Multiplex Bead Assay Research Articles

Analysis of tear fluid proteins: use of multiplex assays in profiling biomarkers of dry eye disease

Aims Dry eye disease (DED) is a distressing condition that is commonly associated with ageing, contact lens wear and autoimmune disease. DED is one of the fastest-growing eye disorders in older populations (over-50s) and is significantly underdiagnosed. Multiplex studies of tear fluids from DED patient have suggested a role for inflammatory cytokines in its pathology. In this study, we investigated tear fluids from DED and normal subjects for a panel of 7 cytokines, using the multiplex bead assay.

Il-8 as an underutilized prognostic factor in metastatic colorectal cancer.

409 Background: Cancer-associated inflammation has been identified as a key determinant of disease progression and survival in colorectal cancer. We investigated the association between circulating inflammatory cytokines and survival in metastatic colorectal cancer (mCRC) patients. Methods: Plasma levels of 47 cytokines were measured using multiplex-bead assays in a cohort of 168 previously untreated mCRC patients. Demographic, clinical-pathological features, body mass index, and mortality data were abstracted from patient medical records. Overall survival (OS) was evaluated by Kaplan-Meier analysis and Cox proportional hazards regression. Results: Using principal component analysis, we identified a subset of cytokines explaining the maximum variance in OS; and found interleukin (IL)-1b, IL-5, IL-8, IL-12 and VEGF to be significantly associated with OS. However, only IL-8 was significantly and independently associated with OS in multivariable-adjusted models. For each 100 pg/ml increase in the level of circulating IL-8, hazard rate for death increased by 1.6 (95% CI 1.24-1.97). IL-8 measurements ranged from <1 to 413 pg/ml with a median value of 22 pg/ml. Median uncensored survival was 26.5 and 15.5 months among patients with IL-8 levels below and above this value, respectively. ROC analysis of IL-8 demonstrated an AUC of 0.69 (95% CI 0.60-0.76), as compared to 0.52 for CEA (95% CI 0.46-0.59). Conclusions: We identified an association between IL-8 and OS in previously untreated mCRC patients, suggesting its potential role as a prognostic inflammatory biomarker. In this dataset, IL-8 outperformed CEA as a prognostic biomarker, a finding which requires validation in subsequent work. Appropriately identifying, monitoring and managing chronic inflammation and the host inflammatory response during colorectal cancer treatment may be important for improving long-term survival.

Association of prior treatment with anti-EGFR monoclonal antibodies with downregulation of circulating TRAIL and upregulation of EGFR and VEGF family ligands in metastatic colorectal cancer.

452 Background: Inhibition of growth factor receptor signaling leads to unintended compensatory pathway activation in preclinical models, which may impact the success of subsequent therapies. We hypothesized that increased circulating levels of receptor ligands after anti-EGFR monoclonal antibody (mAb) therapy, including VEGF ligands or EGFR family ligands, may provide insights into clinically relevant feedback mechanisms. Methods: Plasma was obtained from 96 metastatic colorectal cancer (mCRC) patients: 48 previously treated with anti-EGFR mAbs and 48 mCRC patients previously treated without prior anti-EGFR therapy who were matched for total months of prior chemotherapy and interval since last chemotherapy treatment. Plasma levels of circulating cytokines with emphasis on EGFR and VEGF ligands were determined using suspension bead multiplex assay (BioRad). Comparisons were done by the two-sided nonparametric Wilcoxon signed rank test. Multivariate linear regression analysis was done with forward selection at p<0.1. Results: By Wilcoxon signed rank test, prior anti-EGFR mAb treatment was associated with differences in EGF (3.5-fold increase, p=0.0001), epiregulin (1.6-fold increase, p=0.02), and TGF-alpha (1.4-fold increase, p=0.04). There was no significant association with amphiregulin, betacellulin, or HB-EGF. Prior anti-EGFR mAb treatment was also associated with differences in TRAIL (1.4-fold decrease, p=0.001) and with differences in VEGF-C (1.4-fold increase, p=0.01), and FGF-basic (1.8-fold increase, p=0.02). After multivariate analysis, prior anti-EGFR mAb treatment remained significantly associated with differences in EGF (p=0.003), TRAIL (p=0.045), VEGF-C (p=0.02), and FGF-basic (p=0.02). Conclusions: Prior treatment with anti-EGFR mAbs is associated with an apparent compensatory increase in EGFR ligands and pro-angiogenic factors, and lower levels of the pro-apoptotic cytokine TRAIL. These differences in circulating growth factor receptor ligands may provide insights on resistance mechanisms and optimal treatment sequencing.

