Rapid finger stick test to detect antibodies against SARS-CoV-2 mutant

The current global outbreak of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is devastating, in addition to a phenomenal sacrifice to human life. Has brought about economic and social impacts. Reliable bedside testing of this virus is required for rapid diagnosis, treatment, or isolation of COVID-19-positive cases.

study: Fingerstick blood test to evaluate SARS-CoV-2 neutralizing antibody response after vaccination against mutants.. Image credit: rootstudio / Shutterstock

New preprint reports measurable semi-quantitative test Neutralizing antibody Infect the SARS-CoV-2 pathogen at the Point of Care (POC) using fingertip or venipuncture blood. This test can evaluate serological responses to wild-type viruses and the new variants alpha, beta, gamma, and delta.

This study shows good agreement with previous results, but beta, gamma, and delta variants of the group vaccinated with the Pfizer / BioNTech Messenger Ribonucleic Acid (mRNA) vaccine more than 3 months ago. It also shows a significant reduction in the neutralizing titer of the test compared to those who received it within the last 3 months.

Background

The virus mutates, and as mentioned above, new Concerns (VOCs) with much higher infectivity and antigenic tactics emerge, spread more rapidly in vaccinated populations, and more serious diseases and breaks. It can cause through infections. These became dominant in the next wave, and delta variants are now causing the overwhelming majority of infections.

With virus neutralization test Pseudo virus Neutralizing tests (VNT and pVNT, respectively) are used to assess the protective immune response. Still, it requires skill and a lot of time, so it can only be done at Biosafety Lab Level 2 or 3. Their reproducibility is often limited because many variables affect their results.

An acceptable alternative is a neutralization test based on enzyme-linked immunosorbent assay (ELISA), which also depends on the skill and advanced equipment of the operator. Currently available POC tests are designed to detect total antibody (immunoglobulin) levels, thus providing unreliable correlation of immunoprotection or qualitative imaging only.

Current research, medRxiv* The preprint server aims to help meet this recognized need by explaining an accurate and rapid POC test that can measure the immune antibody response to vaccination, especially protection against VOC infection. ..

A previous paper by the same author presented a paper-based SARS-CoV-2 neutralization assay (Cellulose Pulldown Virus Neutralization Test (cpVNT)). It can identify neutralizing antibodies against the virus within 10 minutes, but it can also be used. Serum or plasma only. This test detected a viral receptor binding domain (RBD) that complexed with the viral angiotensin converting enzyme 2 (ACE2) receptor on host cells. Its destruction by the presence of neutralizing antibodies reduces the overall signal.

This test is suitable for use with whole blood and is better suited for POC testing. It not only showed good agreement with pVNT and ELISA-based assays. This test can also be used to evaluate the neutralization response to SARS-CoV-2VOC in vaccinated populations.

The major changes made included the transition to the use of fluorescent reporter molecules and a series of incubation steps. This improved the neutralization of RBD-ACE2 binding.

What did the study show?

The results show that neutralizing activity differs between vaccinated samples before first dose and 1-2 weeks and 3-4 weeks after initial administration with both Pfizer / BioNTech and Modana vaccine types. I did. The values ​​were ~ 2% vs. 14% and ~ 37%, respectively. After the second dose, the median blocking was 89%.

In the post-vaccination sample, the sample showed different median blocking activity between the two vaccines, while the later sample had more comparable inhibitory activity. The modified test can detect neutralizing activity in a wider range than either sVNT or pVNT, ensuring its clinical applicability within 10 minutes.

The inhibition rate of blood samples is about 0.87 times that observed with plasma samples. The median blocking rate of pre-vaccinated samples was less than one-third of current tests.

Also, this test has over 80% sensitivity and 100% specificity compared to off-the-shelf tests, is less specific and sensitive than ELISA and pVNT, and is probably due to different sample and assay types.

Blood sample types (vein and finger prick) also showed good correlation between pre-vaccination, early vaccination, and late vaccination samples compared to this test, and finger prick sampling. It shows potential usefulness.

The study also showed that mutations in RBD of various VOCs increased binding affinity compared to gamma, which showed a 3-fold increase compared to wild-type, especially ancestral mutants.

They investigated fictitious variants containing both the characteristic N501Y and T478K mutations. Indeed, it had the highest binding affinity for ACE2. All VOCs resulted in high ACE2 affinity compared to wild-type viruses. The variant described here did not appear more than 1.6x 9

Although nAb percent blocking against beta and gamma strains was significantly reduced, unlike the other two VOCs, the engineered variant RBD allowed vaccine-induced neutralizing antibodies to replace ACE2 from RBD. It decreased significantly to 72.4%. The neutralization reaction was most significantly affected by these strains of people vaccinated with the Pfizer vaccine.

Over time, neutralizing activity decreased with wild-type and alpha RBD binding, but more significantly with beta, gamma, and delta mutants, as opposed to the stability of the neutralization response to wild-type virus. Did. This supports recent studies on reduced defense after the Pfizer vaccine, demonstrates the usefulness of trials for assessing vaccine-induced neutralizing immunity, and identifies people who will benefit from a third booster.

What is the impact?

Researchers have pointed out this test as a tool for better analysis of the degree of neutralization and RBD-ACE2 binding to the virus, especially for new mutants. The above fictitious mutants, even with high ACE2-binding affinity, showed no antigenic escape capability with more than 90% blocking by neutralizing antibodies. Therefore, these mutations are not important in neutralizing antibody binding.

Beta had a slightly higher binding affinity, but had a much lower blocking rate of neutralizing antibodies and a much higher antigenic escape compared to wild-type viruses. A strong combination of RBD-ACE2 binding affinity and hyponeutralizing antibody recognition means much greater antigenic escape.

Of course, cell-mediated immunity is the key to providing lasting immunity to the disease, but this test enables reliable detection of immune protection at the POC level. Approximately 90% of neutralizing activity Convalescent plasma Targets immunodominant RBD25-27 and makes this test consistent with standardized percent blocking measurements.

In addition, it is easy to adapt to the identification of new variants, requiring only a single reagent change, and scientists can keep up with the virus as it changes and evolves rapidly. The ability to detect weakened immunity as a POC test is also important, consistent with the increase in breakthrough infections in people vaccinated with the Pfizer vaccine from January to April 2021.

This test is well suited for PoC settings and provides an insightful nAb response in the post-vaccinated population. [This] It will be a valuable tool for public health authorities to manage breakthrough infections and develop effective additional vaccination strategies for more vulnerable individuals. “

*Important Notices

medRxiv publishes unpeer-reviewed preliminary scientific reports and should not be considered definitive, guide clinical / health-related behaviors, or be treated as established information.