Case study observational research: inflammatory cytokines in the bronchial epithelial lining fluid of COVID-19 patients with acute hypoxemic respiratory failure.

BACKGROUND: In this study, the concentrations of inflammatory cytokines were measured in the bronchial epithelial lining fluid (ELF) and plasma in patients with acute hypoxemic respiratory failure (AHRF) secondary to severe coronavirus disease 2019 (COVID-19). METHODS: We comprehensively analyzed the concentrations of 25 cytokines in the ELF and plasma of 27 COVID-19 AHRF patients. ELF was collected using the bronchial microsampling method through an endotracheal tube just after patients were intubated for mechanical ventilation. RESULTS: Compared with those in healthy volunteers, the concentrations of interleukin (IL)-6 (median 27.6 pmol/L), IL-8 (1045.1 pmol/L), IL-17A (0.8 pmol/L), IL-25 (1.5 pmol/L), and IL-31 (42.3 pmol/L) were significantly greater in the ELF of COVID-19 patients than in that of volunteers. The concentrations of MCP-1 and MIP-1ß were significantly greater in the plasma of COVID-19 patients than in that of volunteers. The ELF/plasma ratio of IL-8 was the highest among the 25 cytokines, with a median of 737, and the ELF/plasma ratio of IL-6 (median: 218), IL-1ß (202), IL-31 (169), MCP-1 (81), MIP-1ß (55), and TNF-α (47) were lower. CONCLUSIONS: The ELF concentrations of IL-6, IL-8, IL-17A, IL-25, and IL-31 were significantly increased in COVID-19 patients. Although high levels of MIP-1 and MIP-1ß were also detected in the blood samples collected simultaneously with the ELF samples, the results indicated that lung inflammation was highly compartmentalized. Our study demonstrated that a comprehensive analysis of cytokines in the ELF is a feasible approach for understanding lung inflammation and systemic interactions in patients with severe pneumonia.

Standardized Whole Blood Assay and Bead-Based Cytokine Profiling Reveal Commonalities and Diversity of the Response to Bacteria and TLR Ligands in Cattle.

Recent developments in multiplex technologies enable the determination of a large nu\mber of soluble proteins such as cytokines in various biological samples. More than a one-by-one determination of the concentration of immune mediators, they permit the establishment of secretion profiles for a more accurate description of conditions related to infectious diseases or vaccination. Cytokine profiling has recently been made available for bovine species with the development of a Luminex® technology-based 15-plex assay. Independently from the manufacturer, we evaluated the bovine cytokine/chemokine multiplex assay for limits of detection, recovery rate, and reproducibility. Furthermore, we assessed cytokine secretion in blood samples from 107 cows upon stimulation with heat-killed bacteria and TLR2/4 ligands compared to a null condition. Secretion patterns were analyzed either using the absolute concentration of cytokines or using their relative concentration with respect to the overall secretion level induced by each stimulus. Using Partial Least Square-Discriminant Analysis, we show that the 15-cytokine profile is different under Escherichia coli, Staphylococcus aureus, and Streptococcus uberis conditions, and that IFN-γ, IL-1ß, and TNF-α contribute the most to differentiate these conditions. LPS and E. coli induced largely overlapping biological responses, but S. aureus and S. uberis were associated with distinct cytokine profiles than their respective TLR ligands. Finally, results based on adjusted or absolute cytokine levels yielded similar discriminative power, but led to different stimuli-related signatures.

A rapid bead-based assay for screening of SARS-CoV-2 neutralizing antibodies.

Quantitative determination of neutralizing antibodies against Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) is paramount in immunodiagnostics, vaccine efficacy testing, and immune response profiling among the vaccinated population. Cost-effective, rapid, easy-to-perform assays are essential to support the vaccine development process and immunosurveillance studies. We describe a bead-based screening assay for S1-neutralization using recombinant fluorescent proteins of hACE2 and SARS-CoV2-S1, immobilized on solid beads employing nanobodies/metal-affinity tags. Nanobody-mediated capture of SARS-CoV-2-Spike (S1) on agarose beads served as the trap for soluble recombinant ACE2-GFPSpark, inhibited by neutralizing antibody. The first approach demonstrates single-color fluorescent imaging of ACE2-GFPSpark binding to His-tagged S1-Receptor Binding Domain (RBD-His) immobilized beads. The second approach is dual-color imaging of soluble ACE2-GFPSpark to S1-Orange Fluorescent Protein (S1-OFPSpark) beads. Both methods showed a good correlation with the gold standard pseudovirion assay and can be adapted to any fluorescent platforms for screening.

