in the detection of antibodies against SARS-CoV-2. The nanoparticle-based LFIA AIE490-PS 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 multi-color marker-based LFIA.
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