Microsphere-based Immunoassay Research Articles

Development of a flow cytometric immunoassay for recombinant bovine somatotropin‐induced antibodies in serum of dairy cows

Administration of recombinant bovine somatotropin (rbST) to enhance milk production in dairy cows is banned within the European Union. Therefore, methods for pinpointing rbST abuse are required. Due to the problematic detection of rbST itself in serum, methods are also focused on detecting changes in rbST-related biomarkers. In this study, a fast and easy-to-perform microsphere-based flow cytometric immunoassay (FCIA) for detection of rbST-induced antibodies in serum was developed. Until now, detection of rbST-induced antibodies was also problematic due to non-specific binding of serum proteins resulting in a high rate of false positive results. Therefore, five different sample preparation methods, i.e. dilution, octanoic acid precipitation, filtration, protein G purification, and a previously described generic FCIA sample preparation were critically compared to overcome non-specific binding to the microspheres. Only the generic FCIA sample pretreatment was effective in reducing non-specific binding. As a result, an absolute decision level for detecting rbST antibodies in serum of dairy cows was determined and its applicability was demonstrated. In accordance with biological expectations from literature, rbST antibodies were induced in three out of four rbST-treated dairy cows. These rbST-induced antibodies were successfully detected for up to 4 weeks after the last rbST treatment, whereas no false positive results were obtained for 27 untreated dairy cows. This is the first method, able to overcome the interference of serum proteins and therefore, can be applied with high confidence for screening unknown herds of cattle for rbST antibodies, an important biomarker for pinpointing at rbST abuse in cattle.

Cellular Immune Responses to Nine Mycobacterium tuberculosis Vaccine Candidates following Intranasal Vaccination

BackgroundThe identification of Mycobacterium tuberculosis vaccines that elicit a protective immune response in the lungs is important for the development of an effective vaccine against tuberculosis.Methods and Principal FindingsIn this study, a comparison of intranasal (i.n.) and subcutaneous (s.c.) vaccination with the BCG vaccine demonstrated that a single moderate dose delivered intranasally induced a stronger and sustained M. tuberculosis-specific T-cell response in lung parenchyma and cervical lymph nodes of BALB/c mice than vaccine delivered subcutaneously. Both BCG and a multicomponent subunit vaccine composed of nine M. tuberculosis recombinant proteins induced strong antigen-specific T-cell responses in various local and peripheral immune compartments. Among the nine recombinant proteins evaluated, the alanine proline rich antigen (Apa, Rv1860) was highly antigenic following i.n. BCG and immunogenic after vaccination with a combination of the nine recombinant antigens. The Apa-induced responses included induction of both type 1 and type 2 cytokines in the lungs as evaluated by ELISPOT and a multiplexed microsphere-based cytokine immunoassay. Of importance, i.n. subunit vaccination with Apa imparted significant protection in the lungs and spleen of mice against M. tuberculosis challenge. Despite observed differences in the frequencies and location of specific cytokine secreting T cells both BCG vaccination routes afforded comparable levels of protection in our study.Conclusion and SignificanceOverall, our findings support consideration and further evaluation of an intranasally targeted Apa-based vaccine to prevent tuberculosis.

Multiplex Method for Simultaneous Serological Detection of Porcine Reproductive and Respiratory Syndrome Virus and Porcine Circovirus Type 2

Porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) are major contributors to the porcine respiratory disease complex (PRDC). Routine serological diagnosis and surveillance play an important role in the prevention of PRDC, as it is a leading cause of economic losses to the swine industry. We herein describe an advanced microsphere-based immunoassay that permits the simultaneous detection of antibodies to PCV2 and PRRSV, thereby reducing the time and effort involved in testing. Recombinant PRRSV nucleoprotein antigen and the PCV2 capsid antigen were coupled to fluorophore-dyed beads with distinct spectral addresses. Weekly serum samples from 72 pigs that were experimentally exposed to either PCV2, PRRSV, or both PCV2 and PRRSV were used to validate the microbead assay (MBA) in comparison with the "gold standard" enzyme-linked immunosorbent assays. The kinetics of the PCV2- and PRRSV-specific antibody responses measured by the microbead assay were comparable to those of the standard assays; Spearman's rank correlations were 0.72 (P < 0.001) for PRRSV and 0.80 (P < 0.001) for PCV2. Diagnostic sensitivity and specificity were determined using field sera whose positive or negative status was determined by the standard tests. The diagnostic sensitivity and specificity were both 98% for PCV2 and were 91% and 93%, respectively, for PRRSV (kappa coefficients, 0.85 and 0.67 for PCV2 and PRRSV, respectively). Multiplexing did not interfere with assay performance or diagnostic sensitivity. Therefore, the described study demonstrates proof of concept for the development of more versatile and economical microbead array-based multiplex serological test panels for veterinary use.

RAPID DETECTION OF LILY SYMPTOMLESS VIRUS WITH CdTe QUANTUM DOTS BY FLOW CYTOMETRY

Lily symptomless virus (LSV) is the most common lily virus, being detected in many species and hybrids. We established a microsphere-based fluorescent immunoassay for the determination of LSV, using a polyclonal antibody against LSV covalently bound to carboxy-modified microspheres able to capture LSV antigen. A monoclonal antibody against LSV conjugated to quantum dots (QDs) was used as a fluorescent probe, enabling LSV to be fluorescently detected by a combination of encoded beads and QDs. This method was 16 times more sensitive than ELISA in the detection of LSV, and could potentially be applied to the simultaneous detection of inhomogeneous matter.

Simultaneous Detection of Antibodies to Mouse Hepatitis Virus Recombinant Structural Proteins by a Microsphere-Based Multiplex Fluorescence Immunoassay

We describe a new microsphere-based multiplex fluorescent immunoassay (MFI) using recombinant mouse hepatitis virus (MHV) proteins to detect antibodies to coronaviruses in mouse and rat sera. All the recombinant proteins, including nucleocapsid (N) and 3 subunits of spike protein, S1, S2, and Smid, showed positive reactivity in MFI with mouse antisera to 4 MHV strains (MHV-S, -A59, -JHM, and -Nu67) and rat antiserum to a strain of sialodacryoadenitis virus (SDAV-681). The MFI was evaluated for its diagnostic power, with panels of mouse sera classified as positive or negative for anti-MHV antibodies by enzyme-linked immunosorbent assay (ELISA) using MHV virion antigen and indirect fluorescent antibody assay. The reactivities of 236 naturally infected mouse sera were examined; 227 samples were positive by MFI using S2 antigen (96% sensitivity), and 208 samples were positive using N antigen (88% sensitivity). Based on the assessment by MFI using the S2 and N antigens, only 3 serum samples showed double-negative results, indicating a false-negative rate of 1.3%. In 126 uninfected mouse sera, including 34 ELISA false-positive sera, only 7 samples showed false-positive results by MFI using either the S2 or N antigen (94% specificity). Similarly, the S2 and N antigen-based MFI was 98% sensitive and 100% specific in detecting anticoronavirus antibodies in rat sera. Thus, this MFI-based serologic assay using the S2 and N antigens promises to be a reliable diagnostic method, representing a highly sensitive and specific alternative to traditional ELISA for detection of coronavirus infections in laboratory mouse and rat colonies.

Development and Validation of a Multiplex Microsphere-Based Assay for Detection of Domestic Cat ( Felis catus ) Cytokines

