Low Antigen Concentrations Research Articles

Detection of carcinoembryonic antigen specificity using microwave biosensor with machine learning

Early diagnosis and screening of tumor markers are essential for effective cancer treatment and improve the treatment efficiency and prognosis of tumor recurrence and metastasis. In this study, a split-ring resonator circuit based on an interdigital electrode structure was developed and applied to microwave biosensors along with machine learning to detect extremely low concentrations of Carcinoembryonic Antigen (CEA). CEA was detected using a microwave sensor operating at a resonance frequency of 4.33 GHz. When the microwave sensor is exposed to CEA analytes, it generates a new frequency in the range of 1–2 GHz. The position and intensity of the newly generated frequency can be used to characterize and predict the concentration of the CEA solution. The proposed sensor exhibits excellent resonance linearity for various concentrations of CEA (R2 = 0.999), as well as a very low detection limit (39 pg/mL) and high sensitivity (27.5 MHz/(ng/mL)). A machine learning approach was implemented to predict the CEA concentration in blood samples. The results showed close concurrence with the CEA concentration detected by the sensor. Western Blot (WB) was used to compare the CEA contents of four different cell types, and a biosensor was used for validation; the results of the two experiments showed good agreement. This is the first demonstration of the validation of biosensor reliability at the cellular level. The proposed concept exhibits outstanding detection performance with convenient and rapid tumor marker detection. Hence, it has important implications as an auxiliary diagnosis method for cancer.

Antigen concentration, viral load, and test performance for SARS-CoV-2 in multiple specimen types.

The relationship between N-antigen concentration and viral load within and across different specimens guides the clinical performance of rapid diagnostic tests (RDT) in different uses. A prospective study was conducted in Porto Velho, Brazil, to investigate RDT performance in different specimen types as a function of the correlation between antigen concentration and viral load. The study included 214 close contacts with recent exposures to confirmed cases, aged 12 years and older and with various levels of vaccination. Antigen concentration was measured in nasopharyngeal swab (NPS), anterior nares swab (ANS), and saliva specimens. Reverse transcriptase (RT)-PCR was conducted on the NPS and saliva specimens, and two RDTs were conducted on ANS and one RDT on saliva. Antigen concentration correlated well with viral load when measured in the same specimen type but not across specimen types. Antigen levels were higher in symptomatic cases compared to asymptomatic/oligosymptomatic cases and lower in saliva compared to NPS and ANS samples. Discordant results between the RDTs conducted on ANS and the RT-PCR on NPS were resolved by antigen concentration values. The analytical limit-of-detection of RDTs can be used to predict the performance of the tests in populations for which the antigen concentration is known. The antigen dynamics across different sample types observed in SARS-CoV-2 disease progression support use of RDTs with nasal samples. Given lower antigen concentrations in saliva, rapid testing using saliva is expected to require improved RDT analytical sensitivity to achieve clinical sensitivity similar to rapid testing of nasal samples.

Development of silver nano-based indirect ELISA and Dot-ELISA methods for serological diagnosis of a bacterial fish pathogen Aeromonas veronii

Rapid and accurate detection of bacterial pathogens is critical in controlling disease outbreaks affecting farmed fish. The present study aimed to develop a novel serological diagnostic approach using nano‑silver based Enzyme-linked immunosorbent assay (ELISA) for speedy detection of Aeromonas veronii infections in Nile tilapia. A. veronii isolates used in ELISA assays were recovered from moribund Nile tilapia during a disease outbreak in a private fish farm in Egypt. A. veronii isolates were identified based on alignment analysis of the gyrB and 16S rRNA gene sequences. A. veronii antisera used in ELISA assays were prepared in tilapia, and the bacterial antigens were formalin-killed. The cut-off values were 0.46 and 0.48 in traditional and nano-based ELISA. There were no cross-reactions with bacterial isolates (Aeromonas hydrophila, Aeromonas caviae, Aeromonas sobria, Pseudomonas fluorescens, and Vibrio vulnificus). The lowest antigen concentration that produced positive results after checkerboard titration in indirect-ELISA (i-ELISA) and dot ELISA was 15 μg and 250 ng of prepared antigen, respectively. Nano-ELISA and nano-based dot-ELISA antigen concentration was 10 μg and 100 ng, respectively. Sera concentration was 1:100 in indirect-ELISA and dot-ELISA, while it was 1:50 in nano-based ELISA and nano dot-ELISA. The i-ELISA successfully detected anti-Aeromonas IgG antibodies with 83.33% sensitivity and 66.67% specificity, while in the dot-ELISA, the sensitivity and specificity were 83.33% and 100%, respectively. Nano dot-ELISA had 100% sensitivity, specificity, and accuracy. Nano dot-ELISA assays have higher specificity, sensitivity, and accuracy than traditional ELISAs in detecting A. veronii. Further studies are needed to develop a rapid test kit for on-site field diagnosis.

