New Paper: Clinical evaluation of AffiDX® SARS-CoV-2 Antigen Lateral Flow Test

Testing is a critical tool in monitoring and understanding the prevalence of COVID-19. Quick, cost-effective, and easy-to-use, lateral flow tests (LFTs) are well-positioned to support point-of-care (POC) diagnostics and population screening for the timely identification and isolation of positive cases, to effectively control COVID-19 transmission. A newly published study in the Journal of Biology and Today’s World, by Montoya, J. et al. 2021, is the first to report on the in vitro characterisation and clinical performance of Avacta’s novel Affimer®-based AffiDX® SARS-CoV-2 Antigen LFT within a representative cohort.

In vitro characterisation and candidate selection

To identify a lead biotinylated anti-SARS-CoV-2 S1 Affimer®, candidates were tested against three different concentrations of purified SARS-CoV-2 S1 protein, ranging from 100nM to 3nM, in a label-free surface plasmon resonance imaging (SPRi) system. Affimer® candidate 620_826257 demonstrated good binding kinetics towards the S1 protein, as shown in Fig. 1, and good specificity, as shown in Fig. 2, where its positive response to SARS-CoV-2 S1 is compared with its negative response to equally prepared human coronavirus S1 proteins: MERS-CoV S1, HCov-HKU1 S1, SARS-CoV S1, and HCoV-229E S1.


Fig. 1. SPRi sensorgrams for triplicate spots of Affimer® candidate 620_826257, with injections of 100nM–3nM SARS-CoV-2 S1 protein, overlayed with Association rate constant (ka) (M-1 s-1), Dissociation rate constant (kd) (s-1), and Equilibrium dissociation constant (KD) (nM).


Fig. 2. SPRi sensograms for Affimer® candidate 620_826257 with injections of 50 nM SARS-CoV-2 S1, MERS-CoV S1, HCoV-HKU1 S1, SARS-CoV S1, and HCoV-229E S1.

The team then performed a specificity profiling assay against recombinant S1 coronavirus targets to define the cross-reactivity of the AffiDX® LFT system based on the chosen Affimer® candidate. Cross-reactivity was noted for SARS-CoV only. Interference testing was also carried out on the LFT with three common nasal sprays: Nicorette, Vicks, and Pirinase – only the latter of which correlated to reduced intensity of the control and test lines. Nicorette and Vicks were both shown to have no effect on LFT accuracy and none of the nasal sprays caused false negative or false positive results.

The Limit of Detection (LoD), defined in the study as the lowest concentration of attenuated SARS-CoV-2 virus that results in the positive detection of 18 out of 20 replicates, or 90% of true positives, was confirmed across a series of viral dilutions.

Clinical performance testing

To assess the clinical performance of the Affimer®-based AffiDX® LFT, the team conducted field studies at sites in Madrid on a cohort of consenting patients of any age, gender, and ethnicity who presented a RT-qPCR test result for COVID-19, no older than four days. The RT-qPCR data, comprising 150 PCR-negative and 100 PCR-positive samples, was used as a standard of comparison for the AffiDX® LFT nasal swab samples and results.

The AffiDX® LFT identified all 150 PCR-negative nasal samples as negative, demonstrating a specificity of 100% with no observed false positives. From the cohort of 100 PCR-positive samples with a Ct ≤ 30, 98 nasal samples were identified as positive with the AffiDX® LFT, and all samples with a Ct ≤ 28 tested positive (Fig. 3). This demonstrates a sensitivity of 98% and 100%, respectively, and highlights that Affimer® technology offers effective detection of lower viral loads (higher Ct values). From an epidemiological perspective, Ct ≤ 28, for which the test displayed 100% sensitivity, is estimated to be responsible for the largest proportion of infectious contacts and transmission within a population.

Result

Positive

Negative

RT-qPCR

100 (Ct ≤ 30)

100 (Ct ≤ 28)

150

AffiDX® LFT

98

100

150

Fig. 3. RT-qPCR data compared with AffiDX® LFT results, for both the SARS-CoV-2 positive and negative cohorts.

Affimer® technology offers unique strengths that circumvent the current limitations in antibody-based diagnostics, such as fast production processes, and high specificity and sensitivity capabilities. The strong clinical performance of Avacta’s AffiDX® LFT presented in this study clearly shows that the Affimer® platform can be reliably used for rapid, easy-to-use POC diagnostics and population screening. In addition to the performance data, this novel COVID-19 antigen test is an important addition to other available tests as results can be read in minutes, right off the testing card, so that the patient gets the information almost in real-time. Due to its simpler design, usability and clinical performance within the cohort tested, this new antigen test could mark a pivotal advancement in the global efforts to diagnose the COVID-19 pandemic, as well as the potential for application to other emerging infectious diseases.

A parallel study is under review for publishing, which will further investigate comparative analysis versus a complete panel of lateral flow devices.