Cytokines and Biologics in non-infectious autoimmune uveitis: Bench to Bedside

Intraocular inflammatory eye disease is one of the important causes of ocular morbidity. Even though the prevalence of uveitis is less common in relation to diabetic retinopathy, glaucoma or age related macular degeneration, the complexity and heterogeneity of the disease makes it more unique. Putative uveitogenic retinal antigens incite innate immunity by the process of antigen mimicry and have been shown to be associated in patients with intraocular inflammatory disease by numerous experimental studies. Laboratory diagnostic tools to aid the etiologic association in intraocular inflammatory disease have evolved over the last two decades and we are entering into an era of molecular diagnostic tests. Sophisticated novel technologies such as multiplex bead assays to assess biological signatures have revolutionized the management of complex refractory uveitis. Nevertheless, there is still a long way to go to establish the causal relationship between these biomarkers and specific uveitic entities. Experimental studies have shown the supreme role of infliximab in the management of Behcet's disease. Despite significant experimental and case control studies, the deficiency of randomized clinical trials using these biologic agents has handicapped us in exploring them as a front line therapy in severe refractory uveitis. Studies still need to answer the safety of these potentially life threatening drugs in a selected group of patients and determine when to commence and for how long the treatment has to be given. This review article covers some basic concepts of cytokines in uveitis and their potential application for therapy in refractory uveitis.

Prolidase deficiency breaks tolerance to lupus-associated antigens

Prolidase deficiency is a rare autosomal recessive disease in which one of the last steps of collagen metabolism, cleavage of proline-containing dipeptides, is impaired. Only about 93 patients have been reported with about 10% also having systemic lupus erythematosus (SLE). We studied a large extended Amish pedigree with four prolidase deficiency patients and three heterozygous individuals for lupus-associated autoimmunity. Eight unaffected Amish children served as normal controls. Prolidase genetics and enzyme activity were confirmed. Antinuclear antibodies (ANA) were determined using indirect immunofluorescence and antibodies against extractable nuclear antigens were determined by various methods, including double immunodiffusion, immunoprecipitation and multiplex bead assay. Serum C1q levels were determined by enzyme-linked immunosorbent assay. Two of the four homozygous prolidase deficiency subjects had a positive ANA. One had anti-double-stranded DNA, while another had precipitating anti-Ro. By the simultaneous microbead assay, three of the four had anti-Sm and anti-chromatin. One of the three heterozygous subjects had a positive ANA and immunoprecipitation of a 75 000 molecular weight protein. The unaffected controls had normal prolidase activity and were negative for autoantibodies. Prolidase deficiency may be associated with the loss of immune tolerance to lupus-associated autoantigens even without clinical SLE.

Identification of Immunological Biomarkers Which May Differentiate Latent Tuberculosis from Exposure to Environmental Nontuberculous Mycobacteria in Children

A positive gamma interferon (IFN-γ) response to Mycobacterium tuberculosis early secretory antigenic target-6 (ESAT-6)/culture filtrate protein-10 (CFP-10) has been taken to indicate latent tuberculosis (TB) infection, but it may also be due to exposure to environmental nontuberculous mycobacteria in which ESAT-6 homologues are present. We assessed the immune responses to M. tuberculosis ESAT-6 and cross-reactive responses to ESAT-6 homologues of Mycobacterium avium and Mycobacterium kansasii. Archived culture supernatant samples from children at 3 years post-BCG vaccination were tested for cytokine/chemokine responses to M. tuberculosis antigens. Furthermore, the IFN-γ responses to M. tuberculosis antigens were followed up for 40 children at 8 years post-BCG vaccination, and 15 TB patients were recruited as a control group for the M. tuberculosis ESAT-6 response in Malawi. IFN-γ enzyme-linked immunosorbent assays (ELISAs) on supernatants from diluted whole-blood assays, IFN-γ enzyme-linked immunosorbent spot (ELISpot) assays, QuantiFERON TB Gold-In Tube tests, and multiplex bead assays were performed. More than 45% of the responders to M. tuberculosis ESAT-6 showed IFN-γ responses to M. avium and M. kansasii ESAT-6. In response to M. tuberculosis ESAT-6/CFP-10, interleukin 5 (IL-5), IL-9, IL-13, and IL-17 differentiated the stronger IFN-γ responders to M. tuberculosis ESAT-6 from those who preferentially responded to M. kansasii and M. avium ESAT-6. A cytokine/chemokine signature of IL-5, IL-9, IL-13, and IL-17 was identified as a putative immunological biosignature to differentiate latent TB infection from exposure to M. avium and M. kansasii in Malawian children, indicating that this signature might be particularly informative in areas where both TB and exposure to environmental nontuberculous mycobacteria are endemic.

