Microwave resonator-based sensor system for specific antibody detection.

An antibody-detecting sensor is described that is based on a microwave electrodynamic resonator. A polystyrene film with immobilized bacteria deposited on a lithium niobate plate was placed at one end of the resonator and was used as the sensing element. The second end was electrically shorted. The frequency and depth of the reflection coefficient S11 for three resonances in the range 6.5-8.5 GHz were used as an analytical signal to examine antibody interactions with bacteria and determine the time required for cell immobilization. The sensor distinguished between situations in which bacteria interacted with specific antibodies and those in which no such interaction occurred (control). Although the cell-antibody interaction changed the frequency and depth of the second and third resonance peaks, the parameters of the first resonance peak did not change. The interaction of cells with nonspecific antibodies did not change the parameters of any of the peaks. These results are promising for use in the design of methods to detect specific antibodies, which can supplement the existing methods of antibody analysis.

Gene Profiling of a 3D Psoriatic Skin Model Enriched in T Cells: Downregulation of PTPRM Promotes Keratinocyte Proliferation through Excessive ERK1/2 Signaling.

Psoriasis is a complex, immune-mediated skin disease involving a wide range of epithelial and immune cells. The underlying mechanisms that govern the epidermal defects and immunological dysfunction observed in this condition remain largely unknown. In recent years, the emergence of new, more sophisticated models has allowed the evolution of our knowledge of the pathogenesis of psoriasis. The development of psoriatic skin biomaterials that more closely mimic native psoriatic skin provides advanced preclinical models that will prove relevant in predicting clinical outcomes. In this study, we used a tissue-engineered, two-layered (dermis and epidermis) human skin substitute enriched in T cells as a biomaterial to study both the cellular and molecular mechanisms involved in psoriasis' pathogenesis. Gene profiling on microarrays revealed significant changes in the profile of genes expressed by the psoriatic skin substitutes compared with the healthy ones. Two genes, namely, PTPRM and NELL2, whose products influence the ERK1/2 signaling pathway have been identified as being deregulated in psoriatic substitutes. Deregulation of these genes supports excessive activation of the ERK1/2 pathway in psoriatic skin substitutes. Most importantly, electrophoresis mobility shift assays provided evidence that the DNA-binding properties of two downstream nuclear targets of ERK1/2, both the NF-κB and Sp1 transcription factors, are increased under psoriatic conditions. Moreover, the results obtained with the inhibition of RSK, a downstream effector of ERK1/2, supported the therapeutic potential of inhibiting this signaling pathway for psoriasis treatment. In conclusion, this two-layered human psoriatic skin substitute enriched in T cells may prove particularly useful in deciphering the mechanistic details of psoriatic pathogenesis and provide a relevant biomaterial for the study of potential therapeutic targets.

The impact of immunogenicity on therapeutic antibody pharmacokinetics: A preclinical evaluation of the effect of immune complex formation and antibody effector function on clearance.

The occurrence of an immune response against therapeutic proteins poses a major risk for the development of biologics and for successful treatment of patients. Generation of anti-drug antibodies (ADAs) can lead to formation of immune complexes (ICs), consisting of drug and ADAs, with potential impact on safety, efficacy and exposure. Here, we focus on the effects of IC formation, i.e., specific IC sizes, ADA and drug properties, on drug pharmacokinetics. Pre-formed IC preparations of an IgG1 drug (with wild type or with an ablated effector function at the Fc domain) and different ADA surrogates (directed against the complementarity-determining regions or Fc domain of the drug) were administered to rats and collected serum was analyzed to determine the total drug concentration. A combination of size-exclusion chromatography and ELISA enabled a size-specific evaluation of IC profiles in serum and their changes over time. Within five minutes, total drug concentration decreased by ~20-60% when the drug was complexed. Independent of the ADA surrogate and drug variant used, increasing IC size led to increased clearance. Comparing ICs formed with the same ADA surrogate but different IgG1 variants, we observed that complexed drug with a wildtype Fc domain showed faster clearance compared to immune effector function modified drug. Data generated in this study indicated that clearance of drug due to ADA generation is driven by size and structure of the formed ICs, but also by the immune effector functions of the Fc domains of IgGs.Abbreviations Ab: antibody, ADA: anti-drug antibody, AUC: area under the curve, Bi: biotin, CDR: complementary-determining region, cmax: maximal concentration, Dig: digoxigenin, ELISA: enzyme-linked immunosorbent assay, Fc: fragment crystallizable, FcRn: neonatal Fc receptor, HMW: high molecular weight, IC: immune complex, IC-QC: immune complex quality control, IgG: immunoglobulin G, mAb: monoclonal antibody, mADA: monoclonal ADA, pAb: polyclonal antibody, pADA: polyclonal ADA, PD: pharmacodynamics; PK: pharmacokinetic, QC: quality control, SEC: size-exclusion chromatography, WT: wildtype.