Research on Detection of Ultra-Low Concentration Anthrax Protective Antigen Using Graphene Field-Effect Transistor Biosensor.

Protective antigen (PA) is an important biomarker for the early diagnosis of anthrax, and the accurate detection of protective antigen under extremely low concentration conditions has always been a hot topic in the biomedical field. To complete the diagnosis of anthrax in a timely manner, it is necessary to detect PA at extremely low concentrations, as the amount of PA produced in the early stage of anthrax invasion is relatively small. Graphene field-effect transistor (Gr-FET) biosensors are a new type of material for preparing biosensors, with the advantages of a short detection time and ultra-low detection limit. The effect of different concentrations of diluents on the affinity of PA monoclonal antibodies was determined via an ELISA experiment. Combined with the Debye equation, 0.01 × PBS solution was finally selected as the diluent for the experiment. Then, a PA monoclonal antibody was selected as the bio-recognition element to construct a Gr-FET device based on CVD-grown graphene, which was used to detect the concentration of PA while recording the response time, linear range, detection limit, and other parameters. The experimental results showed that the biosensor could quickly detect PA, with a linear range of 10 fg/mL to 100 pg/mL and a detection limit of 10 fg/mL. In addition, the biosensor showed excellent specificity and repeatability. By constructing a Gr-FET device based on CVD-grown graphene and selecting a PA monoclonal antibody as the bio-recognition element, a highly sensitive, specific, and repeatable Gr-FET biosensor was successfully prepared for detecting extremely low concentrations of anthrax protective antigen (PA). This biosensor is expected to have a wide range of applications in clinical medicine and biological safety monitoring.

Trogocytic-molting of T-cell microvilli controls T-cell clonal expansion

Abstract Internalization of the T-cell antigen receptor (TCR) is intimately linked to T-cell activation: a phenomenon thought to be related to the “exhaustion” of T-cell responses. To date, however, no report has considered that during physical interaction with cognate antigen-presenting cells, T cells release many TCRs via T-cell microvilli particles, which are derived from finger-like membrane structures (microvilli) in a combined process of trogocytosis and enzymatic vesiculation and correspond with the loss of membrane TCRs and many external membrane components. Surprisingly, in contrast to TCR internalization, this event leads to rapid upregulation of surface TCRs and remarkable metabolic reprogramming of cholesterol and fatty acids synthesis to meet the demands of clonal expansion, which drives multiple rounds of division and cell survival. We called this event “trogocytic-molting,” which represents an intrinsic molecular basis of T-cell clonal expansion by which T cells gain increased sensitivity to low antigen concentrations. This work was supported by the Creative Research Initiative Program (2015R1A3A2066253); Bio-Synergy Research Project (2021M3A9C4000991); Bio & Medical Technology Development Program [2020M3A9G3080281] through National Research Foundation (NRF) grants funded by the Ministry of Science and ICT (MSIT), the Basic Science Program (2019R1C1C1009570 & 2022R1A2C4002627) through National Research Foundation (NRF) grants funded by the Ministry of Education (MOE), and supported by Global University Project (GUP), GIST Research Institute (GRI) IBBR grant funded by the GIST (in 2021-2022), and the Joint Research Project of Institutes of Science and Technology (2021–2022), Korea.

Comparison of Anti-rabies Virus Nucleoprotein IgY Prepared by DNA Immunization and Protein Immunization.