A Multiplex Flow Cytometry Bead Assay For Simultaneous Detection Of IgG, IgA, IgM Heparin-Dependent Antibodies

Heparin-Induced Thrombocytopenia (HIT) is an adverse reaction to heparin, in which affected patients produce platelet activating antibodies specific for complexes of platelet-factor 4 (PF4) and heparin (HPF4) resulting in a prothrombotic condition that in severe cases can be life-threatening. HIT is not rare, since as many as 600K people/year are affected. It is therefore important that a quick and accurate diagnosis of HIT be made, which requires both compatible clinical findings and serologic evidence of a heparin dependent antibody. First line testing consists of immunologic assays that detect IgG, IgA and IgM antibodies targeting PF4 in a complex with heparin or another polyanion. The most widely used assay is the PF4 ELISA, in which HPF4 bound to the wells of a microtiter plate is the target antigen. The ELISA has high sensitivity (near 100%) for antibody detection but a low positive predictive value for HIT diagnosis. Therefore, samples giving positive ELISA results are often retested using a functional assay like the serotonin release assay (SRA) that is more specific for HIT diagnosis. However, the SRA is difficult to perform and interpret and is not widely available. Further, the ELISA is time-consuming, costly, and most versions cannot distinguish IgG, IgA, IgM (IgG/A/M) antibodies. Knowledge of the antibody isotype can be useful when ELISA and SRA results are discrepant. For instance, strong ELISA results due to clinically insignificant IgM antibodies would not be detected by SRA, and additional testing to confirm IgM antibody isotype can lead to a delay in HIT diagnosis, and in some cases continuation of high cost treatments with direct thrombin inhibitors (DTI). Moreover, there is evidence that some IgA and IgM antibodies may cause HIT.Despite the high sensitivity of the ELISA and high specificity of the SRA, the shortcomings mentioned for these tests highlight the need for improved methods to support a diagnosis of HIT. We report development of a multiplex flow cytometry bead assay (MFBA) for simultaneous detection of IgG/A/M heparin-dependent antibodies that allows higher throughput and has sensitivity and specificity comparable to the PF4 ELISA.For the MFBA, polystyrene beads, to which chemically depolymerized heparin is covalently linked are incubated with purified platelet PF4 to produce HPF4-bead targets. Lyophilized HPF4 beads are stable for at least 3 months. HPF4-beads are incubated with serum, washed, and incubated with anti-human -IgG, -IgA, and –IgM, each with different fluorescent labels for detection of HIT antibodies by flow cytometry. A total of 164 patient samples could be tested by a single technologist in 8 hours. Serum samples from 300 patients with suspected HIT and previously tested by SRA were tested by both MFBA and an in-house PF4 ELISA including high dose heparin (100 U/L) confirmation step. Studies were approved by institutional IRB. MFBA results showed good sensitivity (87% IgG, 63% IgA, 36% IgM) for IgG, good specificity (96% IgG, 91% IgA, 93% IgM) for all isotypes when compared to the ELISA, and good sensitivity/specificity (93%/91%) compared to SRA. The area under the curve (AUC) calculated for the receiver operating characteristic graphs compared to the SRA were excellent (0.9-1.0) for both MFBA (AUC = 0.95) and PF4 ELISA (AUC = 0.98). All samples positive by MFBA inhibited with high dose heparin suggesting that this extra step is not required, and 3 samples that tested IgG positive in both MFBA and SRA gave indeterminate results (not inhibited with high dose heparin) in ELISA.Overall, these data show that the MFBA has accuracy, sensitivity, and specificity for detection of IgG antibodies comparable to the PF4 ELISA and the “gold standard” SRA with the additional advantage of simultaneously distinguishing IgG/A/M antibody isotypes, which will lead to reduced confirmation testing. Sensitivity of MFBA for detection of IgA and IgM antibodies was significantly lower compared to ELISA, but this reflects the assay design intended to increase specificity of the MFBA for these two isotypes. In addition to its multiplex capabilities, the MFBA allows for higher sample throughput and shortened turn-around times that will contribute to overall improved patient care. Disclosures:No relevant conflicts of interest to declare.