Glomerular Endothelial Cell-Derived microRNA-192 Regulates Nephronectin Expression in Idiopathic Membranous Glomerulonephritis.

BACKGROUND: Autoantibodies binding to podocyte antigens cause idiopathic membranous glomerulonephritis (iMGN). However, it remains elusive how autoantibodies reach the subepithelial space because the glomerular filtration barrier (GFB) is size selective and almost impermeable for antibodies. METHODS: Kidney biopsies from patients with iMGN, cell culture, zebrafish, and mouse models were used to investigate the role of nephronectin (NPNT) regulating microRNAs (miRs) for the GFB. RESULTS: Glomerular endothelial cell (GEC)-derived miR-192-5p and podocyte-derived miR-378a-3p are upregulated in urine and glomeruli of patients with iMGN, whereas glomerular NPNT is reduced. Overexpression of miR-192-5p and morpholino-mediated npnt knockdown induced edema, proteinuria, and podocyte effacement similar to podocyte-derived miR-378a-3p in zebrafish. Structural changes of the glomerular basement membrane (GBM) with increased lucidity, splitting, and lamellation, especially of the lamina rara interna, similar to ultrastructural findings seen in advanced stages of iMGN, were found. IgG-size nanoparticles accumulated in lucidity areas of the lamina rara interna and lamina densa of the GBM in npnt-knockdown zebrafish models. Loss of slit diaphragm proteins and severe structural impairment of the GBM were further confirmed in podocyte-specific Npnt knockout mice. GECs downregulate podocyte NPNT by transfer of miR-192-5p-containing exosomes in a paracrine manner. CONCLUSIONS: Podocyte NPNT is important for proper glomerular filter function and GBM structure and is regulated by GEC-derived miR-192-5p and podocyte-derived miR-378a-3p. We hypothesize that loss of NPNT in the GBM is an important part of the initial pathophysiology of iMGN and enables autoantigenicity of podocyte antigens and subepithelial immune complex deposition in iMGN.

NCS 613, a PDE4 inhibitor, by increasing cAMP level suppresses systemic inflammation and immune complexes deposition in kidney of MRL/lpr lupus- prone mice.

Nephritis remains the most common severe manifestation of systemic lupus erythematosus in which auto-antibodies mediate chronic inflammation and kidney damage. cAMP-phosphodiesterases regulate sodium excretion and inflammation in various tissues. How cAMP elevation can reduce systemic inflammation and suppress kidney inflammation and damage remains elusive. PDE4 signaling and cAMP metabolism were investigated along immune complex depositions in target tissues and kidney damage (histology). SLE disease progression is associated with changes in kidney PDE4 activity and expression. Moreover, lupus prone mice exhibit low kidney cAMP level which is associated to induction and relocation of nuclear and cytoskeleton PDE4 isoforms. Auto-antibodies-induced kidney damage was attested by mesangial proliferation and cellular infiltration. Interestingly, we reported that NCS 613 treatment decreases systemic auto-antibody secretion and their corresponding immune complex deposition in target tissues. Furthermore, NCS 613 is able to increase cAMP levels in the kidney; hence this compound rescues kidney PDE4 alterations in treated mice. NCS 613 overcomes disease progression in lupus prone mice by improving wellbeing and decreasing inflammation in treated mice. The PDE4 inhibitor, NCS 613, is a new anti-inflammatory compound that is believed to be a leading drug candidate for the treatment of inflammatory diseases such as lupus nephritis.

