Measurement of Cetuximab and Panitumumab-Unbound Serum EGFR Extracellular Domain Using an Assay Based on Slow Off-Rate Modified Aptamer (SOMAmer) Reagents

Background

Response to cetuximab (Erbitux®) and panitumumab (Vectibix®) varies among individuals, and even those who show response ultimately gain drug resistance. One possible etiologic factor is differential interaction between the drug and target. We describe the development of an assay based on Slow Off-rate Modified Aptamer (SOMAmer^™) reagents that can distinguish drug-bound from unbound epidermal growth factor receptor (EGFR).

Methods

This quantitative assay uses a SOMAmer reagent specific for EGFR extracellular domain (ECD) as a capturing reagent. Captured SOMAmer is quantitated using PCR. Linearity and accuracy (recovery) of the assay were assessed using normal sera and purified EGFR ECD.

Results

This EGFR ECD assay showed linearity between 2.5 and 600 ng/mL. Average recovery was 101%. The assay detected EGFR but showed little cross-reactivity to other ErbB proteins: 0.4% for ErbB2, 6.9% for ErbB3, and 1.3% for ErbB4. Preincubation of normal serum with either cetuximab or panitumumab resulted in a dose-dependent decrease in EGFR ECD levels measured using the SOMAmer assay; preincubation did not affect measurement with an ELISA.

Conclusions

This SOMAmer-based serum EGFR ECD assay accurately and specifically measures EGFR in serum. Detection of significant amounts of drug-unbound EGFR in patients undergoing cetuximab or panitumumab treatment could be an indicator of poor drug response. Further studies are needed to evaluate the utility of the assay as an indicator of drug efficacy or as a guide to dosing.     

Antibody to Ricin A Chain Hinders Intracellular Routing of Toxin and Protects Cells Even after Toxin Has Been Internalized

Background

Mechanisms of antibody-mediated neutralization are of much interest. For plant and bacterial A-B toxins, A chain mediates toxicity and B chain binds target cells. It is generally accepted and taught that antibody (Ab) neutralizes by preventing toxin binding to cells. Yet for some toxins, ricin included, anti-A chain Abs afford greater protection than anti-B. The mechanism(s) whereby Abs to the A chain neutralize toxins are not understood.

Methodology/Principal Findings

We use quantitative confocal imaging, neutralization assays, and other techniques to study how anti-A chain Abs function to protect cells. Without Ab, ricin enters cells and penetrates to the endoplasmic reticulum within 15 min. Within 45–60 min, ricin entering and being expelled from cells reaches equilibrium. These results are consistent with previous observations, and support the validity of our novel methodology. The addition of neutralizing Ab causes ricin accumulation at the cell surface, delays internalization, and postpones retrograde transport of ricin. Ab binds ricin for >6hr as they traffic together through the cell. Ab protects cells even when administered hours after exposure.

Conclusions/Key Findings

We demonstrate the dynamic nature of the interaction between the host cell and toxin, and how Ab can alter the balance in favor of the cell. Ab blocks ricin’s entry into cells, hinders its intracellular routing, and can protect even after ricin is present in the target organelle, providing evidence that the major site of neutralization is intracellular. These data add toxins to the list of pathogenic agents that can be neutralized intracellularly and explain the in vivo efficacy of delayed administration of anti-toxin Abs. The results encourage the use of post-exposure passive Ab therapy, and show the importance of the A chain as a target of Abs.     

Paracrine IL-33 stimulation enhances lipopolysaccharide-mediated macrophage activation

Background

IL-33, a member of the IL-1 family of cytokines, provokes Th2-type inflammation accompanied by accumulation of eosinophils through IL-33R, which consists of ST2 and IL-1RAcP. We previously demonstrated that macrophages produce IL-33 in response to LPS. Some immune responses were shown to differ between ST2-deficient mice and soluble ST2-Fc fusion protein-treated mice. Even in anti-ST2 antibody (Ab)-treated mice, the phenotypes differed between distinct Ab clones, because the characterization of such Abs (i.e., depletion, agonistic or blocking Abs) was unclear in some cases.

