Microsphere Assay Research Articles

Highly parallel characterization of IgG Fc binding interactions

Because the variable ability of the antibody constant (Fc) domain to recruit innate immune effector cells and complement is a major factor in antibody activity in vivo, convenient means of assessing these binding interactions is of high relevance to the development of enhanced antibody therapeutics, and to understanding the protective or pathogenic antibody response to infection, vaccination, and self. Here, we describe a highly parallel microsphere assay to rapidly assess the ability of antibodies to bind to a suite of antibody receptors. Fc and glycan binding proteins such as FcγR and lectins were conjugated to coded microspheres and the ability of antibodies to interact with these receptors was quantified. We demonstrate qualitative and quantitative assessment of binding preferences and affinities across IgG subclasses, Fc domain point mutants, and antibodies with variant glycosylation. This method can serve as a rapid proxy for biophysical methods that require substantial sample quantities, high-end instrumentation, and serial analysis across multiple binding interactions, thereby offering a useful means to characterize monoclonal antibodies, clinical antibody samples, and antibody mimics, or alternatively, to investigate the binding preferences of candidate Fc receptors.

Flavivirus detection and differentiation by a microsphere array assay

Flaviviruses of the Japanese encephalitis virus (JEV) serocomplex include major human and animal pathogens that have a propensity to spread and emerge in new geographic areas. Different genotypes or genetic lineages have been defined for many of these viruses, and they are distributed worldwide. Tools enabling rapid detection of new or emerging flaviviruses and differentiation of important subgroups have widespread application for arbovirus diagnosis and surveillance, and are crucial for detecting virus incursions, tracking virus emergence and for disease control. A microsphere suspension array assay was developed to identify JEV serocomplex flaviviruses of medical and veterinary importance. Assay performance was evaluated using representative virus strains as well as clinical and surveillance samples. The assay detected all JEV serocomplex viruses tested in this study with an apparent analytical sensitivity equal or better than the reference real-time or conventional RT-PCR assays and was able to identify mixed virus populations. The ability to identify mixed virus populations at a high analytical sensitivity would be pertinent in the Australian context when attempting to detect exotic JEV or West Nile virus (WNV), and differentiate from endemic Murray Valley encephalitis virus and WNV-Kunjin virus. The relatively low cost, the ability to identify mixed virus populations and the multiplex nature makes this assay valuable for a wide range of applications including diagnostic investigations, virus exclusions, and surveillance programs.

Validation of the ligase detection reaction fluorescent microsphere assay for the detection of Plasmodium falciparum resistance mediating polymorphisms in Uganda

BackgroundMalaria remains a major public health problem, and its control has been hampered by drug resistance. For a number of drugs, Plasmodium falciparum single nucleotide polymorphisms (SNPs) are associated with altered drug sensitivity and can be used as markers of drug resistance. Several techniques have been studied to assess resistance markers. The most widely used methodology is restriction fragment length polymorphism (RFLP) analysis. The ligase detection reaction fluorescent microsphere (LDR-FM) assay was recently shown to provide high throughput assessment of P. falciparum SNPs associated with drug resistance. The aim of this study was to validate the reliability and accuracy of the LDR-FM assay in a field setting.MethodsFor 223 samples from a clinical trial in Tororo, Uganda in which P. falciparum was identified by blood smear, DNA was extracted from dried blood spots, genes of interest were amplified by PCR, amplicons were analysed by both RFLP and LDR-FM assays, and results were compared.ResultsSNP prevalence (wild type/mixed/mutant) with RFLP analysis was 8/5/87% for pfcrt K76T, 34/37/29% for pfmdr1 N86Y, 64/17/19% for pfmdr1 Y184F, and 42/21/37% for pfmdr1 D1246Y. These prevalences with the LDR-FM assay were 7/5/88%, 31/24/45%, 62/20/18%, and 48/19/33% for the four SNPs, respectively. Combining mixed and mutant outcomes for analysis, agreement between the assays was 97% (K = 0.77) for pfcrt K76T, 79% (K = 0.55) for pfmdr1 N86Y, 83% (K = 0.65) for pfmdr1 Y184F, and 91% (K = 0.82) for pfmdr1 D1246Y, with most disagreements due to discrepant readings of mixed genotypes.ConclusionThe LDR-FM assay provides a high throughput, relatively inexpensive and accurate assay for the surveillance of P. falciparum SNPs associated with drug resistance in resource-limited countries.