Comparison of acid- versus heat-treatment for immune complex dissociation and detection of Dirofilaria immitis antigen in canine plasma.

Annual antigen testing is a mainstay for diagnosing infection with Dirofiliaria immitis in dogs; yet, it has been documented that some heartworm-infected dogs and cats test false-negative for antigen due to the presence of antigen-antibody complexes. Several studies have reported the use of heat as a reliable means of immune-complex dissociation (ICD) in recent years; however, the data regarding the use of acid as a reliable method of ICD for D. immitis detection are limited. The objective of this study was to compare an acid-based form of ICD to the more established and evaluated method of heat-based ICD in experimentally infected and non-infected dogs. Plasma from class A dogs experimentally infected ∼4 months prior with D. immitis (infected; n = 24) and dogs reared indoors with no history of exposure to mosquitoes (non-infected; n = 75) were evaluated for presence of D. immitis antigen (DiroCHEK® Heartworm antigen test kit). Each sample was divided into three aliquots for testing: [1] Control plasma (no acid- or heat-treatment), [2] acid-treated plasma (trichloroacetic acid (TCA), incubation, centrifugation for 5 min at 16,000 X g, buffer), and [3] heat-treated plasma (104 °C followed by centrifugation at 16,000 X g). Treatments for each aliquot were performed and tested in triplicate; results were determined both visually (color change) and by spectrophotometric analysis (optical density [OD] value). Of the 24 infected dogs, 0/24 tested positive for antigen in the absence of acid- or heat-treatment. Those same plasma samples following processing by either acid- or heat-treatment yielded 18/24 (75.0%) and 19/24 (79.2%) antigen-positive results, respectively. Of the 75 plasma samples from non-infected dogs, neither acid- nor heat-treatment of plasma caused any false-positive color changes or spectrophotometric values. These results indicate that acid as a means of ICD reliably allowed for the detection of D. immitis antigen in infected plasma while not inducing false-positive results in non-infected plasma samples.

A method combining blue native polyacrylamide gel electrophoresis with liquid chromatography tandem-mass spectrometry to detect circulating immune complexes between therapeutic monoclonal antibodies and anti-drug antibodies in animals.

Therapeutic monoclonal antibodies can potentially induce unwanted immune responses, resulting in the production of anti-drug antibodies (ADAs). The binding of ADAs to drugs and subsequent formation of immune complexes (ICs) can trigger various responses, dependent on the size, concentration, and subclass of ADAs. To better understand the impact of ADAs on pharmacokinetics, pharmacodynamics, and toxicological profiles, a bioanalytical method was developed for the detection of ICs between human monoclonal immunoglobulin G (IgG) and ADAs in biological samples. Regarding the experimental procedure, in brief, the human antibody-specific ICs and unbound human antibody in biological samples are separated through blue native polyacrylamide gel electrophoresis (BN-PAGE). The target fractions are then cut from the gel, followed by in-gel trypsin digestion and subsequent liquid chromatography tandem-mass spectrometry (LC-MS/MS) to monitor the human IgG-specific peptide. This method was able to detect various types of human antibodies with a lower limit of detection of 10 µg/mL in monkey serum. The assay performance for the detection of ICs was demonstrated using spiked samples, and pre-incubated ICs in monkey serum were clearly detected. Taken together, these findings indicate that our method enables a semi-quantitative analysis for estimating the ratio of human antibody included ICs in comparison to the total antibody. This method was successfully applied to an in vivo study using mice, and the data helped explain the unexpectedly rapid clearance of a humanized antibody due to the formation of large ICs. The combination of the separation of ICs by BN-PAGE and the detection of the human IgG-specific peptide by LC-MS/MS is a useful general bioanalytical approach for the detection of ICs in animals.