Methodology/Principal Findings

To elucidate the precise role of IL-33, we newly generated neutralizing monoclonal Abs for IL-33. Exogenous IL-33 potentiated LPS-mediated cytokine production by macrophages. That LPS-mediated cytokine production by macrophages was suppressed by inhibition of endogenous IL-33 by the anti-IL-33 neutralizing mAbs.

Conclusions/Significance

Our findings suggest that LPS-mediated macrophage activation is accelerated by macrophage-derived paracrine IL-33 stimulation.     

Common and Distinct Clinical Features in Adult Patients with Anti-Aminoacyl-tRNA Synthetase Antibodies: Heterogeneity within the Syndrome

Objective

To identify similarities and differences in the clinical features of adult Japanese patients with individual anti-aminoacyl-tRNA synthetase antibodies (anti-ARS Abs).

Methods

This was a retrospective analysis of 166 adult Japanese patients with anti-ARS Abs detected by immunoprecipitation assays. These patients had visited Kanazawa University Hospital or collaborating medical centers from 2003 to 2009.

Results

Anti-ARS Ab specificity included anti-Jo-1 (36%), anti-EJ (23%), anti-PL-7 (18%), anti-PL-12 (11%), anti-KS (8%), and anti-OJ (5%). These anti-ARS Abs were mutually exclusive, except for one serum Ab that had both anti-PL-7 and PL-12 reactivity. Myositis was closely associated with anti-Jo-1, anti-EJ, and anti-PL-7, while interstitial lung disease (ILD) was correlated with all 6 anti-ARS Abs. Dermatomyositis (DM)-specific skin manifestations (heliotrope rash and Gottron’s sign) were frequently observed in patients with anti-Jo-1, anti-EJ, anti-PL-7, and anti-PL-12. Therefore, most clinical diagnoses were polymyositis or DM for anti-Jo-1, anti-EJ, and anti-PL-7; clinically amyopathic DM or ILD for anti-PL-12; and ILD for anti-KS and anti-OJ. Patients with anti-Jo-1, anti-EJ, and anti-PL-7 developed myositis later if they had ILD alone at the time of disease onset, and most patients with anti-ARS Abs eventually developed ILD if they did not have ILD at disease onset.

Conclusion

Patients with anti-ARS Abs are relatively homogeneous. However, the distribution and timing of myositis, ILD, and rashes differ among patients with individual anti-ARS Abs. Thus, identification of individual anti-ARS Abs is beneficial to define this rather homogeneous subset and to predict clinical outcomes within the “anti-synthetase syndrome.”     

Sensitive detection of multiplex toxins using antibody microarray

Using a newly developed fluorescent nanoparticle (NP) that gives rise to a high-intensity and stable fluorescent light, a sensitive antibody (Ab) microarray assay system has been developed for specific detection of bioterrorism agents, as exemplified by ricin, cholera toxin (CT), and staphylococcal enterotoxin B (SEB). The Ab microarray uses a sandwich format that consists of capture Abs, analytes (toxins), biotinylated detection Abs, and avidin-conjugated NP. In all three cases, polyclonal Abs (pAbs) displayed superiority over monoclonal antibodies (mAbs) in capturing toxins on microarray slides even when the pAbs and mAbs had similar affinity as determined by enzyme-linked immunosorbent assay (ELISA). The detection system was successfully used to detect toxins spiked in milk, apple cider, and blood samples. We were able to detect ricin at 100 pg/ml in buffer and at 1 ng/ml in spiked apple cider or milk, whereas CT and SEB were detected at 10 pg/ml in buffer and 100 pg/ml in spiked apple cider or milk. High specificities were also demonstrated in the detection of mixed toxin samples with similar sensitivities. The matrix effect of blood samples on the detection of mixed toxins seems to be minimal when the toxin concentration is at or above 100 ng/ml. The current study highlights the significant role of pAb and NP in increasing selectivity and sensitivity of toxin detection in a microarray format.     

Evaluation of high-throughput PCR and microarray-based assay in conjunction with automated DNA extraction instruments for diagnosis of sepsis

Background

High incidence of septic patients increases the pressure of faster and more reliable bacterial identification methods to adapt patient management towards focused and effective treatment options. The aim of this study was to assess two automated DNA extraction solutions with the PCR and microarray-based assay to enable rapid and reliable detection and speciation of causative agents in the diagnosis of sepsis.

