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Rats are used as animal models for many human diseases. Cytokines can serve as biomarkers indicative of these diseases or disease states. Techniques for measuring cytokine expression levels often do not provide the sensitivity needed to measure these biomarkers in biological fluids because the concentrations of many cytokines are below the detection limits of conventional methods. In this paper, we present ultra-sensitive digital immunoassays using Single Molecule Arrays (Simoa) for seven rat cytokines: TNF-α, IL-10, IL-17F, GM-CSF, IFN-γ, IL-4, and IL-1α. These ultra-sensitive immunoassays have limits of detection (LODs) in the femtomolar range and provide the ability to measure rat cytokines in serum below the LODs of conventional immunoassays. We also measured these cytokines in healthy rat serum to obtain baseline levels. The ability to measure cytokines present at low concentrations in rat serum will facilitate future studies of disease using rats as animal models. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: In the Luminex multiplexed immunoassay system, complex samples such as human serum are diluted to minimize disturbing matrix effects with a specific diluent. This diluent has to imitate the sample matrix to allow interpolation and has to provide optimal cytokine–antibody binding for all cytokines. Because diluents influence multiplex immunoassay results, this paper explores several methods to determine the quality of a chosen diluent. Materials and methods: Two commercially available diluents, DY997 and RD6 from R&D Systems, were compared in a 19-plex immunoassay setup from Luminex. Results: Using diluent DY997, multiplex signal intensity was reduced by 55% when spiked samples (chemokines and cytokines at 100 pg/mL) contained 50% v/v human serum, compared to samples containing 25% v/v. When using diluent RD6, signal intensity was reduced by 20% when samples contained 50% v/v human serum, compared to 25% v/v human serum. Diluent DY997 showed decreasing multiplex assay sensitivity with increasing protein concentrations, but not as low as in the presence of 50% v/v human serum. Conclusions: In a 19-plex setup, this paper describes signal intensity, assay sensitivity and background signal levels in relation to the total volume-fraction of serum and protein concentration. For the determination of cytokines in serum samples with the multiplexed system Luminex the diluent RD6 seems more appropriate than the diluent DY997. [Copyright &y& Elsevier]

Nasal allergen challenge of patients with allergic rhinitis results in increased numbers of inflammatory cells and increased production of pro-inflammatory cytokines including interleukin 5 (IL-5). We report a sensitive, noninvasive method to measure changes in the amount of mRNA for IL-5 in nasal epithelium and have used this method to detect alterations of IL-5 mRNA from patients undergoing a nasal allergen challenge. Ten grass or ragweed allergic adults were challenged out of season with appropriate pollen extracts at sufficient dose to give a rhinitis total symptom score of 5 on a scale of 12. After allergen exposure, symptoms were recorded hourly. At 0, 3, and 6 h after allergen exposure, secreted proteins were collected on filter paper strips and two superficial scrapings of nasal epithelium were obtained. The scrapings of epithelium were immediately immersed in 100 μl of RNAlater® (Ambion, Austin, TX) and stored at 4 °C for up to 1 month without loss of RNA quality. Total RNA was isolated and RT-PCR was performed. cDNA for IL-5 was then measured by real-time fluorescence quantitative PCR with Pre-Developed TaqMan® Assay Reagents (PE Biosystems, Foster City, CA). Sufficient RNA was isolated from eight subjects to measure IL-5 mRNA. Data were normalized for content of ribosomal RNA. The relative amount of cDNA for IL-5 was calculated by comparison with internal standards prepared from phytohemagluttinin-stimulated peripheral blood mononuclear cells. Messenger RNA for IL-5 was increased 8.7±2.7-fold at 3 h (p<0.01) and 39.5±20.9-fold at 6 h (p<0.01). Increased IL-5 mRNA levels at 6 h closely correlate with total symptom scores at 6 h (r=0.88; p=0.007). IL-5 protein was measured by ELISA in eluates from the filter papers. At 6 h, there was increased IL-5 protein (7.7±2.8 ng/ml) compared with time zero (1.8±0.5 ng/ml) (p=0.02). The levels of IL-5 protein did not correlate significantly with the symptoms score or with changes in the levels of IL-5 protein with IL-5 mRNA. These data show that changes in IL-5 mRNA in patients with allergic rhinitis undergoing an allergen challenge correlate with total symptom scores better than changes in IL-5 protein eluted from filter paper. Furthermore, these changes can be measured quantitatively in very small amounts of tissue. [Copyright &y& Elsevier]

Abstract: Subcellular fractionation has been an important tool in investigating human eosinophil structure and function, including localizing of cytokine/chemokines within granules, investigating granule protein translocation and intracellular transport during eosinophil secretion, and studying secretory mechanisms of granules. The resolution of organelles obtained by subcellular fractionation was improved considerably after the introduction of nonionic iodinated density-gradient metrizamide and Nycodenz media that, unlike sucrose, exhibit relatively low tonicity throughout the gradient. However, the structure and membrane preservation of isolated organelles were still compromised due to the lack of gradient isoosmolarity. This paper describes a detailed protocol of subcellular fractionation of nitrogen cavitated eosinophils on an isoosmotic iodinated density gradient (iodixanol – OptiPrep) and the isolation of well preserved and functional membrane-bound specific granules. [Copyright &y& Elsevier]