Cytokines are essential signaling molecules that mediate the innate immune response, and therefore their presence can be of diagnostic, prognostic, and pathogenic significance. Microsphere-based immunoassays allow rapid and accurate evaluation of cytokine levels in several species, including humans, dogs, and mice; however, technology to evaluate domestic cat (Felis catus) cytokines has been limited to single-analyte enzyme-linked immunosorbent assays (ELISAs). Microsphere-based immunoassays provide an attractive alternative technology for detecting and quantifying multiple analytes in a single assay using as little as 50 μl of sample. We describe the development and validation of a microsphere-based assay for three commonly analyzed domestic cat cytokines (gamma interferon, interleukin-10, and interleukin-12/interleukin-23 p40) using reagents from commercially available ELISAs. The assay was optimized for capture and detection antibody concentrations, streptavidin-phycoerythrin concentration, and number of microspheres. The validated lower and upper quantitation limits were 31 and 1,000 pg/ml for gamma interferon, 63 and 2,000 pg/ml for interleukin-10, and 39 and 625 pg/ml for interleukin-12/interleukin-23 p40. Cytokine concentrations in peripheral blood mononuclear cell supernatants were measured, and results obtained by the microsphere assay were correlated with values obtained with commercially available ELISA kits. This technology is a convenient and reproducible assay to evaluate domestic cat cytokine responses elicited by a variety of diseases.

53: Neuroprotective fractalkine in fetal alcohol syndrome

Fetal alcohol syndrome (FAS) is associated with mental retardation and neurodevelopmental abnormalities. Neuroprotective peptides (NAP+SAL) can prevent some of the alcohol-induced damage in FAS including fetal death, growth abnormalities and learning impairment. Fractalkine, a chemokine constitutively expressed in neurons throughout the CNS, has a role in neuroprotection reducing neuronal death from activated microglia. Using a model of FAS we evaluated if fractalkine is altered in FAS and if the neuroprotective peptides NAP+SAL work through preventing alterations in fractalkine. Using a well-characterized FAS model, timed, pregnant C57BL6/J mice were treated on gestational day 8 (E8) with alcohol (0.03 mL/g), placebo or alcohol+peptides. Embryos were harvested 24h later on gestational day 9 and 10 days later at E18. Fractalkine was measured using a microsphere-based multiplex immunoassay (Luminex xMAP, Millipore, MA). Statistical analysis included Kruskal-Wallis with P<.05 significant. Six hours after alcohol exposure, fractalkine levels were signficantly elevated(median 341pg/ml, range 263-424pg/ml) vs controls (median 228pg/ml, range 146-332pg/ml; P<.001; Figure). Embryos treated with alcohol and the peptides NAP+SAL prevented the alcohol induced increase in fractalkine (median 137, range 97-255 pg/ml, P<.001). At E18 (10d after exposure), fractalkine levels were similar in all groups (medians 1821, 1852 and 2013 pg/ml respectively, P=0.7). Prenatal alcohol exposure acutely results in elevation of the neuroprotective chemokine fractalkine, perhaps in an effort to counter the alcohol toxicity. Treatment with the peptides NAP+SAL along with the alcohol prevents the acute increase in fractalkine.

Multiplex suspension array for human anti-carbohydrateantibody profiling

Glycan-binding antibodies form a significant subpopulation of both natural and acquired antibodies and play an important role in various immune processes. They are for example involved in innate immune responses, cancer, autoimmune diseases, and neurological disorders. In the present study, a microsphere-based flow-cytometric immunoassay (suspension array) was applied for multiplexed detection of glycan-binding antibodies in human serum. Several approaches for immobilization of glycoconjugates onto commercially available fluorescent microspheres were compared, and as the result, the design based on coupling of end-biotinylated glycopolymers has been selected. This method requires only minute amounts of glycans, similar to a printed glycan microarray. The resulting glyco-microspheres were used for detection of IgM and IgG antibodies directed against ABO blood group antigens. The possibility of multiplexing this assay was demonstrated with mixtures of microspheres modified with six different ABO related glycans. Multiplexed detection of anti-glycan IgM and IgG correlated well with singleplex assays (Pearson's correlation coefficient r = 0.95-0.99 for sera of different blood groups). The suspension array in singleplex format for A/B trisaccharide, H(di) and Le(x) microspheres corresponded well to the standard ELISA (r > 0.94). Therefore, the described method is promising for rapid, sensitive, and reproducible detection of anti-glycan antibodies in a multiplexed format.

