A semi-quantitative visual lateral flow immunoassay for SARS-CoV-2 antibody detection for the follow-up of immune response to vaccination or recovery.

The lateral flow immunoassay (LFIA) technique is largely employed for the point-of-care detection of antibodies especially for revealing the immune response in serum. Visual LFIAs usually provide the qualitative yes/no detection of antibodies, while quantification requires some equipment, making the assay more expensive and complicated. To achieve visual semi-quantification, the alignment of several lines (made of the same antigen) along a LFIA strip has been proposed. The numbering of the reacting lines has been used to correlate with the quantity of some biomarkers in serum. Here, we designed the first semiquantitative LFIA for detecting antibodies and applied it to classify the immune response to SARS-CoV-2 raised by vaccination or natural infection. We used a recombinant spike receptor-binding domain (RBD) as the specific capture reagent to draw two test lines. The detection reagent was selected among three possible ligands that are able to bind to anti-spike human antibodies: the same RBD, staphylococcal protein A, and anti-human immunoglobulin G antibodies. The most convenient detector, adsorbed on gold nanoparticles, was chosen based on the highest correlation with an antibody titre of 171 human sera, measured by a reference serological method, and was the RBD (Spearman's rho = 0.84). Incorporated into the semiquantitative LFIA, it confirmed the ability to discriminate high- and low-titre samples and to classify them into two classes (Dunn's test, P < 0.05). The proposed approach enabled the semiquantification of the immune response to SARS-CoV-2 by the unaided eye observation, thus overcoming the requirement of costly and complicated equipment, and represents a general strategy for the development of semiquantitative serological LFIAs.

The Amount of Cross-Linker Influences Affinity and Selectivity of NanoMIPs Prepared by Solid-Phase Polymerization Synthesis.

The cross-linker methylene-bis-acrylamide is usually present in nanoMIPs obtained by solid-phase polymerization synthesis at 2 mol% concentration, with very few exceptions. Here, we studied the influence of variable amounts of methylene-bis-acrylamide in the range between 0 (no cross-linker) and 50 mol% concentration on the binding properties of rabbit IgG nanoMIPs. The binding parameters were determined by equilibrium binding experiments and the results show that the degree of cross-linking defines three distinct types of nanoMIPs: (i) those with a low degree of cross-linking, including nanoMIPs without cross-linker (0-05 mol%), showing a low binding affinity, high density of binding sites, and low selectivity; (ii) nanoMIPs with a medium degree of cross-linking (1-18 mol%), showing higher binding affinity, low density of binding sites, and high selectivity; (iii) nanoMIPs with a high degree of cross-linking (32-50 mol%), characterized by non-specific nanopolymer-ligand interactions, with low binding affinity, high density of binding sites, and no selectivity. In conclusion, the results are particularly relevant in the synthesis of high-affinity, high-selectivity nanoMIPs as they demonstrate that a significant gain in affinity and selectivity could be achieved with pre-polymerization mixtures containing quantities of cross-linker up to 10-20 mol%, well higher than those normally used in this technique.

Merging Lateral Flow Immunoassay with Electroanalysis as a Novel Sensing Platform: Prostate Specific Antigen Detection as Case of Study.

The COVID-19 pandemic highlighted lateral flow immunoassay (LFIA) strips as the most known point-of-care (POC) devices enabling rapid and easy detection of relevant biomarkers by nonspecialists. However, these diagnostic tests are usually associated with the qualitative detection of the biomarker of interest. Alternatively, electrochemical-based diagnostics, especially known for diabetes care, enable quantitative determination of biomarkers. From an analytical point perspective, the combination of the two approaches might represent a step forward for the POC world: in fact, electrochemical transduction is attractive to be integrated into LFIA strips due to its simplicity, high sensitivity, fast signal generation, and cost effectiveness. In this work, a LFIA strip has been combined with an electrochemical transduction, yielding an electrochemical LFIA (eLFIA). As a proof-of-concept method, the detection of prostate-specific antigen has been carried out by combining a printed-electrochemical strip with the traditional LFIA tests. The electrochemical detection has been based on the measurement of Au ions produced from the dissolution of the gold nanoparticles previously captured on the test line. The analytical performances obtained at LFIA and eLFIA were compared, highlighting how the use of differential pulse voltammetry allowed for a lower detection limit (2.5-fold), respectively, 0.38 and 0.15 ng/mL, but increasing the time of analysis. Although the correlation between the two architectures confirmed the satisfactory agreement of outputs, this technical note has been thought to provide the reader a fair statement with regard to the strength and drawbacks about combining the two (apparently) competitor devices in a diagnostics field, namely, LFIA and electrochemical strips.

