Solid-phase Immunoassay Research Articles

The use of epitope arrays in immunodiagnosis of infectious disease: Hepatitis C virus, a case study

Serodiagnosis of infectious disease is often based on the detection of pathogen-specific antibodies in a patient’s blood. For this, mixtures of pathogen-related antigens are used as bait to capture corresponding antibodies in solid phase immunoassays such as enzyme immunoassay (EIA). Western blots provide improved diagnostic power as compared with EIA due to the fact that the mixture of markers in the EIA well is resolved and tested as individual antigens on the Western blot. Hence, confirmation of EIA results is accomplished using the antigen arrays of Western blots. Here we took this approach one step further and tested the attributes of using epitope arrays in a diagnostic platform coined “combinatorial diagnostics.” As a case in point, we tested a panel of phage-displayed epitope-based markers in the serodiagnosis of hepatitis C virus (HCV). The repertoire of HCV antigens was deconvoluted into panels of distinct linear and conformational epitopes and tested individually by quantitative EIA. Combinatorial diagnostics proved to be effective for the discrimination between positive and negative sera as well as serotyping of HCV.

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Aim In renal transplant (tx) recipients, diagnosis of acute cellular rejection or allograft nephropathy is based on serum creatinine (Cr) level and invasive biopsies. However, serum Cr level usually increases with the onset of any type of allograft injury, and biopsies do not always indicate graft injury. On the other hand, urine is produced and excreted by the kidneys may have a potential to reflect renal allograft injury. We have investigated the presence of osteoprotegerin (OPG), monokine induced by interferon-γ (MIG) and interferon- γ induced protein of 10 kDa (IP-10) in the urine of renal tx patients with acute or chronic cellular or humoral rejection or nephrotoxicity. Methods 51 renal tx recipients who were biopsied for possible rejection were included. Donor-specific HLA-antibodies (DSA) and biomarkers were measured by solid phase immunoassays. Results All patients with clinical and biopsy proven cellular rejection had significantly elevated level of all three OPG, MIG and IP-10 chemokines. OPG alone was elevated in patients with humoral, glumerulonephropathy or tubular injury. Increase level of IP-10 appeared to be more specifically correlated with acute cellular rejection. No correlation was noted with humoral rejection, diagnosed by the presence of DSA and/or C4d staining. Interestingly, all three markers were in the normal range in four patients with serum Cr>2.4, but no indication of cellular rejection. The latter patient’s biopsies showed tubular injuries with no clear cut indication. Conclusions Discussion: This study shows that quantitation of chemokines biomarkers in the urine of renal allograft recipients may be an effective method for detection of cellular rejection. This non-invasive method may be used as an alternative method to reduce the need for invasive biopsies which may cause clinical complications such as bleeding or infection.

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Many Histocompatibility and Immunogenetics laboratories across the world are increasingly using the tool of Virtual crossmatching to predict positive XM based on the information about the presence of donor-specific HLA antibody (HLA-DSA) in patients. However, lab to lab variation in quantifying HLA-DSA is the major limitation to determine a cutoff concentration of DSA that can predict a positive XM. Here, we address this issue by assessing a cutoff value for HLA-DSA detected by solid-phase immunoassay (represented as mean fluorescence intensity, MFI) in a retrospective analysis that can virtually predict a positive CDC-AHG XM. We performed a retrospective study for 61 XM performed for 53 renal transplant recipients. Availability of results for HLA antibodies in patients, HLA typing of donor and patient and result of CDC-AHG XM were the inclusion criteria. The MFI values for DSA in patients were compared with the results of CDC-AHG XM. HLA class I and/or class II DSA were identified in 34 renal transplant patients. DSA strength (MFI value) significantly correlated with the CDC-AHG XM outcome (r = 0.82; p < 0.0001). An MFI value of 11500 showed 100% specificity, 93% sensitivity, 100% positive predictive value and 88% negative predictive value for predicting a positive T cell and/or B cell CDC XM. Our results showed that HLA-DSA strength correlated well the XM outcome. A precise appraisal of HLA-DSA strength (i.e. a cutoff MFI value) can significantly enhance the predictability of virtual crossmatching. The clinical significance of HLA-DSA in cases where DSA is present in strength lower than the cutoff is critical in patient management and requires assessment.