Comparative Impacts Over 5 Years of Artemisinin-Based Combination Therapies on Plasmodium falciparum Polymorphisms That Modulate Drug Sensitivity in Ugandan Children

Artemisinin-based combination therapies, including artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP), are recommended to treat uncomplicated falciparum malaria. Sensitivities to components of AL and DP are impacted by polymorphisms in pfmdr1 and pfcrt. We monitored changes in prevalences of polymorphisms in Tororo, Uganda, from 2008 to 2012. Polymorphic loci in pfmdr1 and pfcrt were characterized in samples from 312 children randomized to AL or DP for each episode of uncomplicated malaria (50 samples per arm for each 3-month interval) utilizing a fluorescent microsphere assay. Treatment outcomes and impacts of prior therapies were also characterized. Prevalence increased significantly over time for pfmdr1 N86 (AL: odds ratio [OR], 2.08 [95% confidence interval {CI}, 1.83-2.38]; DP: 1.41 [95% CI, 1.25-1.57]), pfmdr1 D1246 (AL: 1.46 [95% CI, 1.29-1.64]; DP: 1.36 [95% CI, 1.23-1.50]), and pfcrt K76 (AL: 3.37 [95% CI, 1.85-6.16]; DP: 5.84 [95% CI, 1.94-17.53], and decreased for pfmdr1 Y184 (AL: 0.78 [95% CI, .70-.86]; DP: 0.84 [95% CI, .76-1.50]); changes were consistently greater in the AL arm. Recent AL treatment selected for pfmdr1 N86, D1246, and 184F in subsequent episodes; DP selected for the opposite alleles. Genotypes with decreased sensitivity to AL components increased over time. This increase was greater in children receiving AL, suggesting that the choice of treatment regimen can profoundly influence parasite genetics and drug sensitivity. NCT00527800.

Human genetic polymorphisms in the Knops blood group are not associated with a protective advantage against Plasmodium falciparum malaria in Southern Ghana

BackgroundThe complex interactions between the human host and the Plasmodium falciparum parasite and the factors influencing severity of disease are still not fully understood. Human single nucleotide polymorphisms SNPs associated with Knops blood group system; carried by complement receptor 1 may be associated with the pathology of P. falciparum malaria, and susceptibility to disease.MethodsThe objective of this study was to determine the genotype and haplotype frequencies of the SNPs defining the Knops blood group antigens; Kna/b, McCoya/b, Swain-Langley1/2 and KCAM+/- in Ghanaian patients with malaria and determine possible associations between these polymorphisms and the severity of the disease. Study participants were patients (n = 267) admitted to the emergency room at the Department of Child Health, Korle-Bu Teaching Hospital, Accra, Ghana during the malaria season from June to August in 1995, 1996 and 1997, classified as uncomplicated malaria (n = 89), severe anaemia (n = 57) and cerebral malaria (n = 121) and controls who did not have a detectable Plasmodium infection or were symptomless carriers of the parasite (n = 275). The frequencies were determined using a post-PCR ligation detection reaction-fluorescent microsphere assay, developed to detect the SNPs defining the antigens. Chi-square/Fisher’s exact test and logistic regression models were used to analyse the data.ResultsAs expected, high frequencies of the alleles Kna, McCb, Sl2 and KCAM- were found in the Ghanaian population. Apart from small significant differences between the groups at the Sl locus, no significant allelic or genotypic differences were found between the controls and the disease groups or between the disease groups. The polymorphisms define eight different haplotypes H1(2.4%), H2(9.4%), H3(59.8%), H4(0%), H5(25.2%), H6(0.33%), H7(2.8%) and H8(0%). Investigating these haplotypes, no significant differences between any of the groups were found.ConclusionThe results confirm earlier findings of high frequencies of certain CR1 alleles in Africa; and shed more light on earlier conflicting findings; the alleles McCb, Sl2, Knb and KCAM- or combined haplotypes do not seem to confer any protective advantage against malaria infection or resulting disease severity. Based on these findings, in a very well-characterized population, malaria does not seem to be the selective force on these alleles.