Cytokine response to pregnancy-associated recrudescence of Plasmodium berghei infection in mice with pre-existing immunity to malaria

BackgroundDuring childhood, residents of areas with stable transmission of Plasmodium falciparum parasites acquire substantial protective immunity to malaria, and adults therefore rarely experience clinical disease episodes. However, susceptibility to infection reappears in pregnant women, particularly primigravidae. This is due to appearance of antigenic parasite variants that are restricted to pregnancy. Variant-specific immunity also governs pregnancy-associated recrudescence of Plasmodium berghei infection in pregnant mice. Pregnancy-related changes in the plasma cytokine levels of mice with immunity acquired prior to first pregnancy have not been studied in detail previously, and were the topic of the present study.MethodsA multiplexed bead assay was used to measure plasma levels of IL-5, IL-10, IL-12, IL-13, IFN-γ and TNF in BALB/c mice immunized against P. berghei K173 by repeated infection and drug cure before the first pregnancy. The association between cytokine levels on the one hand and parasitaemia and haemoglobin levels on the other, in mice that had never been pregnant or were pregnant for the first, second or third time were evaluated by Mann–Whitney test and Spearman rank-order correlation analysis.ResultsPregnancy per se did not further increase the already high cytokine levels in mice previously immunized by repeated infection and drug cure. Levels of all the cytokines except IL-10 were correlated with each other, and with parasitaemia and haemoglobin levels. Furthermore, levels of all cytokines were positively correlated with parity, except IL-10, which was negatively correlated with parity. High levels of IL-10 and low levels of the other cytokines were associated with poor pregnancy outcome.ConclusionsHigh levels of IL-10 and low levels of the other cytokines were associated with poor pregnancy outcome in this mouse model of placental malaria. Since the model replicates key parasitological and immunological features of placental P. falciparum malaria, it underpins its usefulness in immunology and pathogenesis studies of this important cause of mother/child morbidity in endemic areas.

Liver protective effect of ursodeoxycholic acid includes regulation of ADAM17 activity

BackgroundUrsodeoxycholic acid (UDCA) is used to treat primary biliary cirrhosis, intrahepatic cholestasis, and other cholestatic conditions. Although much has been learned about the molecular basis of the disease pathophysiology, our understanding of the effects of UDCA remains unclear. Possibly underlying its cytoprotective, anti-apoptotic, anti-oxidative effects, UDCA was reported to regulate the expression of TNFα and other inflammatory cytokines. However, it is not known if this effect involves also modulation of ADAM family of metalloproteinases, which are responsible for release of ectodomains of inflammatory cytokines from the cell surface. We hypothesized that UDCA modulates ADAM17 activity, resulting in amelioration of cholestasis in a murine model of bile duct ligation (BDL).MethodsThe effect of UDCA on ADAM17 activity was studied using the human liver hepatocellular carcinoma cell line HepG2. Untransfected cells or cells ectopically expressing human ADAM17 were cultured with or without UDCA and further activated using phorbol-12-myristate-13-acetate (PMA). The expression and release of ADAM17 substrates, TNFα, TGFα, and c-Met receptor (or its soluble form, sMet) were evaluated using ELISA and quantitative real-time (qRT) PCR. Immunoblotting analyses were conducted to evaluate expression and activation of ADAM17 as well as the level of ERK1/2 phosphorylation after UDCA treatment. The regulation of tissue inhibitor of metalloproteinases-1 (TIMP-1) by UDCA was studied using zymography and qRT-PCR. A mouse model of acute cholestasis was induced by common BDL technique, during which mice received daily orogastric gavage with either UDCA or vehicle only. Liver injury was quantified using alkaline phosphatase (ALP), relative liver weight, and confirmed by histological analysis. ADAM17 substrates in sera were assessed using a bead multiplex assay.ResultsUDCA decreases amount of shed TNFα, TGFα, and sMet in cell culture media and the phosphorylation of ERK1/2. These effects are mediated by the reduction of ADAM17 activity in PMA stimulated cells although the expression ADAM17 is not affected. UDCA reduced the level of the mature form of ADAM17. Moreover, UDCA regulates the expression of TIMP-1 and gelatinases activity in PMA stimulated cells. A BDL-induced acute cholangitis model was characterized by increased relative liver weight, serum levels of ALP, sMet, and loss of intracellular glycogen. UDCA administration significantly decreased ALP and sMet levels, and reduced relative liver weight. Furthermore, hepatocytes of UDCA-treated animals retained their metabolic activity as evidenced by the amount of glycogen storage.ConclusionsThe beneficial effect of UDCA appears to be mediated in part by the inhibition of ADAM17 activation and, thus, the release of TNFα, a strong pro-inflammatory factor. The release of other ADAM17 substrates, TGFα and sMet, are also regulated this way, pointing to a general impact on the release of ADAM17 substrates, which are pivotal for liver regeneration and function. In parallel, UDCA upregulates TIMP-1 that in turn inhibits matrix metalloproteinases, which destroy the hepatic ECM in diseased liver. This control of extracellular matrix turnover represents an additional beneficial path of UDCA treatment.