Synthesis and characterization of a new polymer–drug conjugate with pH-induced activity

A well-defined, stimuli-responsive tetrapolymer with pH-responsive characteristics and targeting specificity has been synthesized by radical copolymerization of methacrylic acid, N-(2-hydroxypropyl)methacrylamide, methacryloyl glycylglycyl sulfamethoxazole, and N-(methacryloyl)glycylglycine 4-nitrophenyl ester. The structure and properties of tetrapolymer were investigated by NMR, FT-IR, UV–visible absorption, TEM and gel permeation chromatography. Incorporation of maleimide linker into tetrapolymer facilitates its conjugation with antibody fragments, as demonstrated by the solid-phase immunoassay experiments. The TEM image shows that tetrapolymer had self-assembled a spherical micelle with a diameter ranging from 50 to 150 nm. Altering the pH of the solution leads to a different extent of aggregation at pH 6.5–3.5, responding in accordance with the properties associated with the extracellular environment of solid tumors and endocytosis. Furthermore, fluorescence spectroscopy indicated a critical micelle concentration (CMC) of 1 mg/mL. Because of the solvation and ionization effects, the tetrapolymer showed considerably enhanced antibacterial activities against Escherichia coli in the presence of DMSO and the antibacterial activity increased with decreasing pH value.

Cutoff values and data handling for solid-phase testing for antibodies to HLA: Effects on listing unacceptable antigens for thoracic organ transplantation

Application of single-antigen solid-phase immunoassay (SPI) in virtual crossmatch-based organ allocation has been hindered by continued debate over the biologic relevance of detected antibodies and the relationship between cutoff mean fluorescence intensity (MFI) values with crossmatch testing results. To define SPI parameters accurately predicting crossmatch testing, we analyzed a series of anti-HLA antibodies from highly-sensitized patients awaiting lung or heart transplantation. Serial dilution of serum for SPI and cytotoxic crossmatch (CXM) enabled comparison over a wide spectrum of antibody "strengths". Receiver operating characteristic (ROC) analysis identified predictive cutoff values for HLA Class I and DR-specific antibodies. However, antibodies to HLA-DQ antigens demonstrated a significantly different characteristic, highlighting difficulties in interpretation of clinical significance. We also quantitatively characterized two data handling methods, MFI ratio (MR) and relative ratio (RR), to examine their potential impact on identifying unacceptable antigens. In combination with user defined cutoff values, MFI, MR and RR lead to discordant identification of antibodies. Establishment of cutoff values for MR and RR that are comparable to MFI demonstrated increased consistency in antibody identification. This single laboratory experience is an example of establishing statistically robust cutoff values and validation across different data handling methods for use of SPI in virtual crossmatch.

Effects of quercetin and epigallocatechin gallate on the inflammatory response of stimulated human peripheral blood mononuclear cells (PBMC)

This study evaluated the ability of epigallocatechin gallate enriched green tea extract (EGCG) to augment quercetin's (Q) inhibition of lipopolysaccharide (LPS)‐induced pro‐inflammatory cytokine secretion in vitro. PBMCs isolated from healthy male adults (n=5) were incubated for one hour with either 25 μM of Q or 5 μM of EGCG alone or in combination, followed by 23 hours of LPS (0.1 μg/ml) stimulation. Inflammatory effects were assessed by quantifying concentrations of three cytokines (TNF‐α, IL‐6, and IL‐1β) in cell supernatant using a chemoluminescence based solid‐phase sandwich immunoassay (Meso Scale Discovery, Gaithersburg, MD, USA). Q alone or in combination with EGCG significantly decreased TNF‐α (P=0.012 and 0.006, respectively), IL‐6 (P&lt;0.001 and P=0.001, respectively), and Il‐1β (P=0.013 and 0.006, respectively), however Q combined with EGCG did not significantly differ from Q alone. In conclusion, EGCG did not effectively augment the anti‐inflammatory effects of Q on LPS‐stimulated cytokine production by PBMC in vitro.