Immunization of egg-laying hens with viral antigens efficiently produces large amounts of virus-specific IgY antibodies from egg yolks. A supply of practical and economical antibodies against the rabies virus is being desired worldwide. We immunized hens with the antigen gene DNA of the rabies virus, purified specific IgY antibodies from the egg yolk, and characterized the immuno-protein chemistry for use as a diagnosis. To prepare specific IgY antibodies against rabies virus nucleoprotein (RV-N) by DNA immunization, laying hens were pre-injected with λ-carrageenan or Freund's complete adjuvant to increase local immune activity (pre-immune stimulation), and then immunized with RV-N recombinant plasmid DNA. RV-N-specific IgY antibodies were prepared from egg yolks of immunized hens. For comparison, conventional protein antigen immunization was also used to induce the production of RV-N-specific IgY antibodies. Laying hens were immunized with an RV-N protein antigen and RV-N-specific IgY was purified from egg yolks. The binding activity against RV-N antigens was examined using IgY samples prepared by DNA (with pre-immune stimulation) and protein immunization. Immunohistochemical staining showed that IgY antibodies prepared by protein immunization strongly detected viral antigens in the brain sections of dogs infected with the virus, whereas IgY antibodies prepared by DNA immunization did not. Enzyme-linked immunosorbent assay was performed using a commercially available rabies vaccine (inactivated virus) treated with 10% formalin and heating (60°C, 30 min and 90°C, 5 min). IgY prepared by DNA immunization had weaker reactivity with denatured antigens and lower antigen concentrations than IgY prepared by protein immunization. These results suggest that it is necessary to develop a DNA immunization method for inducing IgY antibodies against the rabies virus that strongly bind to native and denatured antigens to prepare specific IgYs that can be used for antigen detection in clinical tests.

Diagnosis of Schistosomiasis without a Microscope: Evaluating Circulating Antigen (CCA, CAA) and DNA Detection Methods on Banked Samples of a Community-Based Survey from DR Congo.

Detection of Schistosoma eggs in stool or urine is known for its low sensitivity in diagnosing light infections. Alternative diagnostics with better sensitivity while remaining highly specific, such as real-time PCR and circulating antigen detection, are progressively used as complementary diagnostic procedures but have not yet replaced microscopy. This study evaluates these alternative methods for the detection of Schistosoma infections in the absence of microscopy. Schistosomiasis presence was determined retrospectively in 314 banked stool and urine samples, available from a previous survey on the prevalence of taeniasis in a community in the Democratic Republic of the Congo, using real-time PCR, the point-of-care circulating cathodic antigen (POC-CCA) test, as well as the up-converting particle lateral flow circulating anodic antigen (UCP-LF CAA) test. Schistosoma DNA was present in urine (3%) and stool (28%) samples, while CCA (28%) and CAA (69%) were detected in urine. Further analysis of the generated data indicated stool-based PCR and the POC-CCA test to be suitable diagnostics for screening of S. mansoni infections, even in the absence of microscopy. A substantial proportion (60%) of the 215 CAA-positive cases showed low antigen concentrations, suggesting that even PCR and POC-CCA underestimated the “true” number of schistosome positives.

Suppression of Coffee-Ring Effect on Nitrocellulose Membrane: Effect of Polyethylene Glycol

In the development of the diagnostic kit, it was favorable to have a low antigen concentration due to the difficulty of antigen preparedness and purification. However, it can cause the coffee-ring effect, producing different pattern formations on the selected membrane. It can lead to a false interpretation of the result. Thus, the immobilization of protein solution (lysozyme) as a model protein for antigen, with the addition of hydrosoluble polymer additive onto a membrane, was evaluated to suppress the coffee-ring effect. This research aims to evaluate the effect of polyethylene glycol on the protein solution for coffee-ring effect suppression and to analyze the image of the coffee-ring effect. From the experimental studies, 5 different concentrations (v/v%) of PEG which are 3.0, 2.0, 1.0, 0.1 and 0.01 v/v% is added at 4.0 mg/mL of lysozyme solution before being spotted onto nitrocellulose membrane. The color intensity of the dried spot, together with the formation of the coffee-ring effect, is analyzed by Image-J software. It is the approach to measure the suppression of the ring effect, in which 0.01 v/v% concentration portrays the most faded ring effect on nitrocellulose membrane. This effect occurs due to a surface tension gradient that causes the solute particles to accumulate at the edge of the droplet. As Marangoni flow has been altered, the coffee-ring effect is successfully suppressed; thus, uniform pattern deposition is achieved.

Sensitively detecting antigen of SARS-CoV-2 by NIR-II fluorescent nanoparticles.