Methodology/Principal Findings

We evaluated two automated DNA instruments NucliSENS® easyMAG® and NorDiag Arrow for the preparation of blood culture samples. A set of 91 samples flagged as positive during incubation was analyzed prospectively with the high-throughput generation of Prove-it™ Sepsis assay designed to identify over 60 Gram-negative and Gram-positive bacterial species as well as methicillin resistance marker from a blood culture. Bacterial findings were accurately reported from 77 blood culture samples, whereas 14 samples were reported as negative, containing bacteria not belonging to the pathogen panel of the assay. No difference was observed between the performance of NorDiag Arrow or NucliSENS® easyMAG® with regard to the result reporting of Prove-it™ Sepsis. In addition, we also assessed the quality and quantity of DNA extracted from the clinical Escherichia coli isolate with DNA extraction instruments. We observed only minor differences between the two instruments.

Conclusions

Use of automated and standardized sample preparation methods together with rapid, multiplex pathogen detection offers a strategy to speed up reliably the diagnostics of septic patients. Both tested DNA extraction devices were shown to be feasible for blood culture samples and the Prove-it™ Sepsis assay, providing an accurate identification of pathogen within 4,5 hours when the detected pathogen was in the repertoire of the test.     

Selected Reaction Monitoring (SRM) Analysis of Epidermal Growth Factor Receptor (EGFR) in Formalin Fixed Tumor Tissue

Background

Analysis of key therapeutic targets such as epidermal growth factor receptor (EGFR) in clinical tissue samples is typically done by immunohistochemistry (IHC) and is only subjectively quantitative through a narrow dynamic range. The development of a standardized, highly-sensitive, linear, and quantitative assay for EGFR for use in patient tumor tissue carries high potential for identifying those patients most likely to benefit from EGFR-targeted therapies.

Methods

A mass spectrometry-based Selected Reaction Monitoring (SRM) assay for the EGFR protein (EGFR-SRM) was developed utilizing the Liquid Tissue®-SRM technology platform. Tissue culture cells (n = 4) were analyzed by enzyme-linked immunosorbent assay (ELISA) to establish quantitative EGFR levels. Matching formalin fixed cultures were analyzed by the EGFR-SRM assay and benchmarked against immunoassay of the non-fixed cultured cells. Xenograft human tumor tissue (n = 10) of non-small cell lung cancer (NSCLC) origin and NSCLC patient tumor tissue samples (n = 23) were microdissected and the EGFR-SRM assay performed on Liquid Tissue lysates prepared from microdissected tissue. Quantitative curves and linear regression curves for correlation between immunoassay and SRM methodology were developed in Excel.

Results

The assay was developed for quantitation of a single EGFR tryptic peptide for use in FFPE patient tissue with absolute specificity to uniquely distinguish EGFR from all other proteins including the receptor tyrosine kinases, IGF-1R, cMet, Her2, Her3, and Her4. The assay was analytically validated against a collection of tissue culture cell lines where SRM analysis of the formalin fixed cells accurately reflects EGFR protein levels in matching non-formalin fixed cultures as established by ELISA sandwich immunoassay (R² = 0.9991). The SRM assay was applied to a collection of FFPE NSCLC xenograft tumors where SRM data range from 305amol/μg to 12,860amol/μg and are consistent with EGFR protein levels in these tumors as previously-reported by western blot and SRM analysis of the matched frozen tissue. In addition, the SRM assay was applied to a collection of histologically-characterized FFPE NSCLC patient tumor tissue where EGFR levels were quantitated from not detected (ND) to 670amol/μg.

Conclusions

This report describes and evaluates the performance of a robust and reproducible SRM assay designed for measuring EGFR directly in FFPE patient tumor tissue with accuracy at extremely low (attomolar) levels. This assay can be used as part of a complementary or companion diagnostic strategy to support novel therapies currently under development and demonstrates the potential to identify candidates for EGFR-inhibitor therapy, predict treatment outcome, and reveal mechanisms of therapeutic resistance.     