Predictive value of the Luminex single antigen panel for detecting flow cytometry cross-match positivity

Anti-human leukocyte antigen (HLA) antibodies are a major cause of allograft loss. Solid-phase immunoassays, notably Luminex technology, have lately begun to replace traditional techniques for detecting these antibodies. This platform, however, carries some restrictions in the type of antibodies it detects. For this reason, results using these new technologies must be correlated with results using traditional techniques that have proven clinical significance. We have correlated flow cytometry cross-match (FCXM) outcomes with results from Luminex assays. Serum samples from patients awaiting transplantation who had known anti-HLA antibodies as detected by Luminex were incubated with lymphocytes expressing (a) 1 of the HLA antigens detected by the sera or (b) several of them. Of the 169 T-cell FCXMs we performed, in 92 cases the target cell expressed only 1 of the HLA antigens detected by the serum. The results obtained correlated well with Luminex data (r = 0.84). A cutoff mean fluorescence intensity value of 6,500 for the Luminex single antigen assay yielded a sensitivity of 85% and specificity of 82% for detecting a positive FCXM. In the other 77 cases, the target cell expressed 2 or more of the HLA antigens detected by the serum. In this situation, the same cutoff proved a useful tool for differentiating negative from positive FCXMs.

Usefulness of a semi‐quantitative procalcitonin test and the A‐DROP Japanese prognostic scale for predicting mortality among adults hospitalized with community‐acquired pneumonia

The solid-phase immunoassay, semi-quantitative procalcitonin (PCT) test (B R A H M S PCT-Q) can be used to rapidly categorize PCT levels into four grades. However, the usefulness of this kit for determining the prognosis of adult patients with community-acquired pneumonia (CAP) is unclear. A prospective study was conducted in two Japanese hospitals to evaluate the usefulness of this PCT test in determining the prognosis of adult patients with CAP. The accuracy of the age, dehydration, respiratory failure, orientation disturbance, pressure (A-DROP) scale proposed by the Japanese Respiratory Society for prediction of mortality due to CAP was also investigated. Hospitalized CAP patients (n = 226) were enrolled in the study. Comprehensive examinations were performed to determine PCT and CRP concentrations, disease severity based on the A-DROP, pneumonia severity index (PSI) and confusion, urea, respiratory rate, blood pressure, age ≥65 (CURB-65) scales and the causative pathogens. The usefulness of the biomarkers and prognostic scales for predicting each outcome were then examined. Twenty of the 170 eligible patients died. PCT levels were strongly positively correlated with PSI (ρ = 0.56, P < 0.0001), A-DROP (ρ = 0.61, P < 0.0001) and CURB-65 scores (ρ = 0.58, P < 0.0001). The areas under the receiver operating characteristic curves (95% CI) for prediction of survival, for CRP, PCT, A-DROP, CURB-65, and PSI were 0.54 (0.42-0.67), 0.80 (0.70-0.90), 0.88 (0.82-0.94), 0.88 (0.82-0.94), and 0.89 (0.85-0.94), respectively. The 30-day mortality among patients who were PCT-positive (≥0.5 ng/mL) was significantly higher than that among PCT-negative patients (log-rank test, P < 0.001). The semi-quantitative PCT test and the A-DROP scale were found to be useful for predicting mortality in adult patients with CAP.

Frequency of HLA-DP-specific antibodies and a possible new cross-reacting group

Clinical studies have demonstrated that HLA-DP-specific antibodies can be detrimental to a transplanted kidney. The number of patients affected is proportional to the frequency of DP antibodies. We determined the frequency of HLA-DP-specific antibodies en toto and in the absence of cross-reactive DR antibodies. Of 650 waitlisted renal patients, 271 (42%) were reactive with HLA-DP antigens in solid-phase immunoassays. Of these 271 sera, 58 (21%) were negative for reactivity with cross-reactive DR antigens, and 16 (5.9%) had no class II antibody other than DP. Eliminating sera containing DR cross-reactive antibodies reduced the frequency but not the overall strength of DP antibodies. Although most DP antibodies were not expected to yield a positive cytotoxicity crossmatch, 2 DP-specific antibodies yielded cytotoxic crossmatch tests with titers of >512. The occurrence of HLA-DP-specific antibody differed significantly between previously transplanted (62%) and nontransplanted (38%) patients, but no difference was observed among patients categorized by race or sex. One serum demonstrated strong cross-reactivity between DP and DRB1*01:03 in the absence of DR1 or DR11 reactivity. Sequence alignments were performed and a possible new cross-reactivity between DRB1*01:03 and DP2, DP9, DP10, DP13, DP16, and DP17 was defined. Two additional sera confirmed this cross-reactivity.

Are We Doing Too Many HIT Tests?