Early detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is an efficient way to prevent the spread of coronavirus disease 2019 (COVID-19). Detecting SARS-CoV-2 antigen can be rapid and convenient, but it is still challenging to develop highly sensitive methods for effective diagnosis. Herein, a lateral flow assay (LFA) based on fluorescent nanoparticles emitting in the second near-infrared (NIR-II) window is developed for sensitive detection of SARS-CoV-2 antigen. Benefiting from the NIR-II fluorescence with high penetration and low autofluorescence, such NIR-II based LFA allows enhanced signal-to-background ratio, and the limit of detection is down to 0.01 ng·mL−1 of SARS-CoV-2 antigen. In the clinical swab sample tests, the NIR-II LFA outperforms the colloidal gold LFA with higher overall percent agreement with the polymerase chain reaction test. The clinical samples with low antigen concentrations (∼ 0.015−∼ 0.068 ng·mL−1) can be successfully detected by the NIR-II LFA, but fail for the colloidal gold LFA. The NIR-II LFA can provide a promising platform for highly sensitive, rapid, and cost-effective method for early diagnosis and mass screening of SARS-CoV-2 infection.Electronic Supplementary MaterialSupplementary material (the operation procedure and cost of the materials needed of NIR-II lateral flow assays, the dynamic light scattering spectrum of the NIR-II nanoparticles, the components and testing principle, optimization of main parameters pertaining to the LFA performance, the colloidal gold LFA strip, the fluorescence intensity distribution curves and the T/C values of the strips for clinical samples by NIR-II LFA, and results of clinical swab samples detected by colloidal gold LFA) is available in the online version of this article at 10.1007/s12274-022-4351-1.

Sub-picomolar lateral flow antigen detection with two-wavelength imaging of composite nanoparticles

Lateral flow tests, commonly based on metal plasmonic nanoparticles, are rapid, robust, and low-cost. However, improvements in analytical sensitivity are required to allow detection of low-abundance biomarkers, for example detection of low antigen concentrations for earlier or asymptomatic diagnosis of infectious diseases. Efforts to improve sensitivity often require changes to the assay. Here, we developed optical methods to improve the sensitivity of absorption-based lateral flow tests, requiring no assay modifications to existing tests. We experimentally compared five different lock-in and subtraction-based methods, exploiting the narrow plasmonic peak of gold nanoparticles for background removal by imaging at different light wavelengths. A statistical framework and three fitting models were used to compare limits of detection, giving a 2.0–5.4-fold improvement. We then demonstrated the broad applicability of the method to an ultrasensitive assay, designing 530 nm composite nanoparticles to increase the particle volume, and therefore light absorption per particle, whilst retaining the plasmonic peak to allow background removal and without adding any assay steps. This multifaceted, modular approach gave a combined 58-fold improvement in the fundamental limit of detection using a biotin-avidin model over 50 nm gold nanoparticles with single-wavelength imaging. Applying to a sandwich assay for the detection of HIV capsid protein gave a limit of detection of 170 fM. Additionally, we developed an open-source software tool for performing the detection limit analysis used in this work.

Blind Spot: A Braille Patterned Novel Multiplex Lateral Flow Immunoassay Sensor Array for the Detection of Biothreat Agents.

Lateral flow immunoassays (LFIs) are simple, point-of-care diagnostic devices used for detecting biological agents or other analytes of interest in a sample. LFIs are predominantly singleplex assays, interrogating one target analyte at a time. There is a need for multiplex LFI devices, e.g., a syndromic panel to differentiate pathogens causing diseases exhibiting similar symptoms. Multiplex LFI devices would be especially valuable in instances where sample quantity is limiting and reducing assay time and costs is critical. There are limitations to the design parameters and performance characteristics of a multiplex LFI assay with many horizontal test lines due to constraints in capillary flow dynamics. To address some of the performance issues, we have developed a spot array multiplex LFI using Braille format (hence called Blind Spot) and a sensor, MACAW (Modular Automated Colorimetric Analyses Widget), that can analyze and interpret the results. As a proof of concept, we created a multiplex toxin panel, for detecting three toxins, using two letter codes for each. The results indicated that the six-plex, triple toxin assay performs as well as singleplex assays. The sensor-based calls are better compared to human interpretation in discriminating and interpreting ambiguous test results correctly especially at lower antigen concentrations and from strips with blemishes.