Outcomes of immunoglobulin-associated mesangiocapillary glomerulonephritis: A South African experience.

AIM: Immunoglobulin-associated mesangiocapillary glomerulonephritis is currently the most common biopsy-confirmed glomerulonephritis in Cape Town, South Africa. We aimed to determine the outcome of patients with a biopsy-confirmed diagnosis of immunoglobulin-associated mesangiocapillary glomerulonephritis at our centre. METHODS: A retrospective cohort study of adult patients was conducted from January 1, 2000 to December 31, 2016. The endpoint was a composite of doubling of creatinine and/or end-stage renal disease and/or death. Cox univariable and multivariable proportional hazards models were used to examine the association between the composite endpoint and predictor variables. Survival curves were made with the use of Kaplan-Meier estimates. RESULTS: A total of 70 patients were included in the study and their median duration of follow-up was 30.4 months. Forty-eight (68.6%) patients reached the composite endpoint. The proportion reaching this endpoint at 1, 3 and 5 years were 37.5%, 64.6% and 81.3%, respectively. Cox multivariable proportional hazards model identified a serum creatinine concentration > 200 µmol/L at the time of biopsy, moderate to severe interstitial fibrosis, ≥50% crescents and cyclophosphamide therapy as predictors of the composite endpoint. CONCLUSION: Immunoglobulin-associated mesangiocapillary glomerulonephritis remains a common glomerular pathological diagnosis in our setting and has poor outcomes. This may partially be explained by late presentation. Future research needs to focus on identifying the possible cause(s) of this common glomerular disease so that more targeted therapeutic approaches can be offered.

Use of polyclonal IgY antibodies to detect serum immune complexes in patients with active hookworm infection.

Definitive diagnosis of hookworm infection is usually based on the microscopic detection of eggs in a stool sample; however, several cases display a low or irregular egg output. Serodiagnosis can be a useful tool to identify these cases, but conventional tests do not differentiate past from active infections. The aim of this study was to obtain and apply egg yolk polyclonal immunoglobulin (IgY) antibodies to detect immune complexes (ICs) in serum samples from patients infected with hookworm. Hens were immunized with Ancylostoma ceylanicum saline extract, their eggs were collected and then IgY antibodies were extracted and purified. Antibody purity was tested by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis and specificity was assessed by immunoblotting and immunofluorescence. IgY production was evaluated by kinetics enzyme-linked immunosorbent assay (ELISA). Sandwich ELISA tested the ability of IgY to detect ICs in serum samples, from which diagnostic parameters were calculated. Antibody responses increased steadily from day 7 to 42. In the immunoblotting assay, IgY recognized two protein complexes. The immunofluorescence assay showed no staining in control samples. The sandwich ELISA presented a very high diagnostic value, with a sensitivity of 90% and a specificity of 86.7%. Our pioneer strategy highlights the potential use of egg yolk IgY as a diagnostic test to detect active hookworm infection.

Formation and glomerular deposition of immune complexes in mice administered bovine serum albumin: Evaluation of dose, frequency, and biomarkers.