The Fluctuating Natural Course of CCSVI in MS Patients and Controls,a Prospective Follow-Up

Objectives

A new treatable venous disorder, chronic cerebrospinal venous insufficiency (CCSVI), has been proposed in patients with multiple sclerosis. The natural course of CCSVI has not been examined yet. This is crucial given the fact that surgical procedures are increasingly offered to MS patients to treat venous stenosis.

Methods

To document the natural course of venous haemodynamics we performed extra- and transcranial echo colour Doppler (ECD) in 52 multiple sclerosis patients and 28 healthy controls (HC) and re-examined this group after a median period of 16 weeks. The reexamination was done being blinded to the initial findings and the patients did not undergo any intervention.

Results

The ECD examination at baseline showed CCSVI in 5 (9.6%) of the 52 multiple sclerosis patients and 0 HC (P = 0.26). At follow-up the diagnosis CCSVI could not be reconfirmed in 3 out of 5 patients at follow-up, while 2 new CCSVI-positive multiple sclerosis patients were detected.

Conclusions

ECD examination shows a fluctuating natural course of the extracranial venous haemodynamics, which makes determination of CCSVI by ECD examination unreliable.     

Exacerbation of cigarette smoke-induced pulmonary inflammation by Staphylococcus aureus Enterotoxin B in mice

Background

Cigarette smoke (CS) is a major risk factor for the development of COPD. CS exposure is associated with an increased risk of bacterial colonization and respiratory tract infection, because of suppressed antibacterial activities of the immune system and delayed clearance of microbial agents from the lungs. Colonization with Staphylococcus aureus results in release of virulent enterotoxins, with superantigen activity which causes T cell activation.

Objective

To study the effect of Staphylococcus aureus enterotoxin B (SEB) on CS-induced inflammation, in a mouse model of COPD.

Methods

C57/Bl6 mice were exposed to CS or air for 4 weeks (5 cigarettes/exposure, 4x/day, 5 days/week). Endonasal SEB (10 μg/ml) or saline was concomitantly applied starting from week 3, on alternate days. 24 h after the last CS and SEB exposure, mice were sacrificed and bronchoalveolar lavage (BAL) fluid and lung tissue were collected.

Results

Combined exposure to CS and SEB resulted in a raised number of lymphocytes and neutrophils in BAL, as well as increased numbers of CD8⁺ T lymphocytes and granulocytes in lung tissue, compared to sole CS or SEB exposure. Moreover, concomitant CS/SEB exposure induced both IL-13 mRNA expression in lungs and goblet cell hyperplasia in the airway wall. In addition, combined CS/SEB exposure stimulated the formation of dense, organized aggregates of B- and T- lymphocytes in lungs, as well as significant higher CXCL-13 (protein, mRNA) and CCL19 (mRNA) levels in lungs.

Conclusions

Combined CS and SEB exposure aggravates CS-induced inflammation in mice, suggesting that Staphylococcus aureus could influence the pathogenesis of COPD.     

Systematic spatial bias in DNA microarray hybridization is caused by probe spot position-dependent variability in lateral diffusion

Background

The hybridization of nucleic acid targets with surface-immobilized probes is a widely used assay for the parallel detection of multiple targets in medical and biological research. Despite its widespread application, DNA microarray technology still suffers from several biases and lack of reproducibility, stemming in part from an incomplete understanding of the processes governing surface hybridization. In particular, non-random spatial variations within individual microarray hybridizations are often observed, but the mechanisms underpinning this positional bias remain incompletely explained.

Methodology/Principal Findings

This study identifies and rationalizes a systematic spatial bias in the intensity of surface hybridization, characterized by markedly increased signal intensity of spots located at the boundaries of the spotted areas of the microarray slide. Combining observations from a simplified single-probe block array format with predictions from a mathematical model, the mechanism responsible for this bias is found to be a position-dependent variation in lateral diffusion of target molecules. Numerical simulations reveal a strong influence of microarray well geometry on the spatial bias.

Conclusions

Reciprocal adjustment of the size of the microarray hybridization chamber to the area of surface-bound probes is a simple and effective measure to minimize or eliminate the diffusion-based bias, resulting in increased uniformity and accuracy of quantitative DNA microarray hybridization.     