Viral Vector Vaccines

Viral vector vaccines use harmless, non-replicating or replicating viruses to deliver genetic material for production of vaccine antigens into host cell cytoplasm. While viral vector vaccines may theoretically induce life-long immunity with low antigen concentrations, their attenuation, safety and spread to the community are of concern. Vaccines based on recombinant viral vectors can induce both humoral and cellular immune responses. Adenovirus vectors are versatile gene transfer vectors that can be easily manufactured, and which may allow simultaneous expression of multiple antigens by a single vector construct. Adenovirus vector vaccines based on the adenovirus Ad26 vector have been widely used as vaccines against Ebola and COVID19 (see Chapters 44 and 56). A common concern of using viral vector vaccines is pre-existing immunity or induction of immunity against the vector itself, but in some circumstances it has no meaningful impact and it can be resolved in several ways. Several harmless viruses are already used as vectors for innovative vaccines and many more are in research.

Structurally distinct endocytic pathways for B cell receptors in B lymphocytes.

B lymphocytes play a critical role in adaptive immunity. On antigen binding, B cell receptors (BCR) cluster on the plasma membrane and are internalized by endocytosis. In this process, B cells capture diverse antigens in various contexts and concentrations. However, it is unclear whether the mechanism of BCR endocytosis changes in response to these factors. Here, we studied the mechanism of soluble antigen-induced BCR clustering and internalization in a cultured human B cell line using correlative superresolution fluorescence and platinum replica electron microscopy. First, by visualizing nanoscale BCR clusters, we provide direct evidence that BCR cluster size increases with F(ab’)2 concentration. Next, we show that the physical mechanism of internalization switches in response to BCR cluster size. At low concentrations of antigen, B cells internalize small BCR clusters by classical clathrin-mediated endocytosis. At high antigen concentrations, when cluster size increases beyond the size of a single clathrin-coated pit, B cells retrieve receptor clusters using large invaginations of the plasma membrane capped with clathrin. At these sites, we observed early and sustained recruitment of actin and an actin polymerizing protein FCHSD2. We further show that actin recruitment is required for the efficient generation of these novel endocytic carriers and for their capture into the cytosol. We propose that in B cells, the mechanism of endocytosis switches to accommodate large receptor clusters formed when cells encounter high concentrations of soluble antigen. This mechanism is regulated by the organization and dynamics of the cortical actin cytoskeleton.

Primary immunization using low antigen dosages and immunological tolerance in rainbow trout

Enteric redmouth disease (ERM), caused by the Gram negative enterobacterium Yersinia ruckeri, affects farming of salmonids, but vaccination against ERM confers a certain degree of protection dependent on the administration route. Recent studies on oral vaccination of rainbow trout suggest that immunological tolerance may be induced by primary immunization using a low antigen dosage. We have examined if low dosages of Y. ruckeri antigens, applied in feed or bath exposure over a prolonged period of time, leave rainbow trout more susceptible to infection. Groups of rainbow trout were immunized, either by immersion or feeding using different vaccine dosages, and subsequently challenged by live Y. ruckeri. Survival was recorded and immune reactions in surviving fish were evaluated (ELISA and qPCR). Trout, bath-vaccinated in a highly diluted vaccine or fed the same amount of bacterin in feed over 10 days, were not protected against Y. ruckeri challenge infection and in some cases these sub-optimally immunized fish experienced lower survival compared to non-primed controls. Genes encoding FoxP3 and immune-suppressive cytokines were down-regulated in fish vaccinated with a high antigen dosage when compared to groups exposed to low antigen dosages, suggesting a higher regulatory T cell activity in the latter fish groups. The study suggests that repeated exposure to low antigen concentrations induces some degree of immune tolerance in rainbow trout and we recommend application of high antigen dosages for primary immunization of trout.

Development and validation of a simplified serotyping ELISA based on monoclonal antibodies for the diagnosis of foot-and-mouth disease virus serotypes O, A, C and Asia 1.