Signal-on/signal-off bead-based assays for the multiplexed monitoring of base excision repair activities by flow cytometry.

As the support of all living kingdoms' genetic information, the integrity of the DNA biomolecule must be preserved. To that goal, cells have evolved specific DNA repair pathways to thwart a large diversity of chemical substances and radiations that alter the DNA structure and lead to the development of pathologies such as cancers or neurodegenerative diseases. When dysregulated, activity rates of various actors of DNA repair can play a key role in carcinogenesis as well as in drugs resistance or hypersensitivity mechanisms. For the last 10 years, new complementary treatments have aimed at targeting specific enzymes responsible for such resistances. It is therefore crucial for biomedical research and clinical diagnosis to develop fast and sensitive tools able to measure the activity rate of DNA repair enzymes. In this work, a new assay for measuring enzymatic activities using microbeacons (µBs) is expounded. µB refers to microsphere functionalized by hairpin-shaped nucleic acid probes containing a single site-specific lesion in the stem and modified with chromophores. Following the processing of the lesion by the targeted protein, µB is cleaved and either lights off (signal-off strategy) or on (signal-on), depending on the use of fluorescent or profluorescent probes, respectively. After an optimization phase of the assay, we reported the combined analysis of restriction enzyme, AP-endonuclease, and DNA N-glycosylase by real-time monitoring followed by a flow cytometry measurement. As proofs of concept, we demonstrated the potential of the biosensor for highlighting DNA repair inhibitors and discriminating cell lines from their enzymatic activities.

New mAbs facilitate quantification of secreted equine TNF-α and flow cytometric analysis in monocytes and T cells.

Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine, that is involved in acute inflammation and is employed as a biomarker of inflammatory diseases in several species for which reliable quantification is available. We aimed to develop suitable tools to quantify TNF-α in equine samples. We generated two new mAbs against equine TNF-α (clones 48 and 292), evaluated their specificity for this cytokine, and confirmed detection of native TNF-α in stimulated equine PBMC. The TNF-α mAbs were paired in a fluorescent bead-based assay for quantification of equine TNF-α. The TNF-α assay had a wide quantification range of 12 pg/mL - 38.4 ng/mL. In addition, TNF-α mAb 48 was used for a detailed analysis of TNF-α production in PBMC by intracellular staining and flow cytometry. TNF-α was expressed by CD14+ monocytes after LPS stimulation and by monocytes and lymphocytes after polyclonal stimulation with PMA and ionomycin in vitro. TNF-α expressing lymphocytes consisted mainly of CD4+ T cells. CD8+ T cells and other lymphocytes also expressed TNF-α. The new mAbs evaluated here for soluble and intracellular TNF-α will enable the detailed analysis of this important pro-inflammatory cytokine during equine immune responses and inflammatory diseases of the horse.

Survival and Inactivation by Advanced Oxidative Process of Foodborne Viruses in Model Low-Moisture Foods.

Enteric viruses, such as human norovirus (NoV) and hepatitis A virus (HAV), are the major causes of foodborne illnesses worldwide. These viruses have low infectious dose, and may remain infectious for weeks in the environment and food. Limited information is available regarding viral survival and transmission in low-moisture foods (LMF). LMFs are generally considered as ready-to-eat products, which undergo no or minimal pathogen reduction steps. However, numerous foodborne viral outbreaks associated with LMFs have been reported in recent years. The objective of this study was to examine the survival of foodborne viruses in LMFs during 4-week storage at ambient temperature and to evaluate the efficacy of advanced oxidative process (AOP) treatment in the inactivation of these viruses. For this purpose, select LMFs such as pistachios, chocolate, and cereal were inoculated with HAV and the norovirus surrogates, murine norovirus (MNV) and feline calicivirus (FCV), then viral survival on these food matrices was measured over a four-week incubation at ambient temperature, by both plaque assay and droplet-digital RT-PCR (ddRT-PCR) using the modified ISO-15216 method as well as the magnetic bead assay for viral recovery. We observed an approximately 0.5 log reduction in viral genome copies, and 1 log reduction in viral infectivity for all three tested viruses following storage of select inoculated LMFs for 4 weeks. Therefore, the present study shows that the examined foodborne viruses can persist for a long time in LMFs. Next, we examined the inactivation efficacy of AOP treatment, which combines UV-C, ozone, and hydrogen peroxide vapor, and observed that while approximately 100% (4 log) inactivation can be achieved for FCV, and MNV in chocolate, the inactivation efficiency diminishes to approximately 90% (1 log) in pistachios and 70% (< 1 log) in cereal. AOP treatment could therefore be a good candidate for risk reduction of foodborne viruses from certain LMFs depending on the food matrix and surface of treatment.

