µFBI: A Microfluidic Bead-Based Immunoassay for Multiplexed Detection of Proteins from a µL Sample Volume

BACKGROUND: Over the last ten years, miniaturized multiplexed immunoassays have become robust, reliable research tools that enable researchers to simultaneously determine a multitude of parameters. Among the numerous analytical protein arrays available, bead-based assay systems have evolved into a key technology that enables the quantitative protein profiling of biological samples whilst requiring only a minimal amount of sample material. METHODOLOGY/PRINCIPAL FINDINGS: A microfluidic bead-based immunoassay, µFBI, was developed to perform bead-based multiplexed sandwich immunoassays in a capillary. This setup allows the simultaneous detection of several parameters and only requires 200 ng of tissue lysate in a 1 µL assay volume. In addition, only 1 µL of detection antibodies and 1 µL of the reporter molecule Streptavidin-Phycoerythrin were required. The µFBI was used to compare the expression of seven receptor tyrosine kinases and their degree of tyrosine phosphorylation in breast cancer tissue and in normal tissue lysates. The total amount of HER-2, as well the degree of tyrosine phosphorylation was much higher in breast cancer tissue than in normal tissue. µFBI and a standard bead-based assay led to identical protein expression data. Moreover, it was possible to reduce the quantity of sample material required by a factor of 100 and the quantity of reagents by a factor of 30. CONCLUSIONS/SIGNIFICANCE: The µFBI, microfluidic bead-based immunoassay, allows the analysis of multiple parameters from a very small amount of sample material, such as tumor biopsies or tissue sections

Performance of a Multiplex Serological Helicobacter pylori Assay on a Novel Microfluidic Assay Platform

International audienceInfection with Helicobacter pylori (H. pylori) occurs in 50% of the world population, and is associated with the development of ulcer and gastric cancer. Serological diagnostic tests indicate an H. pylori infection by detecting antibodies directed against H. pylori proteins. In addition to line blots, multiplex assay platforms provide smart solutions for the simultaneous analysis of antibody responses towards several H. pylori proteins. We used seven H. pylori proteins (FliD, gGT, GroEL, HpaA, CagA, VacA, and HP0231) and an H. pylori lysate for the development of a multiplex serological assay on a novel microfluidic platform. The reaction limited binding regime in the microfluidic channels allows for a short incubation time of 35 min. The developed assay showed very high sensitivity (99%) and specificity (100%). Besides sensitivity and specificity, the technical validation (intra-assay CV = 3.7 ± 1.2% and inter-assay CV = 5.5 ± 1.2%) demonstrates that our assay is also a robust tool for the analysis of the H. pylori-specific antibody response. The integration of the virulence factors CagA and VacA allow for the assessment of the risk for gastric cancer development. The short assay time and the performance of the platform shows the potential for implementation of such assays in a clinical setting

Development of a Bead-Based Multiplex Assay for the Analysis of the Serological Response against the Six Pathogens HAV, HBV, HCV, CMV, T. gondii, and H. pylori

The spread of infectious diseases and vaccination history are common subjects of epidemiological and immunological research studies. Multiplexed serological assays are useful tools for assessing both current and previous infections as well as vaccination efficacy. We developed a serological multi-pathogen assay for hepatitis A, B and C virus, cytomegalovirus (CMV), Toxoplasma gondii, and Helicobacter pylori using a bead-based multiplex assay format. The multi-pathogen assay consisting of 15 antigens was utilized for the analysis of the serological response in elderly individuals of an influenza vaccination study (n = 34). The technical assay validation revealed a mean intra-assay precision of coefficient of variation (CV) = 3.2 ± 1.5% and a mean inter-assay precision of CV = 8.2 ± 5.3% across all 15 antigens and all tested samples, indicating a robust test system. Furthermore, the assay shows high sensitivities (ranging between 94% and 100%) and specificities (ranging between 93% and 100%) for the different pathogens. The highest seroprevalence rates in our cohort were observed for hepatitis A virus (HAV; 73.5%), followed by CMV (70.6%), T. gondii (67.6%) and H. pylori (32.4%). Seroprevalences for hepatitis B virus (HBV, 8.8%) and hepatitis C virus (HCV, 0%) were low. The seroprevalences observed in our study were similar to those from other population-based studies in Germany. In summary, we conclude that our multiplex serological assay represents a suitable tool for epidemiological studies

Serologic Responses in Childhood Pulmonary Tuberculosis.