Angiopoietin-2 Is Critical for Cytokine-Induced Vascular Leakage

Genetic experiments (loss-of-function and gain-of-function) have established the role of Angiopoietin/Tie ligand/receptor tyrosine kinase system as a regulator of vessel maturation and quiescence. Angiopoietin-2 (Ang-2) acts on Tie2-expressing resting endothelial cells as an antagonistic ligand to negatively interfere with the vessel stabilizing effects of constitutive Ang-1/Tie-2 signaling. Ang-2 thereby controls the vascular response to inflammation-inducing as well as angiogenesis-inducing cytokines. This study was aimed at assessing the role of Ang-2 as an autocrine (i.e. endothelial-derived) regulator of rapid vascular responses (within minutes) caused by permeability-inducing agents. Employing two independent in vivo assays to quantitatively assess vascular leakage (tracheal microsphere assay, 1–5 min and Miles assay, 20 min), the immediate vascular response to histamine, bradykinin and VEGF was analyzed in Ang-2-deficient (Ang-2−/−) mice. In comparison to the wild type control mice, the Ang2−/− mice demonstrated a significantly attenuated response. The Ang-2−/− phenotype was rescued by systemic administration (paracrine) of an adenovirus encoding Ang-2. Furthermore, cytokine-induced intracellular calcium influx was impaired in Ang-2−/− endothelioma cells, consistent with reduced phospholipase activation in vivo. Additionally, recombinant human Ang-2 (rhAng-2) alone was unable to induce vascular leakage. In summary, we report here in a definite genetic setting that Ang-2 is critical for multiple vascular permeability-inducing cytokines.

Multidetection of Paralytic, Diarrheic, and Amnesic Shellfish Toxins by an Inhibition Immunoassay Using a Microsphere-Flow Cytometry System

The presence of paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP), and amnesic shellfish poisoning (ASP) toxins in seafood is a severe and growing threat to human health. In order to minimize the risks of human exposure, the maximum content of these toxins in seafood has been limited by legal regulations worldwide. The regulated limits are established in equivalents of the main representatives of the groups: saxitoxin (STX), okadaic acid (OA), and domoic acid (DA), for PSP, DSP, and ASP, respectively. In this study a multidetection method to screen shellfish samples for the presence of these toxins simultaneously was developed. Multiplexing was achieved using a solid-phase microsphere assay coupled to flow-fluorimetry detection, based on the Luminex xMap technology. The multidetection method consists of three simultaneous competition immunoassays. Free toxins in solution compete with STX, OA, or DA immobilized on the surface of three different classes of microspheres for binding to specific monoclonal antibodies. The IC50 obtained in the buffer was similar in single- and multidetection: 5.6 ± 1.1 ng/mL for STX, 1.1 ± 0.03 ng/mL for OA, and 1.9 ± 0.1 ng/mL for DA. The sample preparation protocol was optimized for the simultaneous extraction of STX, OA, and DA with a mixture of methanol and acetate buffer. The three immunoassays performed well with mussel and scallop matrixes displaying adequate dynamic ranges and recovery rates (around 90% for STX, 80% for OA, and 100% for DA). This microsphere-based multidetection immunoassay provides an easy and rapid screening method capable of detecting simultaneously in the same sample three regulated groups of marine toxins.