Polystyrene Microparticles with Convergently Grown Mesoporous Silica Shells as a Promising Tool for Multiplexed Bioanalytical Assays.

Functional core/shell particles are highly sought after in analytical chemistry, especially in methods suitable for single-particle analysis such as flow cytometry because they allow for facile multiplexed detection of several analytes in a single run. Aiming to develop a powerful bead platform of which the core particle can be doped in a straightforward manner while the shell offers the highest possible sensitivity when functionalized with (bio)chemical binders, polystyrene particles were coated with different kinds of mesoporous silica shells in a convergent growth approach. Mesoporous shells allow us to obtain distinctly higher surface areas in comparison with conventional nonporous shells. While assessing the potential of narrow- as well as wide-pore silicas such as Mobil composition of matter no. 41 (MCM-41) and Santa Barbara amorphous material no. 15 (SBA-15), especially the synthesis of the latter shells that are much more suitable for biomolecule anchoring was optimized by altering the pH and both, the amount and type of the mediator salt. Our studies showed that the best performing material resulted from a synthesis using neutral conditions and MgSO4 as an ionic mediator. The analytical potential of the particles was investigated in flow cytometric DNA assays after their respective functionalization for individual and multiplexed detection of short oligonucleotide strands. These experiments revealed that a two-step modification of the silica surface with amino silane and succinic anhydride prior to coupling of an amino-terminated capture DNA (c-DNA) strand is superior to coupling carboxylic acid-terminated c-DNA to aminated core/shell particles, yielding limits of detection (LOD) down to 5 pM for a hybridization assay, using labeled complementary single-stranded target DNA (t-DNA) 15mers. The potential of the use of the particles in multiplexed analysis was shown with the aid of dye-doped core particles carrying a respective SBA-15 shell. Characteristic genomic sequences of human papillomaviruses (HPV) were chosen as the t-DNA analytes here, since their high relevance as carcinogens and the high number of different pathogens is a relevant model case. The title particles showed a promising performance and allowed us to unequivocally detect the different high- and low-risk HPV types in a single experimental run.

Evaluating SARS-CoV-2 spike and nucleocapsid proteins as targets for antibody detection in severe and mild COVID-19 cases using a Luminex bead-based assay.

Large-scale serosurveillance of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) will only be possible if serological tests are sufficiently reliable, rapid and affordable. Many assays are either labour-intensive and require specialised facilities (e.g. virus neutralization assays), or are expensive with suboptimal specificity (e.g. commercial ELISAs and RDTs). Bead-based assays offer a cost-effective alternative and allow for multiplexing to test for antibodies against multiple antigens and against other pathogens. Here, we compare the performance of spike (S) and nucleocapsid (NP) antigens for the detection of SARS-CoV-2 specific IgG, IgM and IgA antibodies in a panel of sera that includes recent (up to six weeks after symptom onset, severe n = 44; and mild cases n = 52) and old infections (five months after symptom onset, mild n = 104), using a Luminex-bead based assay and comparison to a virus neutralization test. While we show that neutralizing antibody levels are significantly lower in mild than in severe cases, we demonstrate that a combination of the recombinant nucleocapsid protein (NP) and receptor-binding domain (RBD) results in highly specific (99 %) IgG antibody detection five months after infection in 96 % of cases. Although most severe Covid-19 cases developed a clear IgM and IgA response, titers fell below the detection threshold in more than 20 % of mild cases in our bead-based assay. In conclusion, our data supports the use of RBD and NP for the development of SARS-CoV-2 serological IgG bead-based assays.

Detection of specific Atlantic salmon antibodies against salmonid alphavirus using a bead-based immunoassay.