In preclinical toxicity studies, species-foreign proteins administered to animals frequently leads to formation of anti-drug antibodies (ADA). Such antibodies may form circulating immune complexes (CIC) with the administered protein. These CIC can activate the classical complement pathway, thereby forming complement-bound CIC (cCIC); if large of amounts of CIC or cCIC is formed, the clearance mechanism may become saturated which potentially leads to vascular immune complex (IC) deposition and inflammation. Limited information is available on the effect of different treatment related procedures as well as biomarkers of IC-related vascular disease. In order to explore the effect of different dose regimens on IC formation and deposition, and identification of possible biomarkers of IC deposition and IC-related pathological changes, C57BL/6J and BALB/c mice were dosed subcutaneously twice weekly with bovine serum albumin (BSA) for 13 weeks without adjuvant. After 6 and 13 weeks, CIC and cCIC were detected in plasma; after 13 weeks, IC deposition was detected in kidney glomeruli. In particular immunohistochemistry double-staining was shown to be useful for detection of IC deposition. Increasing dosing frequency or changing BSA dose level on top of an already established CIC and cCIC response did not cause changes in IC deposition, but CIC and cCIC concentrations tended to decrease with increased dose level, and increased cCIC formation was observed after more frequent dosing. The presence of CIC in plasma was associated with glomerular IC deposits in the dose regimen study; however, the use of CIC or cCIC as potential biomarkers for IC deposition and IC-related pathological changes, needs to be explored further.

Immuno-Affinity Mass Spectrometry: A Novel Approaches with Biomedical Relevance.

Identifying antigen-antibody interactions have been shown as a critical step in understanding the proteins biological functions and their involvement in various pathological conditions. While many techniques have been developed to characterize antigen-antibody interactions, one strategy that has gained considerable momentum over the last decade for the identification and quantification of antigen-antibody interactions, is immune affinity-chromatography followed by mass spectrometry. Moreover, the combination of enzymatic digestion of antigens and mass spectrometric identification of specific binding peptide(s) to the corresponding anti-antigen antibody has become a versatile and clinical relevant method for mapping epitopes by mass spectrometry. In this chapter, the development and applications of novel immunoaffinity mass spectrometric methodologies for elucidating biomedical aspects will be presented. First, a simplified mass spectrometric approach that maps an epitope from a digested antigen solution without immobilizing the anti-antigen antibody on a solid support will be reported. iMALDI (from immunoaffinity and MALDI, matrix-assisted laser desorption/ionization), a technique that involves immunoaffinity capture of specific peptides and direct MALDI measurements was used for absolute quantification of serine/threonine-specific protein kinase (AKT) peptides from breast cancer and colon cancer cell lines and flash-frozen tumor lysates. The intact transition epitope mapping (ITEM) was shown as a rapid and accurate epitope mapping method by using Ion mobility mass spectrometry (IMS-MS) for analysing the antigen peptide-containing immune complex previously generated by in solution epitope extraction/excision procedures.

Generation, Characterization, and Quantitative Bioanalysis of Drug/Anti-drug Antibody Immune Complexes to Facilitate Dedicated In Vivo Studies.

PURPOSE: Immunogenicity against biotherapeutics can lead to the formation of drug/anti-drug-antibody (ADA) immune complexes (ICs) with potential impact on safety and drug pharmacokinetics (PK). This work aimed to generate defined drug/ADA ICs, characterized by quantitative (bio) analytical methods for dedicated determination of IC sizes and IC profile changes in serum to facilitate future in vivo studies. METHODS: Defined ICs were generated and extensively characterized with chromatographic, biophysical and imaging methods. Quantification of drug fully complexed with ADAs (drug in ICs) was performed with an acid dissociation ELISA. Sequential coupling of SEC and ELISA enabled the reconstruction of IC patterns and thus analysis of IC species in serum. RESULTS: Characterization of generated ICs identified cyclic dimers, tetramers, hexamers, and larger ICs of drug and ADA as main IC species. The developed acid dissociation ELISA enabled a total quantification of drug fully complexed with ADAs. Multiplexing of SEC and ELISA allowed unbiased reconstruction of IC oligomeric states in serum. CONCLUSIONS: The developed in vitro IC model system has been properly characterized by biophysical and bioanalytical methods. The specificity of the developed methods enable discrimination between different oligomeric states of ICs and can be bench marking for future in vivo studies with ICs.

Immunobinding-induced alteration in the electrophoretic mobility of proteins: An approach to studying the preconcentration of an acidic protein under cationic isotachophoresis.