Development of an experimental model for assessing the effects of cigarette smoke and virus infections on inflammatory responses to bacterial antigens

Interactions among major risk factors associated with bacterial infections were assessed in a model system using surrogates for virus infection; IFN-g, and exposure to cigarette smoke; cigarette smoke extract (CSE), nicotine and cotinine. Cytokine responses elicited by LPS from THP-1 cells in the presence of these components, or combinations of components, were assessed by multiplex bead assay, i.e. IL-1β, IL-6, IL-8, IL-10, TNF-α and IFN-γ. IFN-γ-priming significantly increased pro-inflammatory cytokines induced by LPS. CSE suppressed production of pro-inflammatory cytokines IL-1β, TNF-α and IFN-γ, but enhanced production of IL-8. Nicotine and cotinine suppressed all cytokine responses. In combination, IFN-γ masked the inhibitory effects of CSE. In relation to the objectives of the study, we concluded that (a) IFN-γ at biologically relevant concentrations significantly enhanced pro-inflammatory responses; (b) CSE, nicotine and cotinine dysregulated the inflammatory response and that the effects of CSE were different from those of the individual components, nicotine and cotinine; (c) when both IFN-γ and CSE were present, IFN-γ masked the effect of CSE. There is a need for clinical investigations on the increase in IL-8 responses in relation to exposure to cigarette smoke and increased pro-inflammatory responses in relation to recent viral infection.

INTRAOCULAR GROWTH FACTORS AND CYTOKINES IN PATIENTS WITH DRY AND NEOVASCULAR AGE-RELATED MACULAR DEGENERATION

To analyze intraocular growth factor and cytokine concentrations in eyes with different stages of age-related macular degeneration (AMD) compared with controls. The Clinical Age-Related Maculopathy Staging (CARMS) system was used for assignment of patients into the respective categories. Aqueous humor specimens were taken before cataract surgery in 21 controls (CARMS 1) and in 17 early (CARMS 2) and 16 intermediate (CARMS 3) AMD patients. In 18 neovascular (CARMS 5) AMD patients, specimens were taken immediately before anti-vascular endothelial growth factor intravitreal therapy. Luminex multiplex bead assays were conducted for endostatin, angiogenin, vascular endothelial growth factor, platelet-derived growth factor AA, placental growth factor, thrombospondin 2, and fibroblast growth factor a. Vascular endothelial growth factor concentrations were elevated in CARMS 3 (P = 0.037) and tended to be elevated in CARMS 5 (P = 0.093), whereas levels in CARMS 2 (P = 0.425) were similar to CARMS 1. Platelet-derived growth factor levels were diminished in CARMS 2 (P = 0.020), with a trend to lower levels for CARMS 3 (P = 0.099) and CARMS 5 (P = 0.082) compared with CARMS 1. For CARMS 5, antiangiogenic endostatin was elevated (P < 0.002), while antiangiogenic thrombospondin 2 was reduced (P = 0.029). Clinical Age-Related Maculopathy Staging 3 dry AMD was associated with higher vascular endothelial growth factor levels than CARMS 5 neovascular AMD. Therefore, intraocular vascular endothelial growth factor concentrations do not seem to reflect choroidal neovascularization activity in neovascular AMD directly. Platelet-derived growth factor was decreased in most forms of AMD. The antiangiogenic endostatin was exclusively elevated in neovascular AMD, while thrombospondin 2 was reduced. Age-related macular degeneration disease seems to be associated with a generally altered cytokine system.

Interleukin-12p40 in the spinal fluid as a biomarker for clinically isolated syndrome