Use of cross-reactive serological assays for detecting novel pathogens in wildlife: Assessing an appropriate cutoff for henipavirus assays in African bats

Reservoir hosts of novel pathogens are often identified or suspected as such on the basis of serological assay results, prior to the isolation of the pathogen itself. Serological assays might therefore be used outside of their original, validated scope in order to infer seroprevalences in reservoir host populations, until such time that specific diagnostic assays can be developed. This is particularly the case in wildlife disease research. The absence of positive and negative control samples and gold standard diagnostic assays presents challenges in determining an appropriate threshold, or ‘cutoff’, for the assay that enables differentiation between seronegative and seropositive individuals. Here, multiple methods were explored to determine an appropriate cutoff for a multiplexed microsphere assay that is used to detect henipavirus antibody binding in fruit bat plasma. These methods included calculating multiples of ‘negative’ control assay values, receiver operating characteristic curve analyses, and Bayesian mixture models to assess the distribution of assay outputs for classifying seropositive and seronegative individuals within different age classes. As for any diagnostic assay, the most appropriate cutoff determination method and value selected must be made according to the aims of the study. This study is presented as an example for others where reference samples, and assays that have been characterised previously, are absent.

Optimization of a Ligase Detection Reaction-Fluorescent Microsphere Assay for Characterization of Resistance-Mediating Polymorphisms in African Samples of Plasmodium falciparum

Genetic polymorphisms in the malaria parasite Plasmodium falciparum mediate alterations in sensitivity to important antimalarial drugs. Surveillance for these polymorphisms is helpful in assessing the prevalence of drug resistance and designing strategies for malaria control. Multiple methods are available for the assessment of P. falciparum genetic polymorphisms, but they suffer from low throughput, technical limitations, and high cost. We have optimized and tested a multiplex ligase detection reaction-fluorescent microsphere (LDR-FM) assay for the identification of important P. falciparum genetic polymorphisms. For 84 clinical samples from Kampala, Uganda, a region where both transmission intensity and infection complexity are high, DNA was extracted from dried blood spots, genes of interest were amplified, amplicons were subjected to multiplex ligase detection reactions to add bead-specific oligonucleotides and biotin, fragments were hybridized to magnetic beads, and polymorphism prevalences were assessed fluorometrically in a multiplex format. A total of 19 alleles from the pfcrt, pfmdr1, pfmrp1, pfdhfr, and pfdhps genes were analyzed by LDR-FM and restriction fragment length polymorphism (RFLP) analyses. Considering samples with results from the two assays, concordance between the assays was good, with 78 to 100% of results identical at individual alleles, most nonconcordant results differing only between a mixed and pure genotype call, and full disagreement at individual alleles in only 0 to 3% of results. We estimate that the LDR-FM assay offers much higher throughput and lower cost than RFLP. Our results suggest that the LDR-FM system offers an accurate high-throughput means of classifying genetic polymorphisms in field samples of P. falciparum.

Variation in human β‐defensin genes: new insights from a multi‐population study

Human β-defensin 2 (hBD-2) and hBD-3, encoded by DEFB4 and DEFB103A, respectively, have shown anti-HIV activity, and both genes exhibit copy number variation (CNV). Although the role of hBD-1, encoded by DEFB1, in HIV-1 infection is less clear, single nucleotide polymorphisms (SNPs) in DEFB1 may influence viral loads and disease progression. We examined the distribution of DEFB1 SNPs and DEFB4/103A CNV, and the relationship between DEFB1 SNPs and DEFB4/103A CNV using samples from two HIV/AIDS cohorts from the United States (n = 150) and five diverse populations from the Coriell Cell Repositories (n = 46). We determined the frequencies of 10 SNPs in DEFB1 using a post-PCR, oligonucleotide ligation detection reaction-fluorescent microsphere assay, and CNV in DEFB4/103A by real-time quantitative PCR. There were noticeable differences in the frequencies of DEFB1 SNP alleles and haplotypes among various racial/ethnic groups. The DEFB4/103A copy numbers varied from 2 to 8 (median, 4), and there was a significant difference between the copy numbers of self-identified whites and blacks in the US cohorts (Mann-Whitney U-test P = 0.04). A significant difference was observed in the distribution of DEFB4/103A CNV among DEFB1 -52G/A and -390T/A genotypes (Kruskal-Wallis P = 0.017 and 0.026, respectively), while not in the distribution of DEFB4/103A CNV among -52G/A_-44C/G_-20G/A diplotypes. These observations provide additional insights for further investigating the complex interplay between β-defensin genetic polymorphisms and susceptibility to, or the progression or severity of, HIV infection/disease.