Mycotoxins-Imprinted Polymers: A State-of-the-Art Review.

Mycotoxins are toxic metabolites of molds which can contaminate food and beverages. Because of their acute and chronic toxicity, they can have harmful effects when ingested or inhaled, posing severe risks to human health. Contemporary analytical methods have the sensitivity required for contamination detection and quantification, but the direct application of these methods on real samples is not straightforward because of matrix complexity, and clean-up and preconcentration steps are needed, more and more requiring the application of highly selective solid-phase extraction materials. Molecularly imprinted polymers (MIPs) are artificial receptors mimicking the natural antibodies that are increasingly being used as a solid phase in extraction methods where selectivity towards target analytes is mandatory. In this review, the state-of-the-art about molecularly imprinted polymers as solid-phase extraction materials in mycotoxin contamination analysis will be discussed, with particular attention paid to the use of mimic molecules in the synthesis of mycotoxin-imprinted materials, to the application of these materials to food real samples, and to the development of advanced extraction methods involving molecular imprinting technology.

Experimental design for the development of a multiplex antigen lateral flow immunoassay detecting the Southern African Territory (SAT) serotypes of foot-and-mouth disease virus.

Antigenic lateral flow immunoassays (LFIAs) rely on the non-competitive sandwich format, including a detection (labelled) antibody and a capture antibody immobilised onto the analytical membrane. When the same antibody is used for the capture and the detection (single epitope immunoassay), the saturation of analyte epitopes by the probe compromises the capture and lowers the sensitivity. Hence, several factors, including the amount of the probe, the antibody-to-label ratio, and the contact time between the probe and the analyte before reaching the capture antibody, must be adjusted. We explored different designs of experiments (full-factorial, optimal, sub-optimal models) to optimise a multiplex sandwich-type LFIA for the diagnosis and serotyping of two Southern African Territory (SAT) serotypes of the foot-and-mouth disease virus, and to evaluate the reduction of the number of experiments in the development. Both assays employed single epitope sandwich, so most influencing variables on the sensitivity were studied and individuated. We upgraded a previous device increasing the sensitivity by a factor of two and reached the visual limit of detection of 103.7 and 104.0 (TCID/mL) for SAT 1 and SAT 2, respectively. The positioning of the capture region along the LFIA strip was the most influent variable to increase the detectability. Furthermore, we confirmed that the 13-optimal DoE was the most convenient approach for designing the device.

MIP-based immunoassays: A critical review.

Molecularly imprinted polymers, MIPs, are man-made receptors mimicking the thermodynamic and kinetic binding behaviour of natural antibodies. Therefore, it is not surprising that many researchers have thought about MIPs as artificial receptors in immunoassay-like analytical applications, where the general machinery of the assay is maintained, but the molecular recognition is no longer assured by an antibody but by an artificial receptor. However, the number of papers devoted explicitly to applications of MIPs in the immunoassay field is quite limited if compared to the huge number of papers covering the multifaceted molecular imprinting technology. For this reason, this critical review wants to give a general view of MIP-based immunoassays, trying to highlight the critical points that have so far prevented a wider application of molecular imprinting technology in the immunoassay field and, possibly, try to suggest strategies to overcome them.

Development and In-House Validation of an Enzyme-Linked Immunosorbent Assay and a Lateral Flow Immunoassay for the Dosage of Tenofovir in Human Saliva.