Profiling of Humoral Response to Influenza A(H1N1)pdm09 Infection and Vaccination Measured by a Protein Microarray in Persons with and without History of Seasonal Vaccination

Background

The influence of prior seasonal influenza vaccination on the antibody response produced by natural infection or vaccination is not well understood.

Methods

We compared the profiles of antibody responses of 32 naturally infected subjects and 98 subjects vaccinated with a 2009 influenza A(H1N1) monovalent MF59-adjuvanted vaccine (Focetria®, Novartis), with and without a history of seasonal influenza vaccination. Antibodies were measured by hemagglutination inhibition (HI) assay for influenza A(H1N1)pdm09 and by protein microarray (PA) using the HA1 subunit for seven recent and historic H1, H2 and H3 influenza viruses, and three avian influenza viruses. Serum samples for the infection group were taken at the moment of collection of the diagnostic sample, 10 days and 30 days after onset of influenza symptoms. For the vaccination group, samples were drawn at baseline, 3 weeks after the first vaccination and 5 weeks after the second vaccination.

Results

We showed that subjects with a history of seasonal vaccination generally exhibited higher baseline titers for the various HA1 antigens than subjects without a seasonal vaccination history. Infection and pandemic influenza vaccination responses in persons with a history of seasonal vaccination were skewed towards historic antigens.

Conclusions

Seasonal vaccination is of significant influence on the antibody response to subsequent infection and vaccination, and further research is needed to understand the effect of annual vaccination on protective immunity.     

Utility of survival motor neuron ELISA for spinal muscular atrophy clinical and preclinical analyses

Objectives

Genetic defects leading to the reduction of the survival motor neuron protein (SMN) are a causal factor for Spinal Muscular Atrophy (SMA). While there are a number of therapies under evaluation as potential treatments for SMA, there is a critical lack of a biomarker method for assessing efficacy of therapeutic interventions, particularly those targeting upregulation of SMN protein levels. Towards this end we have engaged in developing an immunoassay capable of accurately measuring SMN protein levels in blood, specifically in peripheral blood mononuclear cells (PBMCs), as a tool for validating SMN protein as a biomarker in SMA.

Methods

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed and validated for measuring SMN protein in human PBMCs and other cell lysates. Protocols for detection and extraction of SMN from transgenic SMA mouse tissues were also developed.

Results

The assay sensitivity for human SMN is 50 pg/mL. Initial analysis reveals that PBMCs yield enough SMN to analyze from blood volumes of less than 1 mL, and SMA Type I patients'' PBMCs show ∼90% reduction of SMN protein compared to normal adults. The ELISA can reliably quantify SMN protein in human and mouse PBMCs and muscle, as well as brain, and spinal cord from a mouse model of severe SMA.

Conclusions

This SMN ELISA assay enables the reliable, quantitative and rapid measurement of SMN in healthy human and SMA patient PBMCs, muscle and fibroblasts. SMN was also detected in several tissues in a mouse model of SMA, as well as in wildtype mouse tissues. This SMN ELISA has general translational applicability to both preclinical and clinical research efforts.     

A Polyclonal Antibody Based Immunoassay Detects Seven Subtypes of Shiga Toxin 2 Produced by Escherichia coli in Human and Environmental Samples

Background

Shiga toxin-producing Escherichia coli (STEC) are frequent causes of severe human diseases ranging from diarrhea to hemolytic uremic syndrome. The existing strategy for detection of STEC relies on the unique sorbitol-negative fermentation property of the O157 strains, the most commonly identified serotype has been E. coli O157. It is becoming increasingly evident, however, that numerous non-O157 STEC serotypes also cause outbreaks and severe illnesses. It is necessary to have new methods that are capable of detecting all STEC strains.

Methods and Findings

Here we describe the development of a sandwich ELISA assay for detecting both O157 and non-O157 STECs by incorporating a novel polyclonal antibody (pAb) against Stx2. The newly established immunoassay was capable of detecting Stx2a spiked in environmental samples with a limit of detection between 10 and 100 pg/mL in soil and between 100 and 500 pg/mL in feces. When applied to 36 bacterial strains isolated from human and environmental samples, this assay detected Stx2 in all strains that were confirmed to be stx2-positive by real-time PCR, demonstrating a 100% sensitivity and specificity.