Abstract 1240 Background:Heparin-induced thrombocytopenia (type II-HIT) is a serious immune mediated clinicopathologic syndrome that may lead to significant arterial or venous thromboembolism and moderate to severe thrombocytopenia. Type II-HIT is estimated to occur with a frequency of 0.2 to 5.0 % after exposure to heparin. Objective:To determine the incidence of type II-HIT and the necessity of its testing within a single, 450-bed, urban, teaching, community hospital. Methods:A retrospective review of the hospital database of inpatient and laboratory medical records was performed for a one-year period from July 2009 to July 2010. Medical records were reviewed to determine how many patients were tested for HIT (i.e., solid phase immunoassay or H-PF4-ELISA) - with the test results and how many confirmatory Serotonin Release Assays (SRA) were performed and the test results. Data was categorized into subgroups depending on the type of care, diagnosis, type of heparin and the dosage of heparin. The percentage of SRA positivity for different strengths of H-PF4-ELISA positivity was also investigated with optical density (OD) at 405 nm (weakly positive with O.D 0.4–0.99, intermediately positive with O.D. 1–1.99 and strongly positive if O.D. equal or more than 2). Pretest probability was also calculated by using 4T’s score for all patients with positive ELISA. Results:A total of 19,474 patient admissions occurred over the one year period. An estimated 213 patients had H-PF4-ELISA tests done. Overall H-PF4-ELISA testing incidence was 1% (213 out of 19,474), overall incidence of H-PF4-ELISA positivity out of all admissions during that year was 0.2% (42 out if 19,474). In this study higher frequency of HIT (H-PF4 ELISA) testing was observed in patients who were treated in ICU and cardiac care unit as well as patients treated for sepsis and venous thromboembolism (VTE). The most common type of heparin associated with higher rate of HIT testing was unfractionated heparin-UFH (prophylactic dose more than therapeutic dose) as compared to low molecular weight heparin-LMWH. The incidence of ELISA test positivity was greatest among patients in the ICU and cardiac care unit and patients with sepsis and VTE. Higher incidence of ELISA positivity was associated with UFH. Out of all performed HIT tests ELISA positivity was 19.71% (42 out of 213) and 171 ELISA tests were negative. 36 of the positive ELISA tests were weakly positive, indicating very low probability of HIT. Out of 42 positive ELISA tests 50% (total 21 of 42) had SRA and none of the SRA returned positive. For all 42 patients with positive ELISA tests, the pretest probability was calculated using the 4T score. These show that of the 42 positive ELISA tests, 52.38% (22 of 42) had a low probability, 28.57 (12 of 42) had an intermediate probability, and only 11.9% (5 of 42) had a high probability. Conclusions:From this study, we conclude that in our institute we are doing too many HIT studies. To avoid unnecessary testing and improve cost effectiveness, this study emphasizes the importance of considering clinical situation and pretest probability very carefully, prior to testing. Disclosures:No relevant conflicts of interest to declare.

Desensitization for Mismatched Hematopoietic Stem Cell Transplantation (HSCT)