Background: The cerebrospinal (CSF) constitutes a specific immune micro-environment to the central nervous system, thus it may contain specific biomarkers involved in the pathogenesis of multiple sclerosis (MS). Objectives: We aimed to study a large array of inflammatory CSF biomarkers in patients with suspected MS to recognize potential early diagnostic markers. Methods: CSF samples were obtained from 115 patients who presented with neurological symptomatology suggestive of MS as follows: clinically isolated syndrome (CIS) = 49, relapsing–remitting multiple sclerosis (RRMS) = 29, and other neurologic disorders (OND) = 37. Protein expression profiles of 30 inflammatory biomarkers were measured by multiplex Luminex bead assay and further analyzed by group comparison statistics, correlation studies and receiver-operating characteristic (ROC) analysis. Results: Interleukin-12 subunit p40 (IL12p40) demonstrated a significant differential expression pattern between the groups (CIS vs OND: p = 1.17*10−7; RRMS vs OND: p = 4.98*10−5), with higher levels in CIS and RRMS patients. ROC analysis demonstrated excellent diagnostic performance of IL12p40 for discrimination between CIS and OND patients (area under the curve = 0.87 (95% CI 0.78–0.93), p = 0.0001). No associations were found with disease activity or severity measures. Conclusions: An increased IL12p40 level characterizes the CSF of MS patients and appears to be helpful in identifying CIS and OND patients early in the process of clinical diagnostic assessment.

Upregulation of TGF-ß1 in experimental proliferative vitreoretinopathy is accompanied by epithelial to mesenchymal transition

Proliferative vitreoretinopathy (PVR) is characterized by epithelial to mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells and consecutive formation of fibrous membranes, leading to retinal redetachment. Transforming growth factor beta (TGF-ß) has been suggested to play an important role in this process, but the role of TGF-ß isoforms is unknown. In pigmented rabbits (n = 14), PVR was induced by cryopexy and a full-thickness limbus-parallel incision. PVR was evaluated by indirect ophthalmoscopy. Concentrations of TGF-ß isoforms were determined by multiplex bead assay analysis in aqueous humor (AH) and vitreous samples. EMT marker vimentin was analyzed by western blot. Masson's-trichrome, haematoxilin and eosine (H&E), and immunohistochemical analysis for EMT marker alpha SMA were performed on cross-sections of eyes. PVR was induced in all treated eyes. The number of quadrants affected by PVR was 1 (n = 5), 2 (n = 2), 3 (n = 2), 4 (n = 5). Vimentin and alpha SMA were expressed during PVR development. During PVR development, both TGF-ß1 levels (AH: p = 0.001; vitreous: p = 0.002) and TGF-ß2 levels increased (AH: p = 0.027; vitreous: p = 0.02), while TGF-ß3 was not detected at any timepoint. The increase was more pronounced for TGF-ß1 than for TGF- ß2 (AH: p = 0.002; vitreous: p = 0.0005), and only TGF-ß1 correlated with the amount of PVR (p = 0.024, r = 0,723). Development of PVR membranes was accompanied by a pronounced upregulation of TGF-ß1, rather than TGF-ß2. Therefore TGF-ß1 could be a promising target for inhibition of PVR.

Pediatric Reference Ranges for Proinflammatory and Anti-Inflammatory Cytokines in Cerebrospinal Fluid and Serum by Multiplexed Immunoassay

To define cytokine concentrations and detectability in children with noninflammatory neurological disorders (NIND). The multiplex bead assay technology was used for simultaneous measurement of 34 soluble cytokines/chemokines in cerebrospinal fluid (CSF) from 73 NIND. Sera from 36 healthy children and 37 NIND also were analyzed. In CSF, CXCL10 had the highest concentration; CCL2, CXCL10, and interleukin (IL)-6 were detectable in all samples, and CXCL8, CCL22, CXCL1, IL-16, and IL-1 receptor antagonist were found in ≥50% of the samples. In serum, CXCL1 had the highest concentration; sIL-2Ra, CXCL1, CXCL10, and CCL22 were detectable in all samples, and CCL2, IL-12, CCL5, and granulocyte monocyte colony-stimulating factor (GM-CSF) were found in ≥50% of the samples. The mean CSF:serum ratio for CCL2 was several-fold higher than the rest, with the CXCL10 and CXCL8 ratios also >1. Intercorrelations between CSF cytokines included CCL2 versus CXCL8 and IL-6, and CXCL1 versus CCL22, reflecting both T-helper-1 (Th1)/Th1 and Th1/Th2 relations. Serum correlations included CCL11 versus CCL2, GM-CSF, and IL-4. For serum cytokines, the agreement between healthy children and NIND was good, with the exception of higher CCL4 in NIND. Cytokines in children varied greatly in concentration and detectability, with chemokines predominating in the CSF. These data allow investigators to select their own kit cytokines, instead of manufacturer-selected cytokines, for greater cost-effectiveness and interpretability.