Highly active antiretroviral therapy (HAART) includes very potent drugs that are often characterized by high toxicity. Tenofovir (TFV) is a widely used drug prescribed mainly for pre-exposure prophylaxis (PreP) and the treatment of human immunodeficiency virus (HIV). The therapeutic range of TFV is narrow, and adverse effects occur with both underdose and overdose. The main factor contributing to therapeutic failure is the improper management of TFV, which may be caused by low compliance or patient variability. An important tool to prevent inappropriate administration is therapeutic drug monitoring (TDM) of compliance-relevant concentrations (ARCs) of TFV. TDM is performed routinely using time-consuming and expensive chromatographic methods coupled with mass spectrometry. Immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and lateral flow immunoassays (LFIAs), are based on antibody-antigen specific recognition and represent key tools for real-time quantitative and qualitative screening for point-of-care testing (POCT). Since saliva is a non-invasive and non-infectious biological sample, it is well-suited for TDM. However, saliva is expected to have a very low ARC for TFV, so tests with high sensitivity are required. Here, we have developed and validated a highly sensitive ELISA (IC50 1.2 ng/mL, dynamic range 0.4-10 ng/mL) that allows the quantification of TFV in saliva at ARCs and an extremely sensitive LFIA (visual LOD 0.5 ng/mL) that is able to distinguish between optimal and suboptimal ARCs of TFV in untreated saliva.

Development of molecular and antigenic-based rapid tests for the identification of African swine fever virus in different tissues.

African swine fever (ASF) is a severe haemorrhagic infectious disease affecting suids, thus representing a great economic concern. Considering the importance of the early diagnosis, rapid point of care testing (POCT) for ASF is highly demanded. In this work, we developed two strategies for the rapid onsite diagnosis of ASF, based on Lateral Flow Immunoassay (LFIA) and Recombinase Polymerase Amplification (RPA) techniques. The LFIA was a sandwich-type immunoassay exploiting a monoclonal antibody directed towards the p30 protein of the virus (Mab). The Mab was anchored onto the LFIA membrane to capture the ASFV and was also labelled with gold nanoparticles for staining the antibody-p30 complex. However, the use of the same antibody for capturing and as detector ligand showed a significant competitive effect for antigen binding, so required an experimental design to minimize reciprocal interference and maximize the response. The RPA assay, employing primers to the capsid protein p72 gene and an exonuclease III probe, was performed at 39 °C. The limit of detection of the method was assessed using a plasmid encoding the target gene and resulted in 5 copy/µL. The new LFIA and RPA were applied for ASFV detection in the animal tissues usually analysed by conventional assays (i.e., real-time PCR), such as kidney, spleen, and lymph nodes. A simple and universal virus extraction protocol was applied for sample preparation, followed by DNA extraction and purification for the RPA. The LFIA only required the addition of 3% H2O2 to limit matrix interference and prevent false positive results. The two rapid methods (25 min and 15 min were needed to complete the analysis for RPA and LFIA, respectively) showed high diagnostic specificity (100%) and sensitivity (93% and 87% for LFIA and RPA, respectively) for samples with high viral load (Ct < 27). False negative results were observed for samples with low viral load (Ct > 28) and/or also containing specific antibodies to ASFV, which decreased antigen availability and were indicative of a chronic, poorly transmissible infection. The simple and rapid sample preparation and the diagnostic performance of the LFIA suggested its large practical applicability for POC diagnosis of ASF.

Effect of Surfactants on the Binding Properties of a Molecularly Imprinted Polymer.

In molecularly imprinted polymers, non-specific interactions are generally based on weak forces between the polymer surface and the sample matrix. Thus, additives able to interfere with such interactions should be able to significantly reduce any non-specific binding effect. Surfactants represent an interesting class of substances as they are cheap and easily available. Here, we present a study of the effect of three surfactants (the anionic sodium dodecylsulphate, SDS, the cationic cetyltrimethylammonium bromide (CTAB) and the non-ionic polyoxyethylene-(20)-sorbitan monolaurate Tween 20) on the binding affinity of a 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-imprinted polymer for the template and its analogue 2,4-dichlorophenoxyacetic acid (2,4-D). The experimental results indicate that increasing amounts of surfactant decrease the binding affinity for the ligands strongly for the ionic ones, and more weakly for the non-ionic one. This effect is general, as it occurs for both 2,4,5-T and 2,4-D and for both the imprinted and the not-imprinted polymers. It also proves that the magnitude of this effect mainly depends on the presence or absence of an ionic charge, and that the hydrophobic "tail" of surfactants plays only a minor role.