This study describes the development and validation of a simplified enzyme-linked immunosorbent assay (ELISA) for the detection and discrimination of foot-and-mouth disease virus (FMDV) serotypes O, A, C and Asia 1. The multiplex ELISA was designed using selected, type-specific monoclonal antibodies (MAbs) coated onto ELISA plates as catching antibodies and a unique pan-FMDV MAb (1F10) as detector conjugate. Capture MAbs with the broadest intratypic reactivity were selected for each of the four FMDV serotypes by screening large panels of candidate MAbs with a wide spectrum of representative FMDV isolates. An additional pan-FMDV ELISA using 1F10 MAb for both capture and detection was used to complement the specific typing ELISAs to detect virus isolates, which might escape binding to the selected serotype-specific MAbs. This multiplex ELISA was prepared in a stabilized format, with immunoplates pre-coated with six MAbs and positive antigen controls already trapped by the relevant MAb, with the view to make available a diagnostic kit. Diagnostic performance of the MAbs-multiplex ELISA was analysed using 299 FMDV-positive epithelial suspensions representative of the antigenic and genomic variability within each serotype. Overall, the results provided evidence that the diagnostic performance of this assay platform is improved compared to the more complex polyclonal-based antigen detection ELISA; combining data from different serotypes and referring to the gold standard tests (i.e. virus isolation and/or RT-PCR), the MAbs-multiplex ELISA showed a sensitivity of 79% for the serotype-specific ELISA, compared to 72% for the polyclonal ELISA. The absence of reactivity of a minority of FMDV strains using the MAbs-multiplex ELISA can largely be attributed to deteriorated or low antigen concentration in the sample. This multiplex ELISA is simple, rapid and stable. FMDV antigenic diversity was adequately covered by the selected MAbs. Therefore, it can be used to replace existing polyclonal ELISAs for FMDV detection and serotyping.

Cross/Tangential Flow Filtration of Antisera on an Industrial Scale

Abstract Purification of antisera, on an industrial scale, to date has been performed by SEC or affinity chromatography, both of which are costly. The method of cross/tangential flow filtration feeds solution across a filter surface rather than pushing product through it. Cross flow membranes are currently used in the manufacturing of serum proteins in order to dialyze and/or concentrate these solutions. Solvent, solutes, and particles smaller than the membrane’s pore size pass through the membrane, while larger molecules are retained and concentrated. This study was designed to apply membrane purification to increase the titers of polyclonal antisera on an industrial scale, as an alternative method to traditional chromatography techniques. Membranes with varying molecular weight cut offs were used to filter and concentrate neat antisera. The reduction of serum fluids and salts increase the concentration of antibodies, and therefore raise the titers. Concentrated antisera titers were then compared to neat antisera titers, to determine if a significant increase or decrease occurred, or if the method had no effect. This method has a marked resemblance to SEC, but without the need for buffers; other characteristics of the antisera remain unchanged. As the current availability of purified polyclonal antisera is scarce, the goal of this study was to produce a more effective product for use in immunology and medical device research and manufacturing across multiple testing platforms, by producing antisera with higher titers. This could potentially result in being able to process antisera to higher titers using lower concentrations of antigen in the host species, which is important from an industrial perspective for reducing manufacturing costs.

Nanobody LAMPoles: Novel tools for dengue virus and Zika virus diagnosis

Abstract Detection of antibodies by ELISA for verifying exposure to dengue virus (DENV) and Zika virus (ZIKV) has been a challenge. Point-of-care (POC) diagnostics with detection of very low concentrations of viral antigens would improve critical disease staging and surveillance to reduce morbidity and mortality. LAMPoles are protein-DNA hybrids where a ‘DNA barcode’ is covalently linked to a single site on a protein. Recombinant camelid nanobodies (Nb), which are variable region of the heavy chain antigen binding domain (VHH), can be tailored to bind almost any molecular target to maximize assay sensitivity and specificity. LAMPole fusions displaying Nb that bind ZIKV and DENV antigens will improve the sensitivity and specificity of virus antigen measurement at the POC. Llama were vaccinated with recombinant DENV envelope or ZIKV NS1 proteins and VHH phagemid libraries constructed and screened for DENV and ZIKV antigen-specific Nb. For DENV 70 full-length Nb were identified, belonging to 26 different CDR3 groups. For ZIKV 63 full-length anti-ZIKV Nb were identified, belonging to 42 different CDR3 groups. Nb screened by surface plasmon resonance spectroscopy with Koff kinetics in the 10−3 sec range were conjugated to chalcone synthase specific DNA elements using psoralen-based photocross-linkers. In a pilot experiment, LAMP targeting the CHS DNA tail of one Nb-LAMPole detected less than 10 fg of Nb-LAMPole protein. Nb-LAMPole recognized DENV-2 E-protein antigen in sandwich ELISA and in DENV-2 infected cells by immunofluorescence. We assert that LAMPole fusions displaying Nb that bind viral antigens may improve the sensitivity and specificity of DENV and ZIKV antigen detection that will facilitate markedly improved POC diagnosis.