BACKGROUND: Identification of the Mycobacterium tuberculosis immunoproteome and antigens associated with serologic responses in adults has renewed interest in developing a serologic test for childhood tuberculosis (TB). We investigated IgG antibody responses against M. tuberculosis antigens in children with well-characterized TB. METHODS: We studied archived sera obtained from hospitalized children with suspected pulmonary TB, and classified as having confirmed TB (culture-confirmed), unlikely TB (clinical improvement without TB treatment), or unconfirmed TB (all others). A multiplexed bead-based assay for IgG antibodies against 119 M. tuberculosis antigens was developed, validated and used to test sera. The area under the curves (AUCs) of the empiric receiver-operator characteristic curves were generated as measures of predictive ability. A cross-validated generalized linear model was used to select the most predictive combinations of antigens. RESULTS: For the confirmed TB versus unlikely TB comparison, the maximal single antigen AUC was 0.63, corresponding to sensitivity 0.60 and specificity 0.60. Older (age: 60+ months old) children's responses were better predictive of TB status than younger (age: 12-59 months old) children's, with a maximal single antigen AUC of -0.76. For the confirmed TB versus unlikely TB groups, the most predictive combinations of antigens assigned TB risk probabilities of 0.33 and 0.33, respectively, when all ages were considered, and 0.57 (interquartile range: 0.48-0.64) and 0.35 (interquartile range: 0.32-0.40) when only older children were considered. CONCLUSION: An antigen-based IgG test is unlikely to meet the performance characteristics required of a TB detection test applicable to all age groups

Serum Inflammatory Profile for the Discrimination of Clinical Subtypes in Parkinson's Disease

Background: Blood levels of immune markers have been proposed to discriminate patients with Parkinson's disease (PD) from controls. However, differences between clinical PD subgroups regarding these markers still need to be identified. Objective: To investigate whether clinical phenotypes can be predicted by the assessment of immune marker profiles in the serum of PD patients. Methods: Phenotypes of clinical PD from Tübingen, Germany (n = 145) and Toronto, Canada (n = 90) were defined regarding clinical subtype, disease onset, severity, and progression as well as presence of cognitive and/or autonomic dysfunction. A panel of serum immune markers was assessed using principal component analysis (PCA) and regression models to define the marker(s) that were associated with clinical phenotypes after adjusting for potential confounders. Findings of both centers were compared for validation. Further, a [18F] FEPPA-PET was performed in a group of patients with high and low values of candidate markers for the assessment of in vivo brain microglial activation. Results: Overall, serum immune markers did not cluster to define a pro/anti-inflammatory profile in PCA. Out of 25 markers only IL-12p40 showed a trend to discriminate between PD subgroups in both cohorts which could not be replicated by [18F] FEPPA-PET. Conclusions: Assessment of cytokines in serum does not reliably differentiate clinical PD subtypes. Accompanying subtype-irrelevant inflammation in PD, dual activity, and lack of specificity of the immune markers, the complex function of microglia, probable effects of treatment, disease stage, and progression on inflammation as well as current technical limitations may limit the usefulness of serum immune markers for the differentiation of subtypes

Inflammatory profile in LRRK2-associated prodromal and clinical PD

BACKGROUND There is evidence for a relevant role of inflammation in the pathogenesis of Parkinson's disease (PD). Mutations in the LRRK2 gene represent the most frequent genetic cause for autosomal dominant PD. LRRK2 is highly expressed in macrophages and microglia suggesting an involvement in inflammatory pathways. The objectives are to test (1) whether idiopathic PD and LRRK2-associated PD share common inflammatory pathways or present distinct profiles and (2) whether non-manifesting LRRK2 mutation carriers present with similar aspects of inflammatory profiles as seen in PD-affected patients. METHODS We assessed serum profiles of 23 immune-associated markers and the brain-derived neurotrophic factor in 534 individuals from the MJFF LRRK2 consortium. RESULTS A large proportion of inflammatory markers were gender-dependent. Both PD-affected cohorts showed increased levels of the pro-inflammatory marker fatty-acid-binding protein. Additionally, idiopathic PD but not LRRK2-associated PD patients showed increased levels of the pro-inflammatory marker interleukin-12-p40 as well as the anti-inflammatory species interleukin-10, brain-derived neurotrophic factor, and stem cell factor. Non-manifesting LRRK2 mutation carriers including those with prodromal characteristics of PD presented with control-like inflammatory profiles. CONCLUSIONS Concomitant inflammation seems to be associated with idiopathic and LRRK2-associated PD. Identifying PD patients in whom inflammatory processes play a major role in their pathophysiology might offer a new therapeutic window at least for a subgroup of patients. Since non-manifesting LRRK2 mutation carriers with symptoms of the prodromal phase of PD did not show inflammatory profiles, activation of the immune system seems not an early event in the disease cascade

Serum Inflammatory Profile for the Discrimination of Clinical Subtypes in Parkinson's Disease