Investigation of the "Antigen Hook Effect" in Lateral Flow Sandwich Immunoassay: The Case of Lumpy Skin Disease Virus Detection.

Lumpy skin disease (LSD) is an infectious disease affecting bovine with severe symptomatology. The implementation of effective control strategies to prevent infection outbreak requires rapid diagnostic tools. Two monoclonal antibodies (mAbs), targeting different epitopes of the LSDV structural protein p32, and gold nanoparticles (AuNPs) were used to set up a colorimetric sandwich-type lateral flow immunoassay (LFIA). Combinations including one or two mAbs, used either as the capture or detection reagent, were explored to investigate the hook effect due to antigen saturation by the detector antibody. The mAb-AuNP preparations were optimized by a full-factorial design of experiment to achieve maximum sensitivity. Opposite optimal conditions were selected when one Mab was used for capture and detection instead of two mAbs; thus, two rational routes for developing a highly sensitive LFIA according to Mab availability were outlined. The optimal LFIA for LSDV showed a low limit of detection (103.4 TCID50/mL), high inter- and intra-assay repeatability (CV% < 5.3%), and specificity (no cross-reaction towards 12 other viruses was observed), thus proving to be a good candidate as a useful tool for the point-of-need diagnosis of LSD.

Rabbit IgG-imprinted nanoMIPs by solid phase synthesis: the effect of cross-linkers on their affinity and selectivity.

Solid phase synthesis (SPS) of molecularly imprinted nanopolymers (nanoMIPs) represents an innovative method to prepare nanomaterials with tailor-made molecular recognition properties towards peptides and proteins. The synthesis of nanoMIPs by SPS usually involves a pre-polymerization formulation, where the cross-linker is invariably N,N'-methylen-bis-acrylamide (BIS). To date, the effect of cross-linkers on the binding properties of nanoMIPs prepared using cross-linkers other than BIS has never been reported. In this work, in order to investigate the effect of different cross-linkers in protein-imprinted nanoMIPs prepared by SPS, alongside BIS we considered other similar cross-linkers: N,N'-ethylene dimethacrylamide (EDAM), N,O-bis-methacryloylethanolamine (NOBE), ethylene glycol dimethacrilate (EDMA) and glycerol dimethacrylate (GDMA), replacing BIS with them in pre-polymerization mixtures. The synthetized nanoMIPs were homogeneous, with a polydispersity index of 0.24-0.30 and a mean diameter of 129-169 nm in water. The binding properties of the nanoMIPs were measured via equilibrium partition experiments with the template, rabbit IgG (RIgG), and the selectivity was evaluated with respect to bovine IgG (BIgG), bovine serum albumin (BSA) and hen egg lysozyme (LZM). The experimental results show that all the cross-linkers, with the exception of EDMA, endowed nanoMIPs with high binding affinities for the template (BIS: 16.0 × 106 mol-1 L, EDAM: 8.8 × 106 mol-1 L, NOBE: 15.8 × 106 mol-1 L, and GDMA: 12.8 × 106 mol-1 L), medium to high imprinting factors (BIS: 12.3, EDAM: 5.5, NOBE: 7.2, and GDMA: 11.6) and good selectivity towards other proteins but markedly dependent on the structure of the cross-linker, confirming the importance of the latter in the SPS of imprinted nanopolymers.

Bacterial ligands as flexible and sensitive detectors in rapid tests for antibodies to SARS-CoV-2.