Cox-2 expression, PGE 2 and cytokines production are inhibited by endogenously synthesized n-3 PUFAs in inflamed colon of fat-1 mice

There is great interest in the role of polyunsaturated fatty acids (PUFAs) in promoting ( n-6 class) or inhibiting ( n-3 class) inflammation. Mammalian cells are devoid of desaturase that converts n-6 to n-3 PUFAs. Consequently, essential n-3 fatty acids must be supplied with the diet. We have studied the effect of endogenously produced n-3 PUFAs on colitis development in fat-1 transgenic mice carrying the Caenorhabditis elegans fat-1 gene encoding n-3 desaturase. Colonic cell lipid profile was measured by capillary gas chromatography in fat-1 and wild-type (WT) littermates fed standard diet supplemented with 10% (w/w) safflower oil rich (76%) in n-6 polyunsaturated linoleic acid (LA). Experimental colitis was induced by administrating 3% dextran sodium sulphate (DSS). Colitis was scored by histopatological analysis. Cyclooxygenase-2 ( Cox-2) expression was evaluated by real time polymerase chain reaction. Prostaglandin E 2 (PGE 2) levels and cytokine production were determined by enzyme and microsphere-based immunoassays, respectively. The n-6/ n-3 PUFA ratios in colonic cells of fat-1 mice were markedly lower (9.83±2.62) compared to WT (54.5±9.24, P<.001). Results also showed an attenuation of colonic acute and chronic inflammation in fat-1 mice with significant decreases in PGE 2 production ( P<.01) and Cox -2 expression ( P<.01). High levels of colitis-induced proinflammatory cytokines, interleukin (IL)-18, IL-1α, IL-1β, IL-6, monocytes chemotactic proteins 1, 2 and 3 (MCP 1,2,3), matrix metalloproteinase 9 and tumor necrosis factor α (TNF-α) were down-regulated in DSS acutely and chronically treated fat-1 mice. The expression of fat-1 gene in the colon was associated with endogenous n-3 PUFAs production, decreased Cox-2 expression, increased PGE 2 and cytokine production.

A microsphere-based duplex competitive immunoassay for the simultaneous measurements of aldosterone and testosterone in small sample volumes: Validation in human and mouse plasma

Background The small blood volumes available in rodent studies often limit adequate quantification of all hormones of interest. We report here the development of two new assays combining an extraction step with multiplex immunoassay (MIA) technology for the simultaneous determination of aldosterone and testosterone in 50 μl sample volume. Methods Following solvent extraction, aldosterone and testosterone competitive immunoassays are performed incorporating biotinylated tracers and antibody-coated beads each having a unique fluorescence. Quantification is via addition of streptavidin–R–phycoerythrin (SA–PE). The assays were validated and compared to established methods. Baseline hormone levels in mice from four different strains, and changes after ACTH and HCG stimulation in CD-1 mice are shown. Results The assays are sensitive (aldosterone 15 pg/ml, testosterone 12 pg/ml), reproducible (intra-/inter-assay imprecision aldosterone 5.1–15.6%/9.9–15.8% and testosterone 9.7–10.9%/7.7–11.4%) and correlate significantly to established assays ( r = 0.94–0.95). Baseline aldosterone levels varied between strains, but not between the genders. Testosterone was significantly higher in male of all strains except in C57BL/6× NMRI mice. After ACTH injection, aldosterone (median, interquartile range) rose from 354 (261–396) pg/ml to 2008 (875–2467) in male and from 260 (210–576) to 1120 (734–1528) in female CD-1 mice. HCG injection in the same strain increased testosterone in male mice only (3.5 (0.4–8.3) ng/ml to 31.8 (30.4–33.9) ng/ml, P < 0.01). Conclusions We describe a MIA for the simultaneous measurement of aldosterone and testosterone in small volumes after extraction. In addition to presenting a new tool for steroid research in rodent models, our data show strain-dependent differences in steroid hormone metabolism in rodents.