Evaluation of humoral immune response to donor HLA after implantation of cellularized versus decellularized human heart valve allografts

We have evaluated the development of antibodies in response to donor allograft valve implant in patients who received cellularized and decellularized allografts and determined possible immunogenic epitopes considered responsible for antibodies reactivity. Serum samples from all recipients who received cellularized allografts or decellularized allografts were collected before valve replacement and at 5, 10, 30 and 90 days post-operatively and frozen until required. Tests were performed using the Luminex-based single human leukocyte antigen (HLA)-A, -B, -C and HLA-DR, -DQ antigen microsphere assay. To determine possible immunogenic epitopes, we used the HLAMatchmaker (HLAMM) software if applicable. Decellularized grafts elicited lower levels of anti-HLA class I and II antibody formation after implantation than cellularized allografts. All patients from cellularized group presented donor-specific antibodies class I and II within 3 months of observation period. In HLAMM analysis, the cellularized group had significantly higher numbers of immunogenic epitopes than decellularized group for both class I and II (p: 0.002 - cl I / p: 0.009 - cl II / p: 0.004 - cl I and II). Our findings demonstrate that the anti-HLA antibodies detected in the cellularized group were against donor HLA possible immunogenic epitopes and that in the decellularized group the anti-HLA antibodies were not against donor HLA possible immunogenic epitopes. These findings lead us to suggest that choosing sodium dodecyl sulfate decellularization process is the best alternative to decrease the immunogenicity of allograft valve transplant.

Detection of Paralytic Shellfish Toxins by a Solid-Phase Inhibition Immunoassay Using a Microsphere-Flow Cytometry System

Paralytic shellfish poisoning is a toxic syndrome described in humans following the ingestion of seafood contaminated with saxitoxin and/or its derivatives. The presence of these toxins in shellfish is considered an important health threat and their levels in seafood destined to human consumption are regulated in many countries, as well as the levels of other chemically unrelated toxins. We studied the feasibility of immunodetection of saxitoxin and its analogs using a solid-phase microsphere assay coupled to flow cytometry detection in a Luminex 200 system. The technique consists of a competition assay where the toxins in solution compete with bead-bound saxitoxin for binding to an antigonyautoxin 2/3 monoclonal antibody (GT-13A). The assay allowed the detection of saxitoxin both in buffer and mussel extracts in the range of 2.2-19.7 ng/mL (IC(20)-IC(80)). Moreover, the assay cross-reactivity with other toxins of the group is similar to previously published immunoassays, with adequate detection of most analogs except N-1 hydroxy analogs. The recovery rate of the assay for saxitoxin was close to 100%. This microsphere-based immunoassay is suitable to be used as a screening method, detecting saxitoxin from 260 to 2360 μg/kg. This microsphere/flow cytometry system provided similar sensitivities to previously published immunoassays and provides a solid background for the development of easy, flexible multiplexing of toxin detection in one sample.

Detection of Avian Influenza Viruses and Differentiation of H5, H7, N1, and N2 Subtypes Using a Multiplex Microsphere Assay

Detection of Avian Influenza Viruses and Differentiation of H5, H7, N1, and N2 Subtypes Using a Multiplex Microsphere Assay

Detection of Avian Influenza Viruses and Differentiation of H5, H7, N1, and N2 Subtypes Using a Multiplex Microsphere Assay