Lateral flow immunoassay (LFIA) is widely employed as point-of-care tests (POCT) for the diagnosis of infectious diseases. The accuracy of LFIA largely depends on the quality of the immunoreagents used. Typical LFIAs to reveal the immune response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) employ anti-human immunoglobulin (hIG) antibodies and recombinant viral antigens, which usually are unstable and poorly soluble. Broad selective bacterial proteins, such as Staphylococcal protein A (SpA) and Streptococcal protein G (SpG) can be considered alternatives to anti-hIG to increase versatility and sensitivity of serological LFIAs because of their high binding capacity, interspecies reactivity, and robustness. We developed two colorimetric LFA devices including SpA and SpG linked to gold nanoparticles (GNP) as detectors and explored the use of a specific, stable, and soluble immunodominant fraction of the nucleocapsid protein from SARS-CoV-2 as the capturing agent. The optimal amount of SpA-GNP and SpG-GNP conjugates and the protein-to-GNP ratios were defined through a full factorial experimental design to maximize the diagnostic sensitivity of the LFIAs. The new LFA devices were applied to analyze 105 human serum samples (69 positive and 36 negatives according to reference molecular diagnostic methods). The results showed higher sensitivity (89.9%, 95% CI 82.7-97.0) and selectivity (91.7%, 82.6-100) for the SpA-based compared to the SpG-based LFA. In addition, 18 serum samples from cats and dogs living with COVID-19 patients were analyzed and 14 showed detectable levels of anti-SARS-CoV-2 antibodies, thus illustrating the flexibility of the SpA- and SpG-based LFAs.

Design of multiplexing lateral flow immunoassay for detection and typing of foot-and-mouth disease virus using pan-reactive and serotype-specific monoclonal antibodies: Evidence of a new hook effect.

The foot-and-mouth disease (FMD) is the most important transboundary viral disease of livestock in the international context, because of its extreme contagiousness, widespread diffusion, and severe impact on animal trade and animal productions. The rapid and on-field detection of the virus responsible for the FMD represents an urgent demand to efficiently control the diffusion of the infection, especially in low resource setting where the FMD is endemic. Colorimetric lateral flow immunoassay (LFIA) is largely used for the development of rapid tests, due to the extreme simplicity, cost-effectiveness, and on-field operation. In this work, two multiplex LFIA devices were designed for the diagnosis of FMD and the simultaneous identification of major circulating serotypes of the FMD virus. The LFIAs relied on the sandwich-type immunoassay and combined a set of well-characterised monoclonal antibodies (mAb) pairs. One LFIA aimed at detecting and identifying O, A and Asia-1 serotypes, the second device enabled the detection and differentiation of the SAT 1 and SAT 2 serotypes. Both devices also incorporated a broad-specific test line reporting on infection from FMDV, regardless the strain and the serotype involved. Accordingly, five and four reactive zones were arranged in the two devices to achieve a total of six simultaneous analyses. The development of the two multiplex systems highlighted for the first time the relevance of the mAb positioning along the LFIA strip in connection with the use of the same or different mAb as capture and detector ligands. In fact, the excess of detector mAb typically employed for increasing the sensitivity of sandwich immunoassay induced a new type of hook effect when combined with the same ligand used as the capture. This effect strongly impacted assay sensitivity, which could be improved by an intelligent alignment of the mAb pairs along the LFIA strip. The analytical and diagnostic performances of the two LFIAs were studied by testing reference FMDV strains grown in cell cultures and some representative field samples (epithelium homogenates). Almost equivalent sensitivity and specificity to those of a reference Ag-ELISA kit were shown, except for the serotype SAT 2. These simple devices are suitable in endemic regions for in-field diagnosis of FMD accompanied by virus serotyping and, moreover, could be deployed and used for rapid confirmation of secondary outbreaks after FMD incursions in free-areas, thus contributing to promptly implement control measures.

Ten Years of Lateral Flow Immunoassay Technique Applications: Trends, Challenges and Future Perspectives.

The Lateral Flow Immunoassay (LFIA) is by far one of the most successful analytical platforms to perform the on-site detection of target substances. LFIA can be considered as a sort of lab-in-a-hand and, together with other point-of-need tests, has represented a paradigm shift from sample-to-lab to lab-to-sample aiming to improve decision making and turnaround time. The features of LFIAs made them a very attractive tool in clinical diagnostic where they can improve patient care by enabling more prompt diagnosis and treatment decisions. The rapidity, simplicity, relative cost-effectiveness, and the possibility to be used by nonskilled personnel contributed to the wide acceptance of LFIAs. As a consequence, from the detection of molecules, organisms, and (bio)markers for clinical purposes, the LFIA application has been rapidly extended to other fields, including food and feed safety, veterinary medicine, environmental control, and many others. This review aims to provide readers with a 10-years overview of applications, outlining the trends for the main application fields and the relative compounded annual growth rates. Moreover, future perspectives and challenges are discussed.