A microsphere-based immunoassay for rapid and sensitive detection of bovine viral diarrhoea virus antibodies

This study describes a novel blocking microsphere-based immunoassay for highly sensitive and specific detection of antibodies against bovine viral diarrhoea virus (BVDV). The intra- and inter-assay variability are 4.9% and less than 7%, respectively, and variability of bead conjugations is less than 6.6%. The diagnostic performance of the assay was evaluated by testing a total of 509 serum samples. Based on a negative/positive cut-off value of 30.3%, the assay has a sensitivity of 99.4% and a specificity of 98.3% relative to ELISA. The new microsphere immunoassay provides an alternative to conventional ELISA systems and can be used for high-throughput screening in the BVD control programmes.

A New Microsphere-Based Immunoassay for Measuring the Activity of Transcription Factors

There are several traditional and well-developed methods for analyzing the activity of transcription factors, such as EMSA, enzyme-linked immunosorbent assay, and reporter gene activity assays. All of these methods have their own distinct disadvantages, but none can analyze the changes in transcription factors in the few cells that are cultured in the wells of 96-well titer plates. Thus, a new microsphere-based immunoassay to measure the activity of transcription factors (MIA-TF) was developed. In MIA-TF, NeutrAvidin-labeled microspheres were used as the solid phase to capture biotin-labeled double-strand DNA fragments which contain certain transcription factor binding elements. The activity of transcription factors was detected by immunoassay using a transcription factor-specific antibody to monitor the binding with the DNA probe. Next, analysis was performed by flow cytometry. The targets hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-kappa B (NF-κB) were applied and detected in this MIA-TF method; the results that we obtained demonstrated that this method could be used to monitor the changes of NF-κB or HIF within 50 or 100 ng of nuclear extract. Furthermore, MIA-TF could detect the changes in NF-κB or HIF in cells that were cultured in wells of a 96-well plate without purification of the nuclear protein, an important consideration for applying this method to high-throughput assays in the future. The development of MIA-TF would support further progress in clinical analysis and drug screening systems. Overall, MIA-TF is a method with high potential to detect the activity of transcription factors.

Detection of Fumonisin B1 and Ochratoxin A in Grain Products Using Microsphere-Based Fluid Array Immunoassays

Grain products are a staple of diets worldwide and therefore, the ability to accurately and efficiently detect foodborne contaminants such as mycotoxins is of importance to everyone. Here we describe an indirect competitive fluid array fluoroimmunoassay to quantify the mycotoxins, fumonisin B1 and ochratoxin A. Both toxins were immobilized to the surface of microspheres using a variety of intermediate molecules and binding of biotinylated "tracer" antibody tracers determined through flow cytometry using streptavidin-phycoerythrin conjugates and the Luminex100 flow cytometer. Competitive assays were developed where the binding of biotinylated monoclonal antibodies to fumonisin B and ochratoxin A was competitively inhibited by different concentrations of those toxins in solution. Concentrations of fumonisin giving 50% inhibition were 300 pg/mL in buffer, 100 ng/g in spiked oats, and 1 μg/g in spiked cornmeal; analogous concentrations for ochratoxin A were 30 ng/mL in buffer, 30 ng/g in spiked oats, and 10 ng/g in spiked corn. The future challenge will be to expand the number of mycotoxins tested both individually and in multiplexed format using this platform.

Preparation of fluorescence-encoded microspheres in a core–shell structure for suspension arrays

Fluorescence-encoded microspheres are widely used in the detection and analysis of biological molecules, especially in suspension arrays. Here, we report an efficient strategy for the preparation of fluorescence-encoded polystyrene microspheres with desirable optical and surface properties. The micron-sized, monodisperse polystyrene seed beads were first synthesized by dispersion polymerization. Then, dye molecules and carboxyl functional groups were copolymerized on the surface of the seed beads by forming a core–shell structure. Rhodamine 6G (R6G) was used as a model dye molecule to prepare the fluorescent beads, and the fluorescence intensity of the beads can be precisely controlled by adjusting the quantity of R6G. These fluorescent beads were characterized by environmental scanning electron microscopy, laser scanning confocal microscopy, and spectrofluorometry. The differences of the fluorescence spectra between fluorescent beads and R6G in solution were investigated. Twelve kinds of fluorescent beads encoded with different R6G fluorescence intensities were prepared, and they can be clearly distinguished on a conventional flow cytometer. Furthermore, the encoded beads are stable in water and resistant to photobleaching, which is crucial for their potential applications in diagnostic assays and imaging. Detection of human alpha fetoprotein antigen via a sandwich microsphere-based immunoassay yielded a detection limit of 80 pg mL−1, demonstrating that the fluorescence-encoded microspheres synthesized herein are efficient in serving as the microcarriers in suspension arrays. As both the encoding and functionalizing procedures are made simultaneously, the newly designed technique is extremely simple and time-saving. Moreover, it could be readily applicable to the preparation of a wide size range of fluorescent particles made by polymerization.