Abstract 1955 Introduction:Sensitization to donor HLA antigens is associated with an increased risk of engraftment failure in HLA mismatched hematopoietic stem cell transplantation (HSCT). However, the use of partially mismatched donors is increasing since, at best, only 30% of patients have an HLA identical sibling donor available for transplantation, and many are unable to find a matched unrelated donor in a timely fashion. A non-myeloablative, T cell replete regimen for HSCT that utilizes post-transplant high dose, cyclophosphamide for graft-versus-host-disease (GVHD) prophylaxis was pioneered at Johns Hopkins and has permitted transplantation of over 200 patients with HLA-haploidentical related donors. The use of HLA haplo-identical donors greatly increases the numbers of potential donors for most HSCT candidates. Review of the evaluations of 148 consecutive candidates for haplotransplantation revealed that 95% had at least one haplo-identical donor with an average of 2.7 donors/patient. However donor specific HLA antibody (DSHA) was observed in 10.8% of patients. We report here, successful desensitization of (DHSA) to levels safe for HSCT in six broadly sensitized patients who had poor-risk hematologic malignancies and for whom there were no other donors for whom HLA specific antibodies were not an issue. Methods:The desensitization protocol was modified from that developed for renal transplant patients at the Johns Hopkins University Comprehensive Transplant Center and included alternate day, single volume plasmapheresis (PP) with low dose, 100mg/kg, anti-CMV hyper immune immunoglobulin (IVIg) under immunosuppression with tacrolimus and mycophenolate mofetil. Varying numbers of PP/IVIg treatments were scheduled prior to the non-myeloablative conditioning regimen according to each patient’s DHSA level. PP/IVIg was stopped during conditioning. All but one patient received one additional PP/IVIg at transplant day −1. HLA antibodies were assessed by solid phase immunoassays using panels of pooled HLA antigens, HLA phenotypes, and single HLA antigens in microbead suspension array immunoassays (GenProbe Lifecodes Inc., San Diego, CA; One Lambda, Inc., Canoga Park, CA) Results and conclusions:All six patients prior to desensitization had DHSA at levels sufficient to yield positive flow cytometric crossmatch (FCXM) tests defined as 12K molecules of equivalent soluble fluorochromes (MESFs). The donor specific antibodies were reduced to levels well below a positive FXCM in all six patients by the end of the PP/IVIG treatments and before transplantation through an average of 4.2 PP/IVIg treatments. The average reduction in the donor specific antibody strength was 71.5% (range: 52–91%). In three patients, the DHSA levels were reduced to negative by time of transplant. A fourth patient was transplanted with a DHSA level just below that consistent with a positive FCXM, but by three months post-transplant had completely eliminated the DHSA. Two patients received one additional post-transplant PP/IVIg, resulting in stable DSA levels well below a +FCXM. Sufficient post-transplant follow-up of more than four months was available for four patients of which 3 received grafts from haploidentical donors and 1 from an HLA-mismatched unrelated donor. All four of these fully engrafted with no acute GVHD episodes. These results demonstrate that desensitization can extend the opportunity for HSCT to sensitized patients with no other donor options.Table 1:Degree of sensitization and DSA reduction by PP/IVIg.Donor TypePre-Treatment PRAbAverage # PP/IVIg (range)cEnd of Treatment % DHSA ↓ChildSibMUDaclass I (2)class II (4)31268.5954.2 (3–6)71.5 ± 12.8aTwo patients received grafts from unrelated donors (MUD) matched for all but one HLA allele. These were at the DQB1 and DPB1 loci.bPRA = panel reactive antibody. As indicated, the six patients were broadly sensitized with antibodies that would have excluded the majority of donors without intervention.cThe transplant for one patient was delayed due to unavailability of the unrelated donor. Treatment was extended to prevent antibody rebound. These additional treatments are not included. Disclosures:Luznik:Otsuka Pharmaceuticals: Research Funding.

Serodiagnosis of Syphilis in the Recombinant Era: Reversal of Fortune

Serodiagnosis of Syphilis in the Recombinant Era: Reversal of Fortune

Impaired functioning in euthymic patients with bipolar disorder — HSV-1 as a predictor

There is a possible association between infectious agents and psychiatric disorders. Previous studies in the US provided evidence for cognitive impairment correlated with Herpes simplex virus type 1 (HSV-1) infection. For a replication study in Europe we chosed individuals diagnosed with bipolar disorder to analyse the correlation with HSV-1 infection. Antibody prevalence was analyzed by using solid phase immunoassay techniques. Cognitive functioning was tested with the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) Form A, the Trail Making Test A&B, and two subtests from the WAIS III: the Letter Number Sequencing Task and the subtest on information. History and psychopathology was assessed using structured interviews and validated rating scales (SCID, HRSD-21, YMRS, PANSS). Additionally, we investigated social functioning and quality of life using self-assessment-scales (SAS, LQLP). Prevalence rates of antibodies against diverse infectious agents did not differ significantly between patients and controls. We found a significant correlation between cognitive impairment in patients with bipolar disorder and the prevalence of antibodies directed against HSV-1. Cognitive functions were significantly impaired including language, attention, and immediate memory. The results of this study confirm previous findings suggesting that HSV-1 affects cognitive functions in patients with bipolar disorder. This may also result in more impaired functioning, less quality of life and difficulties in social adjustment.

Co-circulation of G1, G2 and G9 rotaviruses in hospitalized patients in Bangladesh during 2006-2009

Rotavirus strain diversity in Bangladesh has been explored since 1985 and as seen in other parts of the world, rotaviruses have shown tremendous strain diversity overtime. Rotavirus antigen was detected in stool specimens using a solid-phase sandwich-type enzyme immunoassay. A multiplex reverse transcription polymerase chain reaction (RT-PCR) was performed for rotavirus G and P genotypes. This current study was carried out between 2006 and 2009 during which time 1,607 (23%) of 7,058 fecal specimens tested positive for group A rotaviruses with the highest incidence rate being observed in winter each year. Genotyping of rotaviruses showed a sharp decline in G2P[4] between 2008 and 2009 with a gradual increase in G1 and G9 strains. Since the Government of Bangladesh is planning to include rotavirus vaccine in the national immunization program, these data on rotavirus strain diversity should be taken into consideration for vaccine strain selection.