Effect of Polymerization Time on the Binding Properties of Ciprofloxacin-Imprinted nanoMIPs Prepared by Solid-Phase Synthesis.

An innovative approach to imprinted nanoparticles (nanoMIPs) is represented by solid-phase synthesis. Since the polymeric chains grow over time and rearrange themselves around the template, the binding properties of nanoMIPs could depend on the polymerization time. Here we present an explorative study about the effect of different polymerization times on the binding properties of ciprofloxacin-imprinted nanoMIPs. The binding properties towards ciprofloxacin were studied by measuring the binding affinity constants (Keq) and the kinetic rate constants (kd, ka). Furthermore, selectivity and nonspecific binding were valued by measuring the rebinding of levofloxacin onto ciprofloxacin-imprinted nanoMIPs and ciprofloxacin onto diclofenac-imprinted nanoMIPs, respectively. The results show that different polymerization times produce nanoMIPs with different binding properties: short polymerization times (15 min) produced nanoMIPs with high binding affinity but low selectivity (Keq > 107 mol L-1, α ≈ 1); medium polymerization times (30 min-2 h) produced nanoMIPs with high binding affinity and selectivity (Keq ≥ 106 mol L-1, α < 1); and long polymerization times (>2 h) produced nanoMIPs with low binding affinity, fast dissociation kinetics and low selectivity (Keq ≤ 106 mol L-1, kdis > 0.2 min-1, α ≈ 1). The results can be explained as the combined effect of rearrangement and progressive stiffening of the polymer chains around the template molecules.

Recent Advancements in Enzyme-Based Lateral Flow Immunoassays.

Paper-based lateral-flow immunoassays (LFIAs) have achieved considerable commercial success and their impact in diagnostics is continuously growing. LFIA results are often obtained by visualizing by the naked eye color changes in given areas, providing a qualitative information about the presence/absence of the target analyte in the sample. However, this platform has the potential to provide ultrasensitive quantitative analysis for several applications. Indeed, LFIA is based on well-established immunological techniques, which have known in the last year great advances due to the combination of highly sensitive tracers, innovative signal amplification strategies and last-generation instrumental detectors. All these available progresses can be applied also to the LFIA platform by adapting them to a portable and miniaturized format. This possibility opens countless strategies for definitively turning the LFIA technique into an ultrasensitive quantitative method. Among the different proposals for achieving this goal, the use of enzyme-based immunoassay is very well known and widespread for routine analysis and it can represent a valid approach for improving LFIA performances. Several examples have been recently reported in literature exploiting enzymes properties and features for obtaining significative advances in this field. In this review, we aim to provide a critical overview of the recent progresses in highly sensitive LFIA detection technologies, involving the exploitation of enzyme-based amplification strategies. The features and applications of the technologies, along with future developments and challenges, are also discussed.

Smartphone biosensor for point-of-need chemiluminescence detection of ochratoxin A in wine and coffee.

Exposure to mycotoxins, which may contaminate food and feed commodities, represents a serious health risk for consumers. Ochratoxin A (OTA) is one of the most abundant and toxic mycotoxins, thus specific regulations for fixing its maximum admissible levels in foodstuff have been established. Lateral Flow ImmunoAssay (LFIA)-based devices have been proposed as screening tools to avoid OTA contamination along the whole food chain. We report a portable, user-friendly smartphone-based biosensor for the detection and quantification of OTA in wine and instant coffee, which combines the LFIA approach with chemiluminescence (CL) detection. The device employs the smartphone camera as a light detector and uses low-cost, disposable analytical cartridges containing the LFIA strip and all the necessary reagents. The analysis can be carried out at the point of need by non-specialized operators through simple manual operations. The biosensor allows OTA quantitative detection in wine and coffee samples up to 25 µg L-1 and with limits of detection of 0.3 and 0.1 µg L-1, respectively, which are below the European law-fixed limits. These results demonstrate that the developed device can be used for routine monitoring of OTA contamination, enabling rapid and reliable identification of positive samples requiring confirmatory analysis.

Dual lateral flow optical/chemiluminescence immunosensors for the rapid detection of salivary and serum IgA in patients with COVID-19 disease.