In an outbreak of highly pathogenic H5 and H7 avian influenza, rapid analysis of a large number of clinical samples with the potential to rapidly identify the virus subtype is extremely important. Herein, we report on the development of a rapid multiplex microsphere assay for the simultaneous detection of all avian influenza viruses (AIV) as well as the differentiation of H5, H7, N1, and N2 subtypes. A reverse transcriptase-PCR (RT-PCR) reaction, followed by hybridization of the amplified product with specific oligonucleotide probe-coated microspheres, was conducted in a multiplex format. Following incubation with a reporter dye, the fluorescence intensity was measured using a suspension array system. The limit of detection of the probe-coupled microspheres ranged from 1 x 10(5) to 1 x 10(9) copies of RT-PCR amplified product and the sensitivity of the multiplex assay ranged from 1 x 10(2.5) to 1 x 10(3.2) 50% embryo infectious doses of virus. The diagnostic accuracy of the assay, compared to the standard real-time RT-PCR, was evaluated using 102 swab samples from chickens exposed to low pathogenic AIV, and 97.05% of samples gave identical results with both the assays. The calculated specificity of the assay was 97.43%. Although the assay still needs to be validated, it appears to be a suitable diagnostic tool for detection and differentiation of avian influenza virus H5, H7, N1, and N2 subtypes.

Rapid, Simultaneous Detection ofClostridium sordelliiandClostridium perfringensin Archived Tissues by a Novel PCR-Based Microsphere Assay: Diagnostic Implications for Pregnancy-Associated Toxic Shock Syndrome Cases

Clostridium sordellii and Clostridium perfringens are infrequent human pathogens; however, the case-fatality rates for the infections are very high, particularly in obstetric C. sordellii infections (>90%). Deaths from Clostridium sordellii and Clostridium perfringens toxic shock (CTS) are sudden, and diagnosis is often challenging. Formalin-fixed, paraffin-embedded (FFPE) tissues usually are the only specimens available for sudden fatal cases, and immunohistochemistry (IHC) for Clostridia is generally performed but it cannot identify species. A clear need exists for a rapid, species-specific diagnostic assay for FFPE tissues. We developed a duplex PCR-based microsphere assay for simultaneous detection of C. sordellii and C. perfringens and evaluated DNA extracted from 42 Clostridium isolates and FFPE tissues of 28 patients with toxic shock/endometritis (20 CTS, 8 non-CTS, as confirmed by PCR and sequencing). The microsphere assay correctly identified C. sordellii and C. perfringens in all known isolates and in all CTS patients (10 C. sordellii, 8 C. perfringens, 2 both) and showed 100% concordance with PCR and sequencing results. The microsphere assay is a rapid, specific, and cost-effective method for the diagnosis of CTS and offers the advantage of simultaneous testing for C. sordellii and C. perfringens in FFPE tissues using a limited amount of DNA.

Surface Hydrophobicity of Culture and Water Biofilm of Penicillium spp.

Fungal surface hydrophobicity is involved in several functions in fungal growth and development. Water contact angles measurement has been used as a direct and simple approach for its characterisation in solid cultures. Microsphere adhesion assay is said to be the best method to assess cell hydrophobicity of filamentous fungi. This study aimed to apply these two methods to study hydrophobicity of Penicillium expansum and Penicillium brevicompactum grown as mycelial mats in solid culture, liquid culture and water biofilms. As result, both species in solid cultures were classified as hydrophobic with contact angles ≥90º, but in liquid cultures and water biofilms showed different levels of hydrophobicity when microsphere adhesion assay was applied. In addition, was found that biofilms have specific hydrophobic hyphae which may be involved in fungal ecological functions.

A new high-throughput method for simultaneous detection of drug resistance associated mutations in Plasmodium vivax dhfr, dhps and mdr1 genes