Quantifying HLA-specific antibodies in patients undergoing desensitization

Two major desensitization protocols have been used to eliminate or reduce HLA antibodies to a level that allows transplantation with a low risk of antibody-mediated rejection (AMR). This review will focus on the antibody testing methods used to assess changes in the breadth and strength of antibody levels and the relative strength of donor HLA-specific antibodies (DHSAs). Correlations of solid-phase immunoassay (SPI) class I and II levels with the donor-specific T and B cross-match results have shown the acceptable levels of DHSA that correlate with a low risk for AMR. The DSHA levels determined by SPI correlate with cross-match results and with clinical outcome. Therefore, the results of either assay can be used to determine the risk of AMR and when treatment has reduced DSHA to a level safe for transplantation. Monitoring DSHA is important for guiding the number of treatments as well as the timing of additional treatments needed to achieve these acceptable levels. DSHA monitoring, in both protocols, uses the correlation of solid-phase antibody testing and the donor-specific cross-match to determine the efficacy of the protocol and when the acceptable level of DSHA is achieved permitting transplantation with minimal likelihood of AMR.

Clinical Relevance and IgG Subclass Determination of Non-HLA Antibodies Identified Using Endothelial Cell Precursors Isolated From Donor Blood

ABO and human leukocyte antigen (HLA) alloantibodies provide major immunologic barriers to successful transplantation; however, there is increasing recognition for the role of anti-endothelial cell antibodies (AECAs) in allograft rejection. We investigated the relationship between AECAs identified using donor-derived endothelial cell precursors (ECPs) and kidney allograft rejection and function. Sixty live donor kidney recipients were tested pretransplant for AECAs and HLA-antibodies using flow cytometric crossmatch tests and solid-phase bead immunoassays. Renal allograft function was assessed by serum creatinine (SCr) values collected at early (mean, 50 days) and late (mean, 815 days) time points posttransplant and by incidence and type of rejection. Immunoglobulin G (IgG) subtype determination of both AECAs and HLA antibodies bound to ECPs was performed using flow cytometry. Fourteen patients (23%) tested positive for donor-reactive IgG AECAs and had statistically higher SCr values and incidences of cellular rejection early posttransplant compared with 46 patients who tested negative (P=0.014 and P<0.05). SCr values were not statistically different late posttransplant. IgG subclass determination showed AECAs to be enriched for IgG2 and IgG4, subclasses that do not activate complement effectively. Detection of donor-reactive immunoglobulin M (IgM) AECAs did not correlate with increased SCr or incidence of rejection. Crossmatch tests performed using donor-derived ECPs allow for the identification of alloantibodies that are associated with cellular rejection and are distinct from alloantibodies detected using lymphocytes.

The role of Th1/Th2 disbalanced immune response in the determination of clinical features of autoimmune diabetes mellitus