Salmonid alphavirus (SAV) is the etiological cause of pancreas disease (PD) in Atlantic salmon (Salmo salar). Several vaccines against SAV are in use, but PD still cause significant mortality and concern in European aquaculture, raising the need for optimal tools to monitor SAV immunity. To monitor and control the distribution of PD in Norway, all salmonid farms are regularly screened for SAV by RT-qPCR. While the direct detection of SAV is helpful in the early stages of infection, serological methods could bring additional information on acquired SAV immunity in the later stages. Traditionally, SAV antibodies are monitored in neutralization assays, but they are time-consuming and cumbersome, thus alternative assays are warranted. Enzyme-linked immunosorbent assays (ELISAs) have not yet been successfully used for anti-SAV antibody detection in aquaculture. We aimed to develop a bead-based immunoassay for SAV-specific antibodies. By using detergent-treated SAV particles as antigens, we detected SAV-specific antibodies in plasma collected from both a SAV challenge trial and a field outbreak of PD. Increased levels of SAV-specific antibodies were seen after most fish had become negative for viral RNA. The bead-based assay is time saving compared to virus neutralization assays, and suitable for non-lethal testing due to low sample size requirements. We conclude that the bead-based immunoassay for SAV antibody detection is a promising diagnostic tool to complement SAV screening in aquaculture.

Evaluation of Bead-Based Assays for the Isolation of Foodborne Viruses from Low-Moisture Foods.

ABSTRACT: Foodborne viruses such as norovirus and hepatitis A virus (HAV) are highly transmissible, persistent in the environment, and resistant to many conventional inactivation methods. Foods can become contaminated with these viruses either at the source of harvest or during food handling and processing. Multiple lines of evidence suggest that foodborne viruses can survive desiccation and dry conditions. Several foodborne virus outbreaks have been linked to low-moisture foods (LMFs), indicating that these foods can be vehicles of virus transmission. However, the efficiencies of common virus extraction methodologies have not been examined with LMFs. We adapted the International Organization for Standardization (ISO) 15216-1:2017 method for virus recovery for use with chocolate, pistachios, and cornflakes. We also developed a magnetic bead assay for the recovery of HAV from LMFs and used the porcine gastric mucin-coated magnetic beads (PGM-MBs) to extract norovirus surrogates, feline calicivirus (FCV), and murine norovirus (MNV) from the same LMFs. The efficiency of virus recovery using the bead-based assay was then compared with that of the ISO 15216-1:2017 method. In chocolate and pistachios, the recovery rates with the PGM-MB method were 5.6- and 21.3-fold higher, respectively, for FCV and 1.65- and 18-fold higher, respectively, for MNV than those with the ISO 15216-1:2017 method. However, the PGM-MB method failed to recover MNV and FCV from cornflakes. The recovery rates for HAV in chocolate, pistachios, and corn flakes with the magnetic bead method were 11.5-, 3-, and 5.6-fold higher, respectively, than those with the ISO 15216-1:2017 method. Thus, depending upon the food matrix and the target virus, the bead-based assays can be used to efficiently and rapidly extract viruses from LMFs.

Advanced ELISA-like Biosensing Based on Ultralarge-Pore Silica Microbeads.

In this work, functionalized porous silica-based materials, widely used in the literature as drug and biomolecule nanocarriers, were innovatively used as an effective three-dimensional (3D) substrate for the development of a specific biomolecular assay showing great versatility in terms of detection performance. One-pot synthesis of ultralarge-pore silica microbeads was optimized to develop an enzyme-linked immunosorbent (ELISA)-like DNA detection assay. Cocondensation synthesis enabled introducing thiol functionalities into the silica framework while preserving both the high specific surface area (560 m2/g) and large pore size (17 nm average diameter), which are essential to guaranteeing high loading capability. Indeed, the bead-capturing ability was proved by developing an ELISA-like assay for the detection of short DNA sequences (≈20 bp), both in labeled and label-free configurations. In particular, the suppression of unspecific binding on the bead surface by testing two different blocking agents was a matter of interest. The detection performances were evaluated and compared to the ones obtained by following the same detection protocol on a standard flat surface (two-dimensional, 2D), which is most commonly used for this purpose. The bead-based assay showed a limit of detection two times lower than the flat-surface assay, confirming the promising capturing ability due to the larger active surface area. Furthermore, compared to traditional ELISA, the bead-based assay showed an intrinsic larger dynamic range that can be tailored depending on the final amount of beads used for the colorimetric quantification.