Removal of Species Constraints in Antibody Detection

Serum antibodies from myriad species, particularly birds, can provide key information regarding the transmission and the expansion of the territory of emerging pathogens. Expedient antibody analysis is constrained by a lack of species-specific reagents, a deficiency potentially highlighted by the recent swine-origin influenza A virus (H1N1) outbreak. Available methodologies present difficulties that discourage thorough serologic monitoring of potential disease vectors or hosts. Rapid high-throughput procedures that combined serum amine labeling via biotinylation, contaminant removal, and microsphere-based immunoassays for antibodies to three arboviruses were developed. Agent-specific adaptations of this simple format should facilitate expanded surveillance and diagnostic capabilities regarding pathogens of human and veterinary importance.

Over-expression of receptor activator of nuclear factor-κB ligand (RANKL), inflammatory cytokines, and chemokines in periprosthetic osteolysis of loosened total hip arthroplasty

Loosening of total hip arthroplasty (THA) caused by periprosthetic osteolysis induced by ultra-high molecular weight polyethylene (UHMWPE) particles is a major clinical problem. We investigated whether there are differences between loosened THA patients and primary THA patients in (1) receptor activator of nuclear factor-κB ligand (RANKL) expression on periprosthetic bone marrow cells; (2) RANKL levels, osteoprotegerin (OPG)/RANKL ratios, the levels of inflammatory cytokines and chemokines in synovial fluid. We used flow cytometric analysis to detect RANKL expression on periprosthetic bone marrow cells. We used enzyme-linked immunoassay and multiplex microsphere-based immunoassay to measure RANKL, OPG, cytokines, and chemokines in synovial fluid. We found loosened THA patients had higher RANKL expression on osteoblastic stromal cells, higher levels of RANKL, interleukin (IL)-6, IL-8, IL-10, interferon-γ-inducible protein (IP)-10, monocyte chemoattractant protein (MCP)-1, monokine induced by interferon-γ (MIG), and lower OPG/RANKL ratios in synovial fluid than primary THA patients. There was positive correlation between the levels of IL-6, IL-8, IL-10, IP-10, MCP-1, or MIG and RANKL levels in synovial fluid or RANKL expression on osteoblastic stromal cells. These suggest that UHMWPE particles induce over-expression of RANKL, IL-6, IL-8, IP-10, MCP-1, and MIG in human periprosthetic microenvironment. This results in periprosthetic osteolysis and loosening of THA.

Modification and optimization of organosilica microspheres for peptide synthesis and microsphere-based immunoassays

A new generation of optically encoded organosilica microspheres, suitable for both solid phase synthesis and multiplexed microsphere-based assays, has recently been described. One of the challenges of producing this type of dual-purpose solid support is that the particles must maintain their morphology as well as their encoding during exposure to the solvents used for solid phase synthesis. In this article, organosilica microspheres are subjected to ammonia treatment methods for enhancing the condensation of the silica matrix and their subsequent resilience toward organic solvents and peptide synthesis reagents is described. The instability of the untreated supports toward organic synthesis reagents was found to be associated with the swelling and permeability of these microspheres in organic solvents. Post-synthesis ammonia treatment resulted in reduced permeability, as demonstrated by dye uptake studies. The treated microspheres exhibited enhanced stability against organic synthesis conditions and were characterized via a variety of techniques including electron microscopy, (29)Si-nuclear magnetic resonance (NMR) and optical microscopy. The ammonia-treated supports were subjected to an Fmoc peptide synthesis procedure and successfully applied in a model microsphere-based flow cytometric immunoassay.