Aim. To elucidate the role of Th1/Th2 polarization of immune response in LADA patients in the realization of the clinical phenotype of the disease. Materials and methods. 70 patients aged 21-61 (mean 41.3?1.0 yr) with DM diagnosed based on WHO criteria (1999). Groups 1 and 2 included 13 DM1and 57 DM2 patients (34.6?7.2 and 43.6?7.6 yr respectively). 27 DM2 patients (41.2?1.6 yr) presumably had LADA (P. Zimmet's criteria).Serum anti-GAD65, ICA, and IAA antibodies along with C-peptide were measured in fasting sera and 120 min after GTT by solid phase immunoenzymeassays following manufacturer's instructions with the use of a photometer for Multiscan EX microplates (ThermoLabSystems, Finland) at405 nm (for GAG and ICA) and 450 nm (for IAA and C-peptide). GAD, IAA, and C-peptides levels were calculated automatically from calibrationcurves. Mononuclear leukocytes were isolated by centrifugation in the ficoll-verographin density gradient. The cells thus obtained were resuspendedin the complete nutritient medium reducing their concentration to 2.0x10^6/ml. Phytohemagglutinin (Difco, Germany) was added (10 mcg/1 ml) tothe samples to stimulate mononuclear leukocytes; cell suspensions were further incubated for 24 hr. Initial and PGA-induced levels of IL-2, 4, 10 insupernatants of cell cultures were measured by solid phase immunoassay at 450 nm. Results. At least one type of autoantibodies (GAD, ICA or IAA) was identified in 24.3% of all DM patients (17/70) and in 18% of the DM2 patients(10/57). The level of anti-GAD and ICA ABs and percentage of AB-positive patients were higher in the LADA group while that of anti-IAA ABs amongDM1 patients without LADA. Two AB types at a time were found in 17% (4/23) of the patients with autoimmune DM in the absence of significantdifference between LADA and DM1. Patients with LADA had a significantly lower basal C-peptide level than DM2 patients. The was a tendencytoward lower level of stimulated C-peptide secretion in LADA patients compared with DM2 ones. It suggests impairment of beta-cell secretory functionaffected by the autoimmune process. We observed enhanced basal production of IFN-y by blood mononuclear leukocytes in all DM patients in theabsence of significant difference between the groups. Mitogen-activated production in all CD patients was lower than normal without inter-groupdifferences. Patients with DM2 had the inverted type of IL-2 secretion unlike those with autoimmune diabetes. In both cases it was significantly differentfrom normal values. There was a tendency toward higher basal production of IL-4 by mononuclear leukocytes in LADA and DM2 comparedwith CD1 which reflects pathogenetic peculiarities of beta-cell function in LADA differing from those in DM1 and responsible for slower impairment ofbeta-cell function in this condition. Basal and PGA- induced production of IL-10 was higher in LADA and DM2 than in DM1. It suggests enhancedsuppressor activity of leukocytes that may protect beta-cells from autoimmune destruction and determines gradual development of clinical symptoms ofinsulin deficiency. In contrast, low production of IL-10 in DM1 gives evidence of polarization of the immune response. Conclusion. The loss of functional parenchyma and manifestation of insulin deficiency in LADA occur at a relatively low rate due to the peculiarcharacter of cytokine-mediated cell interactions. It suggests the necessity of an active and careful diagnostic strategy with the use of immunologicalmethods for examination of elder patients presenting with a variety of pathogenetic variants of DM.

Poly(dimethylsiloxane) microchip-based immunoassay with multiple reaction zones: Toward on-chip multiplex detection platform

In this work, a poly(dimethylsiloxane) (PDMS) microchip-based immuno-sensing platform with integrated pneumatic micro valves is described. The microchip was fabricated with multiple layer soft lithography technology. By controlling the activation status of corresponding valves, reagent flows in the microchannel network can be well manipulated so that immuno-reactions only take place at designated reaction zones (DRZs). Four DRZs are included in the prototype microchip. Since these DRZs are all isolated from each other by micro valves, cross contamination is prevented. Using the inner surface of the all-PDMS microchannel as immunoassay substrate, on-chip sandwich format solid phase immunoassay was performed to demonstrate the feasibility of this immuno-sensing platform. Mouse IgG and fluorescein isothiocyanate (FITC) were used as the model analyte and the signal reporter respectively. Only 10 μl sample is needed for the assay and low detection limit of 5 ng/ml (≈33 pM) was achieved though low-cost polyclonal antibodies were used in our experiment for feasibility study only. Much lower detection limit may easily be achieved by using high quality monoclonal antibodies. Through a simple yet effective comparison experiment, it was also found that high concentration BSA blocking solution (5% BSA in PBS buffer) can effectively suppress the non-specific binding in the surface of the microfluidic channels. The encouraging results from mouse IgG immunoassay proved the feasibility of our microchip design. With slight modification of the assay protocol, the same chip design can be used for multi-target detection and can provide a simple, cost-effective and integrated microchip solution for multiplex immunoassay applications.