A universal method for the functionalization of dyed magnetic microspheres with peptides.

The need for the functionalization of magnetic, water-soluble dyed microspheres with peptides is apparent with the ever-growing biointeraction capabilities and the increased use of dyed microspheres in multiplex, microsphere-based detection assays. This method describes the attachment of any peptide to dyed magnetic microspheres regardless of peptide length, size, or sequence. The method exploits 'click' chemistry with short reaction times in a mixed organic/water system for simultaneous selective surface functionalization and reduction of microsphere dye leaching. All optimization studies were performed using a Luminex 200 assay platform, but the functionalized microspheres are capable of use in any similar multiplex format.

Quantitation of low abundant soluble biomarkers using high sensitivity Single Molecule Counting technology.

Quantitation of biomarkers in biofluids plays a central role in basic research to management of patient care and is routinely used in clinical laboratories and academic institutions. Standard immunoassays, such as an enzyme-linked immunosorbent assay (ELISA), have provided understanding of both normal and pathological processes for many decades. However, in more recent decades, new immunoassay technologies have uncovered numerous analytes in blood that were once undetectable using traditional ELISAs. To meet this new challenge for quantifying low abundant proteins in biofluids, Single Molecule Counting (SMC™) technology was developed. This new technology is a combination of improvements to both the immunoassay procedure as well as the instrument. The aim of this article is to introduce the new SMCxPRO™ instrument, xPRO Acquisition and Analysis software, and the high sensitivity immunoassay kits validated on this instrument for the detection of low abundant proteins in biofluids, such as serum and plasma. Using this new technology platform, biomarkers that were once unquantifiable can now be quantitated in both normal and diseased biofluids.

Detection and profiling of diarrheic marine biotoxins in shellfish by mRNA analysis of exposed Caco-2 cells using qRT-PCR and multiplex magnetic bead-based assays.

The mouse bioassay for the detection of marine biotoxins in shellfish products is 40 years old and still in use. A full ban or total replacement of this in vivo test has been postponed because of the fear that current chemical-based detection methods could miss a new emerging toxin. In order to fully replace the mouse bioassay, more efforts are needed on the search for functional assays with specific endpoints. Gene expression elicited by diarrheic shellfish poisons in Caco-2 cells allowed us to determine three 'DSP profiles', i.e. OA/DTX, AZA-YTX and PTX profiles. Twelve marker genes were selected to envision the three profiles. qRT-PCR is relatively cheap and easy, and although its multiplex capacity is limited to 5 genes, this turned out to be sufficient to show the three expected profiles. The use of the multiplex magnetic bead-based assay turned out to be even a slightly better alternative, allowing the use of all twelve selected marker genes and 2 reference genes, and resulting in clear profiles with for some genes even higher induction factors as obtained by qRT-PCR. When analysing blank and contaminated shellfish samples with this multiplex magnetic bead-based assay, the contaminated samples could easily be distinguished from the blank samples, showing the expected profiles. This work is one step further on the final replacement of the mouse bioassay, e.g. by combining the neuro-2a bioassay for screening and detection with analytical chemical analyses and the multiplex magnetic bead-based assay for confirmation of known and unknown toxins respectively.

Multifunctional Polystyrene Core/Silica Shell Microparticles with Antifouling Properties for Bead-Based Multiplexed and Quantitative Analysis.

Commercial bead-based assays are commonly built upon polystyrene particles. The polymeric carrier can be encoded with organic dyes and has ideal material properties for cytometric applications such as low density and high refractive index. However, functional groups are conventionally integrated during polymerization and subsequent modification is limited to the reactivity of those groups. Additionally, polystyrene as the core material leads to many hydrophobic areas still being present on the beads' surfaces even after functionalization, rendering the particles prone to nonspecific adsorption during an application. The latter calls for several washing steps and the use of additives in (bio)analytical assays. In this contribution, we show how these limitations can be overcome by using monodisperse polystyrene (PS) core/silica (SiO2) shell particles (SiO2@PS). Two different hydrophobic BODIPY (boron-dipyrromethene) dyes were encapsulated inside a poly(vinylpyrrolidone) (PVP) -stabilized polystyrene core in different concentrations to create 5-plex arrays in two separate detection channels of a cytometer. A subsequent modification of the silica shell with an equimolar APTES/PEGS (aminopropyltriethoxysilane/polyethylene glycol silane) blend added multifunctional properties to the hybrid core/shell microparticles in a single step: APTES provides amino groups for the attachment of a caffeine derivative (as a hapten) to create antigen-coupled microspheres; the PEG moiety effectively suppresses nonspecific binding of antibodies, endowing the surface with antifouling properties. The particles were applied in a competitive fluorescence immunoassay in suspension, and a highly selective wash-free assay for the detection of caffeine in beverages was developed as a proof of concept.