Exogenous Immunoglobulin Downregulates T Cell Receptor Signaling and Cytokine Production

BACKGROUND: Immune globulin, a polyvalent solution of pooled human serum immunoglobulin, when administered intravenously (IGIV) is used as replacement therapy in patients with hypogammaglobulinemia. In addition, IGIV has immunosuppressive properties and is FDA-approved to treat idiopathic thrombocytopenic purpura; it has also been used to treat several other autoimmune diseases including autoimmune hemolytic anemia and Kawasaki Disease. The mechanisms of action underlying IGIVs immunomodulatory effects are poorly understood, although a few reports have shown IGIV can inhibit T cell activation-induced upregulation of CD38 and CD69 in vitro and variably affect cytokine secretion by Th1 and Th2 cells. We hypothesized that the immunosuppressive effects of IGIV might be lymphocyte subset specific and mediated through T cell antigen receptor signaling interference, thus modulating Th1/Th2 cytokine production.METHODS: Mature (CD45RO) and naïve (CD45RA) lymphocytes from adult and umbilical cord blood were cultured for 8 days in the presence of IGIV (Gamunex™, 0–2.0 mg/mL). T, B, and natural killer (NK) lymphocyte subsets were then quantified by flow cytometry (FACS) over a time course. Additionally, the integrity of the T cell antigen receptor signaling pathway was assessed by FACS, quantifying expression of CD25 and CD69 following anti-CD3 cross-linking. Finally, multiplexed microsphere-based immunoassays were used to quantify cytokine secretion from IGIV-treated cells.RESULTS: IGIV, in a dose-dependent manner, reduced lymphocyte proliferation up to 3-fold after 6 days in culture in both cord and adult blood. IGIV also decreased activation-induced upregulation of CD25 and CD69, with the highest concentrations of IGIV leading to a 70% reduction in cells with an activated phenotype. This inhibitory effect was sustained over a 7-day period in both CD4 and CD8 T cell subsets and was independent of maturation markers (CD45RA/CD45RO). The percentage and absolute number of NK cells varied with IGIV treatment, but a marked increase was observed by day 6. IGIV also selectively reduced the absolute number and percentage of CD45RO+ T cells in cord blood over a time course. Direct cell surface Ig binding was not detected on T cell subsets. IGIV, in a dose-dependent manner, significantly decreased Th2 cytokine secretion, specifically IL-5, IL-10, and IL-13. This effect was more pronounced in cord blood lymphocytes but also was observed in adult blood lymphocytes. IGIV treatment also decreased Th1 cytokine secretion, particularly IL-2 and IFN-γ.CONCLUSIONS: IGIV inhibits T cell proliferation and activation as well as Th1 and Th2 cytokine secretion in a dose- and time-dependent manner. These effects may be responsible for the immunophenotypic changes observed—specifically a reduction in mature T cells and expansion of NK cells. While it is not entirely clear how IGIV is mediating its effects, the inhibition of Th1 and Th2 cytokine production and reduction of T cell activation signals in vitro may explain its role in blocking autoimmune responses in the clinical setting. Further studies are required to identify more precisely the molecular target and mechanism of action of IGIV in vivo.

Vitreous Levels of Bevacizumab and Vascular Endothelial Growth Factor-A in Patients with Choroidal Neovascularization