Conclusions

The sandwich ELISA developed in this study will enable any competent laboratory to identify and characterize Stx2-producing O157 and non-O157 strains in human and environmental samples, resulting in rapid diagnosis and patient care. The results of epitope mapping from this study will be useful for further development of a peptide-based antibody and vaccine.     

Repair of Abdominal Wall Defects with Biodegradable Laminar Prostheses: Polymeric or Biological?

Introduction

Biological and synthetic laminar absorbable prostheses are available for the repair of hernia defects in the abdominal wall. They share the important feature of being gradually degraded in the host, resulting in place the formation of a neotissue. This study was designed to assess the host tissue’s incorporation of collagen bioprostheses and a synthetic absorbable prosthesis.

Methods

Partial defects were created in the abdominal walls of 72 New Zealand rabbits and repaired using collagen bioprostheses Tutomesh® and Strattice® or a synthetic prosthesis Bio-A®. Specimens were collected for light microscopy, collagens gene and protein expression, macrophage response and biomechanical resistance at 14, 30, 90 and 180 days post-implantation.

Results

Tutomesh® and Bio-A® were gradually infiltrated by the host tissue and almost completely degraded by 180 days post-implantation. In contrast, Strattice® exhibited material encapsulation, no prosthetic degradation and low cell infiltration at earlier timepoints, whereas at later study time, collagen deposition could be observed within the mesh. In the short term, Bio-A® exhibited higher level of collagen 1 and 3 mRNA expression compared with the two other biological prostheses, which exhibited two peaks of higher expression at 14 and 90 days. The expression of collagen III was homogeneous throughout the study and collagen I deposition was more evident in Strattice®. Macrophage response decreased over time in biomeshes. However, in the synthetic mesh remained high and homogeneous until 90 days. The biomechanical analysis demonstrated the progressively increasing tensile strength of all biomaterials.

Conclusions

The tissue infiltration of laminar absorbable prostheses is affected by the structure and composition of the mesh. The synthetic prosthesis exhibited a distinct pattern of tissue incorporation and a greater macrophage response than did the biological prostheses. Of all of the laminar, absorbable biomaterials that were tested in this study, Strattice® demonstrated the optimal levels of integration and degradation.     

Evaluation of the human IgG antibody response to Aedes albopictus saliva as a new specific biomarker of exposure to vector bites

Background

The spread of Aedes albopictus, a vector for re-emergent arbovirus diseases like chikungunya and dengue, points up the need for better control strategies and new tools to evaluate transmission risk. Human antibody (Ab) responses to mosquito salivary proteins could represent a reliable biomarker for evaluating human-vector contact and the efficacy of control programs.

Methodology/Principal Findings

We used ELISA tests to evaluate specific immunoglobulin G (IgG) responses to salivary gland extracts (SGE) in adults exposed to Aedes albopictus in Reunion Island. The percentage of immune responders (88%) and levels of anti-SGE IgG Abs were high in exposed individuals. At an individual level, our results indicate heterogeneity of the exposure to Aedes albopictus bites. In addition, low-level immune cross-reactivity between Aedes albopictus and Aedes aegypti SGEs was observed, mainly in the highest responders.

Conclusion/Significance

Ab responses to saliva could be used as an immuno-epidemiological tool for evaluating exposure to Aedes albopictus bites. Combined with entomological and epidemiological methods, a “salivary” biomarker of exposure to Aedes albopictus could enhance surveillance of its spread and the risk of arbovirus transmission, and could be used as a direct tool for the evaluation of Aedes albopictus control strategies.     