Background: Blood levels of immune markers have been proposed to discriminate patients with Parkinson's disease (PD) from controls. However, differences between clinical PD subgroups regarding these markers still need to be identified.Objective: To investigate whether clinical phenotypes can be predicted by the assessment of immune marker profiles in the serum of PD patients.Methods: Phenotypes of clinical PD from Tübingen, Germany (n = 145) and Toronto, Canada (n = 90) were defined regarding clinical subtype, disease onset, severity, and progression as well as presence of cognitive and/or autonomic dysfunction. A panel of serum immune markers was assessed using principal component analysis (PCA) and regression models to define the marker(s) that were associated with clinical phenotypes after adjusting for potential confounders. Findings of both centers were compared for validation. Further, a [18F] FEPPA-PET was performed in a group of patients with high and low values of candidate markers for the assessment of in vivo brain microglial activation.Results: Overall, serum immune markers did not cluster to define a pro/anti-inflammatory profile in PCA. Out of 25 markers only IL-12p40 showed a trend to discriminate between PD subgroups in both cohorts which could not be replicated by [18F] FEPPA-PET.Conclusions: Assessment of cytokines in serum does not reliably differentiate clinical PD subtypes. Accompanying subtype-irrelevant inflammation in PD, dual activity, and lack of specificity of the immune markers, the complex function of microglia, probable effects of treatment, disease stage, and progression on inflammation as well as current technical limitations may limit the usefulness of serum immune markers for the differentiation of subtypes

Nitic oxide-evoked accumulation of the tumorsuppressor p53 - molecular mechanism

p53 und NO sind zwei Moleküle mit großer Bedeutung für den menschlichen Organismus. Beide beeinflussen den Zellzyklus und sind entweder Auslöser (NO) oder Vermittler der Apoptose. Seit Beginn der 90er Jahre ist bekannt, dass der Tumorsuppressor p53 unter Einfluss von NO akkumuliert. Dabei spielt es keine Rolle, ob NO exogen über NO-Donoren oder durch Stimulation endogener NO-Synthasen zugeführt wird. In der vorliegenden Arbeit wurde der molekulare Mechanismus der NO-vermittelten Akkumulation von p53 näher untersucht. Im Fokus der Studien stand die Degradation des Tumorsuppressors durch das Ubiquitin-Proteasom-System sowie dessen nukleär-cytoplasmatischer Transport. Es ist bekannt, dass p53 vorrangig über das Ubiquitin-Proteasom-System abgebaut wird. Dabei ist die Interaktion von p53 mit der E3-Ligase Mdm2 für seine Ubiquitinierung unerlässlich. Durch Co-Immunopräzipitationsversuche konnte gezeigt werden, dass p53 auch nach NO-Stimulation noch mit Mdm2 wechselwirkt. Dies ist interessanterweise trotz Phosphorylierung von p53 an Serin 15 der Fall, und steht im Gegensatz zu Daten, die auf einen Abbruch der Interaktion zwischen p53 und Mdm2 nach Serin 15-Phosphorylierung hinweisen. Ein Einfluss auf die Ubiquitinierung von exogenem HA-p53 und auch endogenem p53, wie er durch UV-Bestrahlung oder Hemmung des Proteasoms zu verzeichnen ist, konnte nach NO-Stimulation nicht festgestellt werden. Da weder die Bindung an Mdm2 noch die Ubiquitinierung von p53 nach NO-Stimulation beeinträchtigt war, wurde ein Einfluss der Serin 15-Phosphorylierung von p53 auf den nukleären Export vermutet. Durch Heterokaryon-Analysen konnte gezeigt werden, dass Serin 15-phosphoryliertes p53 nicht mehr in der Lage ist, den Kern zu verlassen. Aufgrund des gehemmten nukleären Exports von p53, in Korrelation mit der Phosphorylierung an Serin 15, kann dieses nicht mehr ins Cytoplasma gelangen, um dort vom Proteasom degradiert zu werden.p53 and nitric oxide (NO) are two molecules with a high impact for the human organism. Both are influencing the cell cycle and induce/mediate apoptosis. Since the early nineties, it is known that the tumorsuppressor p53 accumulates in different cell systems under the impact of NO. Thus, it does not make any difference if NO is applied exogenously by NO-donors or generated endogenously via stimulation/induction of NO-synthases. In the present work the molecular mechanism of p53 accumulation under the impact of NO was examined in detail. In this study the degradation of p53 via the ubiquitin-proteasome system and the nucleo-cytoplasmic transport were focused. It is established that p53 is mainly degraded through the ubiquitin-proteasome system. Therefore, the interaction and ubiquitination of p53 through its E3-ligase Mdm2 is an important step. Interestingly, interaction between p53 and Mdm2 is not influenced by NO as shown by co-immunoprecipitation experiments. Moreover, p53 is found phosphorylated on serine 15 which is in contrast to data showing that this phosphorylation may block Mdm2/p53-interaction. An influence on the ubiquitination of exogenous HA-tagged p53 and also endogenous p53, as it is known for UV-irradiation or inhibition of the proteasome, could not be detected after NO-treatment. Since neither the interaction with Mdm2 nor the ubiquitination of p53 after NO-treatment were affected, an effect of the serine 15-phosphorylation of p53 on the nuclear export was assumed. Heterokaryon analysis, showed that serine 15-phosphorylated p53 is not able to shuttle between nucleus and cytoplasm. Due to inhibition of nuclear export of p53 correlating with serine 15-phosphorylation the proteasomal degradation of p53, which takes place in cytoplasm, is inhibited. This in turn leads to the accumulation of a predominantly nuclear localized p53