Immuno-gold silver staining assays on capillary-driven microfluidics for the detection of malaria antigens.

Accurate and affordable rapid diagnostic tests (RDTs) are indispensable but often lacking for many infectious diseases. Specifically, there is a lack of highly sensitive malaria RDTs that can detect low antigen concentration at the onset of infection. Here, we present a strategy to improve the sensitivity of malaria RDTs by using capillary-driven microfluidic chips and combining sandwich immunoassays with electroless silver staining. We used 5μm fluorescent beads functionalized with capture antibodies (cAbs). These beads are self-assembled by capillary action in recessed "bead lanes", which cross the main flow path of chips microfabricated in Si and SU-8. The binding of analytes to detection antibodies (dAbs) and secondary antibodies (2ndAbs) conjugated to gold nanoparticles (NPs) allows the formation of a silver film on the beads. Such silver film masks the fluorescent core of the bead inversely proportional to the concentration of antigen in a sample. We illustrate this method using the recombinant malaria antigen Plasmodium falciparum histidine-rich-protein 2 (rPfHRP2) spiked in human serum. This antigen was a recombinant HRP2 protein expressed in Escherichia coli, which is also the standard reference material. The limit of detection (LOD) of our immunoassay was found to be less than 6ngmL-1 of rPfHRP2 within 20min, which is approaching the desired sensitivity needed in the Target Product Profile (TPP) for malaria elimination settings. The concept presented here is flexible and may also be utilized for implementing fluorescence immunoassays for the parallel detection of biomarkers on capillary-driven microfluidic chips.

Electrochemical measurement of antibody-antigen recognition biophysics: Thermodynamics and kinetics of human chorionic gonadotropin (hCG) binding to redox-tagged antibodies

The thermodynamics and kinetics of antigen binding under diffusive conditions to an electrode surface modified with ferrocene-tagged antibodies is studied within this work, and realised experimentally for the case of human chorionic gonadotropin (hGC) as the antigen with monoclonal anti-hCG antibodies immobilised on an electrode surface via a molecular wire platform. The formation of the antigen-antibody complex is monitored through the blocking of the ferrocene voltammetry, thereby enabling the fractional coverage of the electrode binding sites to be unravelled as a function of time. It is found that, at low antigen concentrations, a Frumkin adsorption isotherm fits the data, with repulsive interactions between bound antigens playing a significant rôle, with an affinity constant that is an order of magnitude larger than in the case of an untagged antibody, suggesting that the chemical hydrophobicity of the redox tag may encourage stronger binding. Comparison of the experimental temporal data with relevant diffusion-adsorption models under activation control allows for the extraction of the kinetic parameters; at zero coverage, the rate constants for adsorption and desorption are, respectively, larger and smaller than the untagged antibody. The kinetic study enables the confirmation that this type of platform may be utilised for rapid (15 min) and quantitative electroimmunoassay. This is validated through proof-of-concept analytical measurements, yielding a limit of detection around 25 mIU mL−1 (corresponding to 2.7 ng mL−1) – a value used clinically for urine hCG measurements corresponding to around four weeks of gestational age.

Effect of oral booster vaccination of rainbow trout against Yersinia ruckeri depends on type of primary immunization

Vaccination of rainbow trout against Enteric Redmouth Disease (ERM) caused by Yersinia ruckeri can be successfully performed by administering vaccine (a bacterin consisting of formalin killed bacteria) by immersion, bath or injection. Booster immunization is known to increase the protection of fish already primed by one of these vaccination methods. Oral vaccination of trout (administering vaccine in feed) is an even more convenient way of presenting antigen to the fish but the effect of an oral booster has not previously been described in detail. The present work describes to what extent protection may be enhanced by oral boostering following priming with different administration methods. The study confirms that vaccination by 30 s dip into a bacterin (diluted 1:10) may confer a significant protection compared to non-vaccinated fish. The immunity may be optimized by booster immunization either provided as dip (most effective), bath (less effective) or orally (least effective). Oral immunization may be used as booster after dip but applied as a single oral application it induced merely a slight and statistically non-significant response. It is noteworthy that primary oral immunization followed by an oral booster vaccination showed a trend for an even weaker response. It should be investigated if continued exposure to a low antigen concentration – as performed by two oral immunizations - may induce tolerance to the pathogen and thereby leave the fish more vulnerable.