Influence of Banana Versus Sports Beverage Ingestion On 75-Km Cycling Performance And Exercise-Induced Inflammation

PURPOSE: One banana (BAN) contains about 27 gm carbohydrate (half as sugars), is a good source of potassium and antioxidants, and has a low-to-medium glycemic index. The purpose of this project was to compare the effect of ingesting BAN versus a 6% carbohydrate sports drink (CHO) on performance and exercise-induced inflammation in cyclists. Design: In this randomized, crossover study, 14 trained cyclists (age 37±1.9 y, Wattsmax 379±12.5) completed two 75-km cycling time trials (3 weeks apart) while ingesting BAN or CHO. Subjects provided blood samples, ingested 0.4 gm/kg carbohydrate from BAN or CHO, and then cycled on CompuTrainers (RacerMate Inc., Seattle, WA) for 75-km at the fastest pace possible. During the cycling bouts, subjects ingested 0.2 gm/kg carbohydrate from BAN or CHO every 15 min. Blood samples were taken immediately following and 1-h post-exercise, and analyzed for blood lactate and glucose, total blood leukocytes (WBC), and nine inflammatory cytokines (IL-6, TNFα, GM-CSF, IFNγ, IL-1B, IL-2, IL-8, IL-10, IL-12p70) using an electrochemiluminescence based solid-phase sandwich immunoassay (Meso Scale Discovery, Gaithersburg, MD, USA). RESULTS: 75-km cycling performance did not differ between BAN and CHO trials (2.41±0.06 and 2.36±0.05 h, respectively, P=0.258), and blood glucose and lactate did not differ during or after exercise (interaction effects, P=0.439 and 0.849, respectively). Exercise caused significant increases in WBCs, IL-10, IL-2, IL-6, IL-8, and TNFα, with significantly higher post-exercise levels during BAN for IL-10 (84%) and IL-8 (50%) (interaction effects, P=0.003 and 0.004, respectively). CONCLUSIONS: Ingestion of bananas during a 75-km cycling trial supported performance in a similar fashion to a 6% sports drink when carbohydrate intake was equated. Post-exercise elevations in several cytokines were similar between BAN and CHO trials, with significantly higher levels attained during BAN for the anti-inflammatory IL-10 cytokine and chemoattractant IL-8 chemokine. Supported by a grant from Dole Foods, Inc.

The Acute Effect of Ingesting a Quercetin-Based Supplement On Exercise-Induced Inflammation In Runners

PURPOSE: This study tested the acute anti-inflammatory influence of a quercetin-based supplement consumed by endurance athletes 15-min prior to an intense 2-h run. Design: In this randomized, crossover study, 20 long distance runners (N=11 males, N=9 females, age 38.4±2.1 y, VO2max 52.6±2.0) completed two 2-h treadmill runs at 70% VO2max (3 weeks apart) that incorporated a closing 15-min time trial. In double-blinded fashion, subjects ingested either four Q-chews or placebo chews (PL) 15 min prior to the treadmill runs. The four Q-chews provided 1000 mg quercetin, 120 mg epigallocatechin 3-gallate (EGCG), 400 mg isoquercetin, 400 mg eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), 1000 mg vitamin C, and 40 mg niacinamide. Subjects provided blood samples 30 min before, immediately following and 1-h post-exercise, and were analyzed for plasma quercetin concentration, total blood leukocytes (WBC), and nine inflammatory cytokines (IL-6, TNFα, GM-CSF, IFNγ, IL-1B, IL-2, IL-8, IL-10, IL-12p70) using an electrochemiluminescence based solid-phase sandwich immunoassay (Meso Scale Discovery, Gaithersburg, MD, USA). RESULTS: Plasma quercetin was elevated from 80.0±26.0 μg/L (pre-exercise) to 6,337±414 (post-exercise) and 4,324±310 μg/L (1-h post-exercise) after ingestion of Q-chews compared to no change in PL (interaction effect, P<0.001). No difference was measured between Q-chew and PL conditions in the distance run during the 15-min time trial (3.08±0.11 and 3.13±0.11 km, respectively, P=0.370). Exercise caused significant increases in WBCs, GM-CSF, IL-10, IL-1B, IL-2, IL-6, IL-8, and TNFα, but no differences in the pattern of change were measured between Q-chew and PL trials. CONCLUSIONS: Acute ingestion of Q-chews 15 min before heavy exertion caused a strong increase in plasma quercetin levels but did not counter post-exercise inflammation relative to placebo. These data are in contrast to the anti-inflammatory effect measured with this same quercetin-based supplement when consumed for two weeks prior to a 3-day period of intensified exercise (Med Sci Sports Exerc. 2009;41:1467-75). Taken together, these data imply that a prolonged Q-chew supplementation period is needed before anti-inflammatory effects can be achieved.