BackgroundReports of severe cases and increasing levels of drug resistance highlight the importance of improved Plasmodium vivax case management. Whereas monitoring P. vivax resistance to anti-malarial drug by in vivo and in vitro tests remain challenging, molecular markers of resistance represent a valuable tool for high-scale analysis and surveillance studies. A new high-throughput assay for detecting the most relevant markers related to P. vivax drug resistance was developed and assessed on Papua New Guinea (PNG) patient isolates.MethodsPvdhfr, pvdhps and pvmdr1 fragments were amplified by multiplex nested PCR. Then, PCR products were processed through an LDR-FMA (ligase detection reaction - fluorescent microsphere assay). 23 SNPs, including pvdhfr 57-58-61 and 173, pvdhps 382-383, 553, 647 and pvmdr1 976, were simultaneously screened in 366 PNG P. vivax samples.ResultsGenotyping was successful in 95.4% of the samples for at least one gene. The coexistence of multiple distinct haplotypes in the parasite population necessitated the introduction of a computer-assisted approach to data analysis. Whereas 73.1% of patients were infected with at least one wild-type genotype at codons 57, 58 and 61 of pvdhfr, a triple mutant genotype was detected in 65.6% of the patients, often associated with the 117T mutation. Only one patient carried the 173L mutation. The mutant 647P pvdhps genotype allele was approaching genetic fixation (99.3%), whereas 35.1% of patients were infected with parasites carrying the pvmdr1 976F mutant allele.ConclusionsThe LDR-FMA described here allows a discriminant genotyping of resistance alleles in the pvdhfr, pvdhps, and pvmdr1 genes and can be used in large-scale surveillance studies.

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.

Formation of stress-specific p53 binding patterns is influenced by chromatin but not by modulation of p53 binding affinity to response elements †

The p53 protein is crucial for adapting programs of gene expression in response to stress. Recently, we revealed that this occurs partly through the formation of stress-specific p53 binding patterns. However, the mechanisms that generate these binding patterns remain largely unknown. It is not established whether the selective binding of p53 is achieved through modulation of its binding affinity to certain response elements (REs) or via a chromatin-dependent mechanism. To shed light on this issue, we used a microsphere assay for protein–DNA binding to measure p53 binding patterns on naked DNA. In parallel, we measured p53 binding patterns within chromatin using chromatin immunoprecipitation and DNase I coupled to ligation-mediated polymerase chain reaction footprinting. Through this experimental approach, we revealed that UVB and Nutlin-3 doses, which lead to different cellular outcomes, induce similar p53 binding patterns on naked DNA. Conversely, the same treatments lead to stress-specific p53 binding patterns on chromatin. We show further that altering chromatin remodeling using an histone acetyltransferase inhibitor reduces p53 binding to REs. Altogether, our results reveal that the formation of p53 binding patterns is not due to the modulation of sequence-specific p53 binding affinity. Rather, we propose that chromatin and chromatin remodeling are required in this process.

High-throughput molecular diagnosis of circumsporozoite variants VK210 and VK247 detects complex Plasmodium vivax infections in malaria endemic populations in Papua New Guinea

Malaria is endemic in lowland and coastal regions of Papua New Guinea (PNG), and is caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. Infection by P. vivax is attributed to distinct strains, VK210 and VK247, which differ in the sequence of the circumsporozoite protein (pvcsp). Here, based upon sequence polymorphisms in pvcsp, we developed a post-PCR ligation detection reaction-fluorescent microsphere assay (LDR-FMA) to distinguish these P. vivax strains. This diagnostic assay was designed to detect the presence of both VK210 and VK247 P. vivax strains simultaneously in a high-throughput 96-well format. Using this assay, we analyzed human blood samples from the Wosera (n=703) and Mugil (n=986) regions to evaluate the prevalence of these P. vivax strains. VK210 and VK247 strains were found in both study sites. In the Wosera, single infections with VK210 strain were observed to be most common (41.7%), followed by mixed-strain (36.8%) and VK247 single-strain infections (21.5%). Similarly, in Mugil, VK210 single-strain infections were most common (51.6%), followed by mixed-strain (34.4%) and VK247 single-strain infections (14%). These results suggest that the distribution of P. vivax infections was similar between the two study sites. Interestingly, we observed a non-random distribution of these two P. vivax strains, as mixed-strain infections were significantly more prevalent than expected in both study sites (Wosera and Mugil χ2p-value<0.001). Additionally, DNA sequence analysis of a subset of P. vivax infections showed that no individual pvcsp alleles were shared between the two study sites. Overall, our results illustrate that PNG malaria-endemic regions harbor a complex mixture of P. vivax strains, and emphasize the importance of malaria control strategies that would be effective against a highly diverse parasite population.