The objective of this study is to explore an approach for analyzing negatively charged proteins using paper-based cationic ITP. The rationale of electrophoretic focusing the target protein with negative charges under unfavorable cationic ITP condition is to modify the electrophoretic mobility of the target protein through antigen-antibody immunobinding. Cationic ITP was performed on a paper-based analytical device that was fabricated using fiberglass paper. The paper matrix was modified with (3-aminopropyl)trimethoxysilane to minimize sample attraction to the surface for cationic ITP. Negatively charged BSA was used as the model target protein for the cationic ITP experiments. No electrophoretic mobility was observed for BSA-only samples during cationic ITP experimental condition. However, the presence of a primary antibody to BSA significantly improved the electrokinetic behavior of the target protein. Adding a secondary antibody conjugated with amine-rich quantum dots to the sample further facilitated the concentrating effect of ITP, reduced experiment time, and elevated the stacking ratio. Under our optimized experimental conditions, the cationic ITP-based paper device electrophoretically stacked 94% of loaded BSA in less than 7 min. Our results demonstrate that the technique has a broad potential for rapid and cost-effective isotachphoretic analysis of multiplex protein biomarkers in serum samples at the point of care.

Use of hafnium(IV) oxide in biosensors.

Hafnium(IV) oxide is a material with properties that can increase the sensitivity, durability, and reliability of biosensors made from silicon dioxide and other semiconductor materials due to its high dielectric constant, thermodynamic stability, and the simplicity with which it can be deposited. This work describes the use of this material in biosensors based on field-effect transistors to detect ions and DNA, in immunosensors to detect an antigen-antibody complex, its use as a contrast material in computed tomography scans and the possibility of using it in optic biosensors in the infrared region. Its low cost and versatility in the field of biosensors is underscored.

Number of individual ACPA reactivities in synovial fluid immune complexes, but not serum anti-CCP2 levels, associate with inflammation and joint destruction in rheumatoid arthritis.

INTRODUCTION: Individual patients with rheumatoid arthritis (RA) show divergent specific anti-citrullinated protein/peptide antibodies (ACPA) patterns, but hitherto no individual ACPA specificity has consistently been linked to RA pathogenesis. ACPA are also implicated in immune complexes (IC)-associated joint pathology, but until now, there has been no method to investigate the role of individual ACPA in RA IC formation and IC-associated pathogenesis. METHODS: We have developed a new technique based on IC binding to C1q-coated magnetic beads to purify and solubilise circulating IC in sera and synovial fluids (SF) from 77 patients with RA. This was combined with measurement of 19 individual ACPA in serum, SF and in the IC fractions from serum and SF. We investigated whether occurrence of individual ACPA as well as number of ACPA in these compartments was related to clinical and laboratory measures of disease activity and inflammation. RESULTS: The majority of individual ACPA reactivities were enriched in SF as compared with in serum, and levels of ACPA in IC were regulated independently of levels in serum and SF. No individual ACPA reactivity in any compartment showed a dominating association to clinical and laboratory measures of disease activity and severity. Instead, the number of individual ACPA reactivities in the IC fraction from SF associated with a number of markers of joint destruction and inflammation. CONCLUSIONS: Our data highlight the polyclonality of ACPA in joint IC and the possibility that a broad ACPA repertoire in synovial fluid IC might drive the local inflammatory and matrix-degrading processes in joints, in analogy with antibody-induced rodent arthritis models.

The immune complex transfer enzyme immunoassay: Mechanism of improved sensitivity compared with conventional sandwich enzyme immunoassay.