Laborsaving production of site-specific anti-phospho monoclonal antibodies by simultaneous screening for multiple phosphopeptides using multiplex bead assay (P3372)

Abstract Monoclonal antibodies (MAbs) to phosphotyrosines at specific sites of cellular proteins are critical tools to analyze the functional status of proteins. However, time consuming and laborious screening steps in current methods limits production. Here, we introduce a bead-based multiplex flow cytometry method for simultaneous evaluation of the generated MAbs for the cross-reactivity to different peptide sequences. In this proof of concept study, we chose four phosphorylation motifs from different receptors expressed on B cells (Ig-alpha, membrane IgG and IgE, and FCRL5). They were synthesized as a series of 11-mer peptides containing a tyrosine or a phosphotyrosine at the center and surrounded by unique amino acid sequences. They were conjugated with different color-coded beads which can be identified by red laser excitation in flow cytometry. Antibody binding to the peptides was measured as the level of blue laser excitable R-Phycoerythrin fluorescence. Mice immunized with phosphopeptide-KLH conjugates were used for making hybridomas. As expected, the simultaneous screening revealed unique specificities of the 10-30 MAbs generated in each fusion experiment. In particular, we obtained anti-phospho MAbs specific to Ig-alpha and membrane IgG as well as pan anti-phospho tyrosine Ab. Our results suggest that this method is suitable for production of anti-phospho MAbs that need to recognize a specific phosphopeptide among other phosphopeptides with structural similarity.

Quality control, analysis and secure sharing of Luminex® immunoassay data using the open source LabKey Server platform.

BackgroundImmunoassays that employ multiplexed bead arrays produce high information content per sample. Such assays are now frequently used to evaluate humoral responses in clinical trials. Integrated software is needed for the analysis, quality control, and secure sharing of the high volume of data produced by such multiplexed assays. Software that facilitates data exchange and provides flexibility to perform customized analyses (including multiple curve fits and visualizations of assay performance over time) could increase scientists’ capacity to use these immunoassays to evaluate human clinical trials.ResultsThe HIV Vaccine Trials Network and the Statistical Center for HIV/AIDS Research and Prevention collaborated with LabKey Software to enhance the open source LabKey Server platform to facilitate workflows for multiplexed bead assays. This system now supports the management, analysis, quality control, and secure sharing of data from multiplexed immunoassays that leverage Luminex xMAP® technology. These assays may be custom or kit-based. Newly added features enable labs to: (i) import run data from spreadsheets output by Bio-Plex Manager™ software; (ii) customize data processing, curve fits, and algorithms through scripts written in common languages, such as R; (iii) select script-defined calculation options through a graphical user interface; (iv) collect custom metadata for each titration, analyte, run and batch of runs; (v) calculate dose–response curves for titrations; (vi) interpolate unknown concentrations from curves for titrated standards; (vii) flag run data for exclusion from analysis; (viii) track quality control metrics across runs using Levey-Jennings plots; and (ix) automatically flag outliers based on expected values. Existing system features allow researchers to analyze, integrate, visualize, export and securely share their data, as well as to construct custom user interfaces and workflows.ConclusionsUnlike other tools tailored for Luminex immunoassays, LabKey Server allows labs to customize their Luminex analyses using scripting while still presenting users with a single, graphical interface for processing and analyzing data. The LabKey Server system also stands out among Luminex tools for enabling smooth, secure transfer of data, quality control information, and analyses between collaborators. LabKey Server and its Luminex features are freely available as open source software at http://www.labkey.com under the Apache 2.0 license.

Effects Of Modifying Both The Linoleic and Linolenic Acid Content of a 45% Kcal DIO Diet on Body Weight And Metabolic Parameters in C57BL6J Mice

The objective of this study was to test the effects of modifying both the ratio and concentration of linoleic and linolenic acids in a DIO mouse model of metabolic inflammation. B6 mice were fed high‐fat diets (45% kcal) for 18 weeks: AIN‐93G (control); 10% (kcal) n6 1% n3 (HL; 10:1 ratio); 10% n6 5% n3 (HH; 2:1 ratio); 5% n6 1% n3 (LL; 5:1 ratio); 5% n6 5% n3 (LH; 1:1 ratio). Plasma cytokines and metabolic hormones were measured with a multiplexed bead assay. Results indicate the diets did not affect food intake, body mass, liver weight, subcutaneous, gonadal, retroperitoneal and total body fat, fasting glucose, HOMA‐IR, OGTT or plasma LPS. All experimental groups had higher body weight, fat depots, GIP and leptin than control. HH group consumed less food during weeks 3–12 and had lower plasma insulin at the end compared with the other groups. LH group had more mesenteric fat and lower plasma IL10 compared with the other experimental groups. LL group had lower GLP‐1 and MCP‐1 compared with the other 4 groups. Taken together, high n3 PUFA reduced anti‐inflammatory cytokine IL10, low PUFA reduced inflammatory cytokine MCP‐1, high PUFA reduced food intake and plasma insulin. In conclusion, PUFA at 6–15% kcal with different n6:n3 ratios had statistically significant, but seemingly minor effects on metabolic inflammation in a DIO mouse model. This project was funded by a USDA NIFA grant UTA01065.