To investigate the vitreous levels of bevacizumab and vascular endothelial growth factor-A (VEGF-A) after intravitreal injection of the drug in patients with choroidal neovascularization (CNV). Interventional case series. Eleven eyes of 11 patients with submacular hemorrhage and CNV due to age-related macular degeneration (n = 10) or angioid streaks (n = 1). All patients were treatment naïve except for a single dose of intravitreal injection of bevacizumab (1.25 mg/50 muL dose) and subsequent vitrectomy after various intervals (1-101 days) because of active and progressive lesion. Intravitreal free bevacizumab and VEGF-A levels were measured using enzyme-linked immunosorbent assay and microsphere-based immunoassay, respectively. Vitreous VEGF-A isoforms were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting. Intravitreal bevacizumab and VEGF-A levels were measured and pharmacokinetic parameters were calculated. Pharmacokinetics of intravitreal bevacizumab followed a 2-compartment model with initial and terminal half-lives of 0.5 and 6.7 days, respectively. Bevacizumab could be detected in all cases, ranging from 2.63 ng/ml to 165 microg/ml. The peak concentration was observed on the second day after intravitreal bevacizumab injection. Vitreous free VEGF-A levels ranged from 0.2 to 33.9 pg/ml and showed a negative correlation with the bevacizumab concentration (P<0.001; r = -0.955) and a positive correlation with time (P<0.001; r = 0.964). However, the percentage expression of VEGF-A(165) exhibited a positive correlation with the bevacizumab concentration (P = 0.032, r = 0.645) and a negative correlation with time (P = 0.007, r = -0.755). A time-dependent increase was found for the percentage expression of VEGF-A(189) (P = 0.023, r = 0.673). Neither bevacizumab- nor time-related alterations were found for VEGF-A(121). Based on pharmacokinetics, the interval of 6-7 weeks would be appropriate for efficacy, although clinical trials should guide dosing recommendations. Vitreous levels of free VEGF-A showed a negative correlation with the bevacizumab concentration, which confirmed the in vivo binding affinity of bevacizumab to VEGF-A. The analysis of the VEGF-A isoforms suggests differences of interaction between bevacizumab and individual VEGF-A isoforms.

Combining cytokines of different pathways may improve predictive power for radiation-induced lung toxicity

22177 Background: Although Transforming growth factor-beta (TGF-β) is a key player, radiation-induced lung toxicity (RILT) involves numerous cytokines of multiple pathways. We hypothesized that TGF-β combined with cytokines of other pathways such as Interleukin (IL)-1β and Macrophage inflammatory protein (MIP)-1β can predict RILT more accurately. Methods: Eligible subjects included patients with stages I-III non-small cell lung cancer (NSCLC) treated with RT alone or combined chemoradiation. Plasma TGF- β1 and 29 cytokine levels were measured using ELISA and a microsphere-based sandwich immunoassay at pre, 2, and 4 weeks during RT. Logistic Regression was used to test the correlations between cytokine concentration and RILT. Results: There were 25 patients, 6 with and 19 without RILT. Hazard factors TGF-β, MIP-1β, IL-1β, IL-15, GM-CSF, IL-8, IL-12p40, IP-10, IL-12p70, MIP-1α, TGF-α and protective factor IL-1ra were significantly associated with RILT under univariate analysis (X2 all ≥ 3.83, P all ≤ 0.05, -2log likelihood all ≤ 23.73, R2 all ≥ 0.14) at one or more time points. Limited by the case number, the 3 most significant cytokines (TGF-β, MIP-1β and IL-1β) with different functional pathways were chosen for multivariate analysis. The results showed that models using combined cytokines were significantly better than using TGF-β alone at pre and 2 weeks during RT (Table). Conclusions: Multiple cytokines besides TGF-β predict RILT. Models using multiple cytokines are better than using TGF-β alone. Study with a larger sample size is ongoing to validate this encouraging finding. Predictive Power for RILT (P all < 0.05) Time Model ID Cytokines in Model Equation X2 -2log likelihood R2 Pre-RT 1 TGF-β 5.1 22.4 0.18 2 TGF-β, MIP-1β & IL-1β 27.6 0.0 0.66 2 weeks 1 Δ TGF-β 7.3 20.3 0.25 2 Δ TGF-β, MIP-1β & IL-1β 11.4 16.0 0.36 4 weeks 1 Δ TGF-β 12.8 14.8 0.40 2 Δ TGF-β, MIP-1β & IL-1β 14.5 13.0 0.44 Δ TGF-β: relative level to pre-RT No significant financial relationships to disclose.