Immune complex transfer enzyme immunoassay (ICT-EIA) is one of the technologies which enables ultrasensitive measurements of protein biomarkers. The ICT-EIA uses two types of beads and sandwich-shaped immune complexes are transferred from the 1st bead to the 2nd bead in the assay. The purpose of the study is to reveal the reason why the ICT-EIA achieves ultrasensitive measurements by making a detailed comparison between conventional sandwich enzyme immunoassay (Sand-EIA) and ICT-EIA. ICT-EIAs for cytokines were developed and the sensitivities were compared with the sandwich EIAs. ICT-EIAs had about 100 times higher sensitivities because of markedly decreased non-specific signals derived from non-specific binding of detection antibody conjugates onto the polystyrene bead. The results have enabled us to show the importance of reducing non-specific signals in EIAs to obtain higher sensitivities. This methodology should be more valuable if combined with a different label detection system such as digital counting or immuno-PCR, which may enable the detection of single target protein molecules in the near future.

Proteomic approach to profiling immune complex antigens in cerebrospinal fluid samples from patients with central nervous system autoimmune diseases.

BACKGROUND: Immune complexes (ICs) may clearly reflect immunological abnormalities caused by disease, especially for autoimmune diseases. Although ICs have been detected in cerebrospinal fluid (CSF) from patients with CNS autoimmune diseases, identities of antigens in such ICs have not been comprehensively determined. METHODS: We used immune complexome analysis, in which nano-liquid chromatography-tandem mass spectrometry is employed to comprehensively identify antigens incorporated into ICs in biological fluids, to characterize ICs in CSF samples from patients with CNS autoimmune diseases, and to find disease-specific IC antigen to a certain CNS autoimmune disease. Also, we compared the IC antigens we identified with the reported CSF proteome or with the published plasma proteome to examine if the method is distinguished from the conventional CSF proteome analysis. RESULTS: We identified 176 antigens in 78 CSF samples. We then assessed the overlaps among these antigens, the CSF proteome, and the plasma proteome; 140 of the 176 antigens were found to be exclusively detected by our method. Notably, IC-associated suprabasin in CSF was 100% specific to neuropsychiatric systemic lupus erythematosus (NPSLE). CONCLUSIONS: This report is the first to comprehensively identify the antigens incorporated into ICs in CSF. There was limited overlap between the antigens we identified and the CSF proteome or the plasma proteome; therefore, our method can be distinguished from the conventional CSF proteome analysis. Although the sensitivity of disease-specific IC-antigens detected in immune complexome analysis screening, the sensitivity may be improved by developing an ELISA method specifically for detecting the ICs. Immune complexome analysis of CSF may be a new and promising path to biomarker discovery for diagnosis and study for CNS autoimmune diseases.

Detection of platelet autoantibodies to identify immune thrombocytopenia: state of the art.

Immune Thrombocytopenia (ITP) is diagnosed by exclusion of other causes for thrombocytopenia. Reliable detection of platelet autoantibodies would support the clinical diagnosis of ITP and prevent misdiagnosis. We optimized our diagnostic algorithm for suspected ITP using the direct monoclonal antibody immobilization of platelet antigens assay (MAIPA), which evaluates the presence of platelet autoantibodies on the glycoproteins (GP) IIb/IIIa, Ib/IX and V bound on the patient platelets. The direct MAIPA was shown to be a valuable technique for the detection of platelet autoantibodies and could possibly become a guide for optimizing therapy towards a more personalized treatment of ITP.

Comparison between patients with norovirus-related gastroenteritis and asymptomatic carriers with respect to distribution of antibody-complexed viral particles and intestinal flora.

Asymptomatic carriers have a major influence on the spreading of norovirus infections. The objective of this study was to examine the characteristics of patients and asymptomatic carriers affected by norovirus-related community gastroenteritis outbreaks. No significant difference between the two groups was observed in terms of the number of norovirus-antibody complexes with respect to total numbers. Principal coordinates analysis of the intestinal flora based on ß-diversity analysis, revealed a different bacterial composition between patients and asymptomatic carriers, particularly regarding the genera Pseudomonas, Bacteroides, and Erwinia, as well as the Ruminococcaceae family. Although the proportional changes between these intestinal microorganisms were not sufficient to explain gastroenteritis symptoms, they represent possible markers shared by asymptomatic norovirus carriers.