Ebolavirus Nucleoprotein C-Termini Potently Attract Single Domain Antibodies Enabling Monoclonal Affinity Reagent Sandwich Assay (MARSA) Formulation

Background

Antigen detection assays can play an important part in environmental surveillance and diagnostics for emerging threats. We are interested in accelerating assay formulation; targeting the agents themselves to bypass requirements for a priori genome information or surrogates. Previously, using in vitro affinity reagent selection on Marburg virus we rapidly established monoclonal affinity reagent sandwich assay (MARSA) where one recombinant antibody clone was both captor and tracer for polyvalent nucleoprotein (NP). Hypothesizing that the closely related Ebolavirus genus may share the same Achilles'' heel, we redirected the scheme to see whether similar assays could be delivered and began to explore their mechanism.

Methods and Findings

In parallel we selected panels of llama single domain antibodies (sdAb) from a semi-synthetic library against Zaire, Sudan, Ivory Coast, and Reston Ebola viruses. Each could perform as both captor and tracer in the same antigen sandwich capture assay thereby forming MARSAs. All sdAb were specific for NP and those tested required the C-terminal domain for recognition. Several clones were cross-reactive, indicating epitope conservation across the Ebolavirus genus. Analysis of two immune shark sdAb revealed they also targeted the C-terminal domain, and could be similarly employed, yet were less sensitive than a comparable llama sdAb despite stemming from immune selections.

Conclusions

The C-terminal domain of Ebolavirus NP is a strong attractant for antibodies and enables sensitive sandwich immunoassays to be rapidly generated using a single antibody clone. The polyvalent nature of nucleocapsid borne NP and display of the C-terminal region likely serves as a bountiful affinity sink during selections, and a highly avid target for subsequent immunoassay capture. Combined with the high degree of amino acid conservation through 37 years and across wide geographies, this domain makes an ideal handle for monoclonal affinity reagent driven antigen sandwich assays for the Ebolavirus genus.     

TNFAIP3 Facilitates Degradation of Microbial Antigen SEB in Enterocytes

Background and Aims

The enterocytes have the potential to absorb noxious substances, such as microbial products, from the gut lumen. How the enterocytes process the substances to harmless materials is not fully understood. This study aims to elucidate the role of ubiquitin E3 ligase TNFAIP3 (TNFAIP3) in facilitating the degradation of endocytic microbial products in enterocytes.

Methods

Human intestinal epithelial cell line, HT-29 cells, was cultured to monolayers using as an in vitro model to observe the endocytosis and degradation of microbial products, Staphylococcal enterotoxin B (SEB) in epithelial cells. The RNA interference was employed to knock down the TNFAIP3 gene in HT-29 cells to observe the role of TNFAIP3 in the degradation of endocytic SEB. The role of TNFAIP3 in facilitating the endosome/lysosome fusion was observed by immunocytochemistry.

Results

Upon the absorption of SEB, the expression of TNFAIP3 was increased in HT-29 cells. Silencing the TNFAIP3 gene in HT-29 cells resulted in a large quantity of SEB to be transported across the HT-29 monolayers to the transwell basal chambers; the transportation was via the intracellular pathway. TNFAIP3 was required in the fusion of SEB-carrying endosomes and lysosomes.

Conclusions

TNFAIP3 plays a critical role in the degradation of endocytic SEB in enterocytes.     

Analytical Performance of the Roche LightCycler? Mycobacterium Detection Kit for the Diagnosis of Clinically Important Mycobacterial Species

Background

The LightCycler® Mycobacterium Detection Kit based on real-time PCR technology for the detection of Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium kansasii was recently developed. This study evaluated its analytical sensitivity, specificity and reproducibility.

Methodology/Principal Findings

Plasmid standards were prepared and used to determine the limit of detection. The assay was also performed against organisms other than mycobacteria, other mycobacterial strains and interfering substances to exclude cross-reactivity and interference. Reference standards were prepared and tested to assess the assay''s reproducibility. All PCR assays were performed using the LightCycler® 2.0 Instrument. The detection limit for M. tuberculosis was 28 copies per microlitre. Neither cross-reactivity nor interference occurred with non-mycobacterial organisms and substances tested. Overall reproducibility for consecutive measurements, run-to-run, lot-to-lot, day-to-day and laboratory-to-laboratory achieved a coefficient of variance of less than two percent.

Significance

The LightCycler® Mycobacterium Detection kit has shown to be a robust and accurate assay with the potential to be used as a rapid TB diagnostic test.