Determination of human neutrophil antigen‐1, ‐3, ‐4 and ‐5 allele frequencies in English Caucasoid blood donors using a multiplex fluorescent DNA‐based assay

A number of DNA-based methods to genotype the alleles coding for HNA have been described, but all require the separate amplification and analysis of each allele. The aim was to develop a DNA-based method for simultaneous detection of HNA-1, HNA-3, HNA-4 and HNA-5 alleles. An allele-specific primer extension method was used in combination with magnetic beads from Luminex technology. PCR-sequence-specific primers (SSP) was used to resolve the presence of the HNA-1b allele in samples assigned by the Luminex bead assay as HNA-1a/-1b/-1c or HNA-1b/-1c. HNA allele frequencies were determined in a panel of 140 randomly selected English Caucasoid blood donors. HNA allelic types were compared with historical results, and 100% concordance was found. Only eight of the 97 samples used in the validation required additional testing by PCR-SSP. Allele frequencies were determined in the blood donor population as follows: 0·318 for HNA-1a, 0·668 for HNA-1b, 0·014 for HNA-1c, 0·768 for HNA-3a, 0·232 for HNA-3b, 0·882 for HNA-4a, 0·118 for HNA-4b, 0·736 for HNA-5a and 0·264 for HNA-5b. A multiplex Luminex bead assay for the simultaneous detection of HNA-1, HNA-3, HNA-4 and HNA-5 alleles is described that enables rapid typing of donors to support HNA alloimmunized patients who require HNA-compatible blood products.

Profiling biomarkers in gingival crevicular fluid using multiplex bead immunoassay

ObjectiveBiomarkers in gingival crevicular fluid (GCF) have been investigated; however, measurements were limited by the small sample volume available. The aim of this study was to determine the levels of 40 different cytokines and chemokines in GCF samples. DesignEleven patients with generalised chronic periodontitis participating in a supportive periodontal therapy programme with remaining probing pocket depths (PDs) of >5mm were enrolled. One healthy and two diseased sites were sampled in each subject. Forty biomarkers in GCF were examined using a multiplex bead immunoassay. Porphyromonas gingivalis from the diseased sites was quantified by real-time polymerase chain reaction. ResultsTwenty-six biomarkers were detected in the GCF samples using the multiplex bead immunoassay. The levels of nine biomarkers were significantly different between the diseased and healthy sites after adjustment with Bonferroni's correction. The level of 26 biomarkers in diseased sites was compared between bleeding on probing (BOP)-positive and BOP-negative sites. Interleukin (IL)-1β and interferon-inducible protein (IP)-10 levels were significantly higher in BOP-positive diseased sites than BOP-negative diseased sites after adjustment for multiple comparisons (IL-1β, p=0.0007, IP-10; p=0.0009). In addition, the levels of IL-1β in GCF were found to be strongly correlated with the P. gingivalis ratio (r=0.646, p=0.0012). ConclusionIL-1β levels in GCF correlate with the PDs, BOP and the presence of P. gingivalis in subgingival plaque. Multiplex bead assays can be useful in GCF studies. These findings can help in identifying new diagnostic methods in the diagnosis of periodontal disease.

Multiplex Bead Based Immunoassays For The Serodiagnosis of Lyme Borreliosis

Laboratory diagnosis of Lyme borreliosis is based on the detection of specific IgG and IgM antibodies against relevant antigens of Borrelia burgdorferi. Serological diagnosis usually is performed by a two-step procedure. Where immunoassays for antibody screening exhibit high sensitivity, second-line tests, e.g. Western-blot or line-blot assays,show high specificity, and are used for confirmation of positive screening results. In addition to serology, in certain cases Borrelia spp. can be detected directly, for instance in synovial fluid from patients with Lyme arthritis by PCR or bacterial culture. With the development of multiplex technology, e.g. bead-based immunoassays, multiple antibodies to distinct bacterial antigens can be detected in a single run. Detection systems used in this context, e.g. analysers based on flow cytometry, can be highly standardised and automated. Furthermore, these analysers can be connected bidirectionally to an order-entry based laboratory information system, with random access for high throughput analysis. Thus, multiplex bead assays may have the power to replace the current two-step procedure.