To accurately diagnose COVID-19 infection and its time-dependent progression, the rapid, sensitive, and noninvasive determination of immunoglobulins A specific to SARS-CoV-2 (IgA) in saliva and serum is needed to complement tests that detect immunoglobulins G and M. We have developed a dual optical/chemiluminescence format of a lateral flow immunoassay (LFIA) immunosensor for IgA in serum and saliva. A recombinant nucleocapsid antigen specifically captures SARS-CoV-2 antibodies in patient specimens. A labelled anti-human IgA reveals the bound IgA fraction. A dual colorimetric and chemiluminescence detection enables the affordable and ultrasensitive determination of IgA to SARS-CoV-2. Specifically, a simple smartphone-camera-based device measures the colour signal provided by nanogold-labelled anti-human IgA. For the ultrasensitive chemiluminescence transduction, we used a contact imaging portable device based on cooled CCD, and measured the light signal resulting from the reaction of the HRP-labelled anti-human IgA with a H2O2/luminol/enhancers substrate. A total of 25 serum and 9 saliva samples from infected and/or recovered individuals were analysed by the colorimetric LFIA, which was sensitive and reproducible enough for the semi-quantification of IgA in subjects with a strong serological response and in the early stage of COVID-19 infection. Switching to CL detection, the same immunosensor exhibited higher detection capability, revealing the presence of salivary IgA in infected individuals. For the patients included in the study (n = 4), the level of salivary IgA correlated with the time elapsed from diagnosis and with the severity of the disease. This IgA-LFIA immunosensor could be useful for noninvasively monitoring early immune responses to COVID-19 and for investigating the diagnostic/prognostic utility of salivary IgA in the context of large-scale screening to assess the efficacy of SARS-CoV-2 vaccines.

A multi-target lateral flow immunoassay enabling the specific and sensitive detection of total antibodies to SARS COV-2.

A rapid test for detecting total immunoglobulins directed towards the nucleocapsid protein (N) of severe acute syndrome coronavirus 2 (SARS CoV-2) was developed, based on a multi-target lateral flow immunoassay comprising two test lines. Both test lines bound to several classes of immunoglobulins (G, M, and A). Specific anti-SARS immunoglobulins were revealed by a colorimetric probe formed by N and gold nanoparticles. Targeting the total antibodies response to infection enabled achieving 100% diagnostic specificity (95.75-100, C.I. 95%, n = 85 healthy and with other infections individuals) and 94.6% sensitivity (84.9-98.9, C.I. 95%, n = 62 SARS CoV-2 infected subjects) as early as 7 days post confirmation of positivity. Agreeing results with a reference serological ELISA were achieved, except for the earlier detection capability of the rapid test. Follow up of the three seroconverting patients endorsed the hypothesis of the random rise of the different immunoglobulins and strengthened the 'total antibodies' approach for the trustworthy detection of serological response to SARS CoV-2 infection.

Switching from Multiplex to Multimodal Colorimetric Lateral Flow Immunosensor.

Multiplex lateral flow immunoassay (LFIA) is largely used for point-of-care testing to detect different pathogens or biomarkers in a single device. The increasing demand for multitargeting diagnostics requires multi-informative single tests. In this study, we demonstrated three strategies to upgrade standard multiplex LFIA to multimodal capacity. As a proof-of-concept, we applied the strategies to the differential diagnosis of Human Immunodeficiency Virus (HIV) infection, a widespread pathogen, for which conventional multiplex LFIA testing is well-established. In the new two-parameter LFIA (x2LFIA), we exploited color encoding, in which the binding of multiple targets occurs in one reactive band and the color of the probe reveals which one is present in the sample. By combining the sequential alignment of several reactive zones along the membrane of the LFIA strip and gold nanoparticles and gold nanostars for the differential visualization, in this demonstration, the x2LFIA can furnish information on HIV serotype and stage of infection in a single device. Three immunosensors were designed. The use of bioreagents as the capturing ligand anchored onto the membrane or as the detection ligand labelled with gold nanomaterials affected the performance of the x2LFIA. Higher detectability was achieved by the format involving the HIV-specific antigens as capturing agent and labelled secondary bioligands (anti-human immunoglobulins M and protein G) as the probes.