Real-time tracking of complex ubiquitination cascades using a fluorescent confocal on-bead assay.

BACKGROUND: The ubiquitin-proteasome system (UPS) controls the stability, localization and/or activity of the proteome. However, the identification and characterization of complex individual ubiquitination cascades and their modulators remains a challenge. Here, we report a broadly applicable, multiplexed, miniaturized on-bead technique for real-time monitoring of various ubiquitination-related enzymatic activities. The assay, termed UPS-confocal fluorescence nanoscanning (UPS-CONA), employs a substrate of interest immobilized on a micro-bead and a fluorescently labeled ubiquitin which, upon enzymatic conjugation to the substrate, is quantitatively detected on the bead periphery by confocal imaging. RESULTS: UPS-CONA is suitable for studying individual enzymatic activities, including various E1, E2, and HECT-type E3 enzymes, and for monitoring multi-step reactions within ubiquitination cascades in a single experimental compartment. We demonstrate the power of the UPS-CONA technique by simultaneously following ubiquitin transfer from Ube1 through Ube2L3 to E6AP. We applied this multi-step setup to investigate the selectivity of five ubiquitination inhibitors reportedly targeting different classes of ubiquitination enzymes. Using UPS-CONA, we have identified a new activity of a small molecule E2 inhibitor, BAY 11-7082, and of a HECT E3 inhibitor, heclin, towards the Ube1 enzyme. CONCLUSIONS: As a sensitive, quantitative, flexible, and reagent-efficient method with a straightforward protocol, UPS-CONA constitutes a powerful tool for interrogation of ubiquitination-related enzymatic pathways and their chemical modulators, and is readily scalable for large experiments.

Multiplex Analysis on a Single Porous Hydrogel Bead with Encoded SERS Nanotags.

Bead-based assays have drawn more and more attention in biomedical fields. Herein, we proposed a novel approach to achieve multiplex analysis on a single porous hydrogel bead (PHB) with Raman dyes (RDs) encoded core-shell surface-enhanced Raman scattering (SERS) nanotags. Because of the amplified signal of RDs by core-shell metal structure of the nanotag and the high surface area to volume ratio (SVR) of the PHB, the sensitivity and linear dynamic range (LDR) of the as-proposed multiplex analysis method are significantly improved. We anticipate this approach to be widely used in the multiplex assays and biosensors.

Quantification of equine immunoglobulin A in serum and secretions by a fluorescent bead-based assay.

Only few quantitative reports exist about the concentrations and induction of immunoglobulin A (IgA) in mucosal secretions of horses. Despite this, it is widely assumed that IgA is the predominant immunoglobulin on mucosal surfaces in the horse. Here, two new monoclonal antibodies (mAbs) against equine IgA, clones 84-1 and 161-1, were developed and characterized in detail. Both IgA mAbs specifically bound monomeric and dimeric equine IgA in different applications, such as Western blots and fluorescent bead-based assays. Cross-reactivity with other equine immunoglobulin isotypes was not observed. The new IgA mAb 84-1 was used in combination with the previously characterized anti-equine IgA mAb BVS2 for the development and validation of a fluorescent bead-based assay to quantify total IgA in equine serum and various secretions. The IgA assay's linear detection ranged from 64pg/ml to 1000ng/ml. For the quantification of IgA in serum or in secretions an IgA standard was purified from serum or nasal wash fluid (secretory IgA), respectively. The different standards were needed for accurate IgA quantification in the respective samples taking the different signal intensities of monomeric and dimeric IgA on the florescent bead-based assay into account. IgA was quantified by the bead-based assay established here in different equine samples of healthy adult individuals. In serum the median total IgA was 0.45mg/ml for Thoroughbred horses (TB, n=10) and 1.16mg/ml in Icelandic horses (ICH, n=12). In nasopharyngeal secretions of TB (n=7) 0.13mg/ml median total IgA was measured, and 0.25mg/ml for ICH (n=12). Saliva of ICH (n=6) contained a median of 0.15mg/ml, colostrum of Warmbloods (n=8) a median of 1.89mg/ml IgA. Compared to IgG1 and IgG4/7 quantified in the same samples, IgA appeared as the major immunoglobulin isotype in nasopharyngeal secretions and saliva while it is a minor isotype in serum and colostrum. The newly developed monoclonal antibodies against equine IgA and the resulting bead-based assay for quantification of total IgA can notably improve the evaluation of mucosal immunity in horses.