Abstract: Feasibility, amount of sample aliquots, processing time and cost are critical considerations for optimizing and conducting assays for large-population based studies. Well designed statistical approaches that quickly identify optimal conditions for a given assay could assist efficient completion of the laboratory assays for such studies. For example, assessment of the profile of secreted cytokines is important in understanding the immune response after vaccination. To characterize the cytokine immune response following smallpox vaccination, PBMC obtained from recently vaccinated subjects were stimulated with varying doses of live or UV-inactivated vaccinia virus and cultured for up to 8 days. In this paper, we describe a novel statistical method to identify optimal operating conditions for length in culture and virus MOI in order to measure a panel of secreted Th1, Th2, and inflammatory cytokines. This statistical method is comprised of two components. It first identifies a subset of the possible time in culture by virus MOI combinations to be studied. It then utilizes response surface analysis techniques to predict the optimal operating conditions for the measurement of each secreted cytokine. This method was applied, and the predicted optimal combinations of length in culture and virus MOI for maximum vaccinia-specific cytokine secretion were identified. The use of the response surface methodology can be applied to the optimization of other laboratory assays; especially when the number of PBMC available limits the testing of all possible combinations of parameters. [Copyright &y& Elsevier]

Effector T lymphocyte responses are considered critical for controlling human immunodeficiency virus type-1 (HIV-1) infection. The enzyme-linked immunospot (ELISpot) assay has emerged as a primary means of assessing HIV-specific T cell responses, and the development of objective methods that distinguish positive and negative ELISpot responses while properly controlling the rate of false positives is critical. In this paper, we consider several statistical methods that are helpful in defining such a positive criterion. Simulation results under a variety of scenarios suggest that a permutation-based criterion using a resampling adjustment for multiple comparisons yields the desired false positive rate while remaining competitive with other potential criteria in terms of sensitivity. These results also provide guidance on the effect of the number of experimental and negative control replicate wells on assay sensitivity. Application of different potential positive criteria using ELISpot assay results from IFN-γ-secreting T cells of HIV-1 seropositive and seronegative donors confirmed several of the results obtained under simulation. Our findings support the application of statistically-based positive criteria such as the permutation-based resampling approach in assessing HIV vaccine-induced T cell responses. Moreover, the proposed methods have potential utility in related HIV immunopathogenesis studies and in non-HIV clinical vaccine trials. [Copyright &y& Elsevier]

Different single-cell analyses for the detection of antigen-specific T cells based on antigen-triggered induction of cytokine production (elispot, intracellular cytokine staining, cytokine secretion assay, etc.) have been analyzed. In this paper we present the data of a thorough validation of the IFNγ Secretion Assay (ISA, Miltenyi Biotec, Bergisch Gladbach, Germany). In this assay the secreted IFNγ is bound to the cell surface and is then stained as an artificial surface molecule and analyzed by flow-cytometry. The introduction of five quality criteria markedly improved the reproducibility of this assay and made it very reliable (intra-assay variability<5%; inter-assay variability<20%). Recovery experiments further demonstrated that almost 100% of IFNγ+ labeled cells could be detected by this technology. In order to analyze which cell subsets contribute to IFNγ-production, we compared the results obtained in different individuals after VZAg-stimulation. Three different IFNγ-secretion patterns could be discerned. In Pattern 1 there is a predominant and almost equal contribution of T cells and NK cells with a minor contribution of CD3+CD56+ and B cells. Pattern 2, which is most abundant, is characterized by a predominance of NK cells (60–70%). Pattern 3 differs from the previous one in its minor contribution of NK cells. Here T cells predominate the IFNγ secretion. These results clearly demonstrate that the IFNγ+ subset distribution after VZAg-stimulation is not uniform and differs individually. Furthermore, the ISA-technology proves to be very useful in vaccine research. This was demonstrated by testing the IFNγ+ secretion pattern after HBsAg-stimulation in PBMC from HBsAg-vaccinated individuals. [Copyright &y& Elsevier]

Patients treated with interferons, other cytokines, or various biologically active proteins may form neutralizing antibodies, which can adversely affect clinical outcome. It is therefore important to understand how antibodies neutralize such soluble protein antigens and how best to quantitate such antibodies. By applying the mass action law to antigen–antibody reactions, we previously developed a mathematical model applicable in two situations: first, for antibodies having low affinity for the antigen concerned (the Constant Proportion (CP) case), and, second, for antibodies having high affinity (the Fixed Amount (FA) case). The results allowed calculation of neutralization titers which were independent of the particular assay method used. Neutralization by antibodies of intermediate affinity, however, requires different mathematical treatment because the mode of neutralization does not fit the two cases mentioned above.In this paper, theoretical neutralization curves were derived, based on the same mathematical model, for antibodies of intermediate affinity. We show that the slope of the neutralization curve relating residual active antigen to the concentration of antibodies is determined by the antibody association constant and the molar concentration of the effector antigen. It is therefore possible to infer the magnitude of the association constant from the observed neutralization curve. We show that values obtained for the neutralization titer of antibodies of intermediate affinity by the use of the formula previously described for the Fixed Amount and Constant Proportion cases may deviate from the theoretically sound values; the magnitude of the deviation can be estimated by applying the formulas described herein. These relationships should apply generally to antibody neutralization reactions with all biologically active soluble protein effector molecules that have a single and nonrepetitive epitope. [Copyright &y& Elsevier]