Comparison of T24H-his, GST-T24H and GST-Ts8B2 recombinant antigens in western blot, ELISA and multiplex bead-based assay for diagnosis of neurocysticercosis.

BACKGROUND: Currently, the reference standard assay for the serodiagnosis of neurocysticercosis (NCC) is the lentil lectin-bound glycoproteins/enzyme-linked immunoelectrotransfer blot (LLGP-EITB). The main disadvantage of this technique is the complexity of obtaining and purifying the LLGP extract. This could be solved by replacement with highly specific recombinant antigens from Taenia solium. Based on previous studies, we selected and produced the recombinant Ts8B2 and T24H proteins and applied them to three diagnostic techniques: western blot (WB), enzyme-linked immunosorbent assay (ELISA) and the multiplex bead-based assay (MBA). METHODS: The Ts8B2 and T24H cDNA sequences were expressed in a prokaryotic system and the corresponding expression products purified; three recombinant proteins were further characterized: T24H-his, GST-T24H and GST-Ts8B2. The proteins on WB, ELISA and MBA were tested against 149 sera from patients with NCC confirmed by brain imaging, 40 sera from patients with other parasitic diseases, and 131 sera from US. individuals without evidence of neurocysticercosis (clinical/serological/brain imaging). The sensitivity and specificity of each antigen by WB were calculated by counting the number of true positive, false positive, true negative and false negative results. Using the receiver operating characteristic (ROC) curves, the cut-off values for the ELISA and MBA were established as well as the sensitivity and specificity of each assay. RESULTS: All three antigens showed a high sensitivity on WB in active NCC cases with two or more viable cysts and low sensitivity for cases with single viable cyst or calcified lesions and inactive NCC. WB showed the highest specificity and sensitivity out of the three diagnostic techniques. The recombinant T24H-his was the best diagnostic reagent in WB (100% sensitivity, 99.4% specificity), exhibiting similar results to the LLGP-EITB, against the same panel of NCC sera. The GST-T24H antigen worked better than the others in ELISA and MBA protocols (88.3 and 96.1% sensitivity, respectively and 96.5% specificity). CONCLUSIONS: The sensitivity and specificity that we obtained were similar to results from a previous study using a similar recombinant antigen (rT24H), suggesting that recombinant antigens may be good alternatives to crude extracts in a variety of diagnostic techniques. Furthermore, these antigens can be applied in the development of point-of-care tests which would be useful in NCC field studies.

Incorporating Aptamers in the Multiple Analyte Profiling Assays (xMAP): Detection of C-Reactive Protein.

Aptamers are short oligonucleotide sequences used in detection systems because of their high affinity binding to a variety of macromolecules. With the introduction of aptamers over 25 years ago came the exploration of their use in many different applications as a substitute for antibodies. Aptamers have several advantages; they are easy to synthesize, can bind to analytes for which it is difficult to obtain antibodies, and in some cases bind better than antibodies. As such, aptamer applications have significantly expanded as an adjunct to a variety of different immunoassay designs. The Multiple-Analyte Profiling (xMAP) technology developed by Luminex Corporation commonly uses antibodies for the detection of analytes in small sample volumes through the use of fluorescently coded microbeads. This technology permits the simultaneous detection of multiple analytes in each sample tested and hence could be applied in many research fields. Although little work has been performed adapting this technology for use with apatmers, optimizing aptamer-based xMAP assays would dramatically increase the versatility of analyte detection. We report herein on the development of an xMAP bead-based aptamer/antibody sandwich assay for a biomarker of inflammation (C-reactive protein or CRP). Protocols for the coupling of aptamers to xMAP beads, validation of coupling, and for an aptamer/antibody sandwich-type assay for CRP are detailed. The optimized conditions, protocols and findings described in this research could serve as a starting point for the development of new aptamer-based xMAP assays.