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This study aimed to address the effects of frozen storage temperature and duration on the changes in physicochemical properties of beef myofibrillar protein. The beef was stored at −1, −6, −9, −12, and −18°C for 28, 84, 126, 168, and 168 days, respectively. The myofibrillar protein of beef samples denatured gradually with the extention of storage period. Regarding the samples stored at temperature range of −12∼−1°C, higher storage temperature resulted in more severe denaturation (the myofibrillar protein exhibited lower sulfhydryl content, Ca2+-ATPase activity, ionic bonds, hydrogen bonds, and higher surface hydrophobicity). Particularly, difference in −12 and −18°C did not yield significant effects upon the protein properties throughout 168-day storage P>0.05. These results indicated that lowering freezing temperature may not minimize myofibrillar protein denaturation in a limited storage duration, which was also confirmed by the quality properties of beef.

Cooking meat at different temperatures resulted in loss of fluids and alterations in its textures. This work signifies the effect of different cooking temperatures on the volumetric changes and biophysical properties of breast meat blocks obtained from broilers and yellow-feather chicken (YFC). Shrinkage in the length and volume of meat blocks of broilers were higher, whereas YFC showed an increase in cross-sectional area. At each cooking temperature (60°C, 70°C, 80°C, 90°C, and 100°C), mostly there were significant differences (p< .05) in volumetric changes found between the meat block of both breeds. Results from the biophysical properties indicated that the cooking of broilers meat below 80°C increases its hardness, the opposite was observed for YFC. Cooking at 100°C produced more hardness in YFC meat than broilers (34.15 N vs 26.80 N) (p< .05). Moreover, cooking at higher temperature produced early gel properties in broilers than YFC meat. SDS-PAGE results revealed a similar trend for both breeds except cooking at 100°C where broilers have more band intensities than YFC. LF-NMR (low-field nuclear magnetic resonance) and MRI (magnetic resonance imaging) data suggested that after cooking at each temperature YFC reserved more water than broilers. Physical meat quality traits have correlations with volumetric changes and cooking temperatures. Cooking of YFC meat at 90°C and 100°C has demonstrated similar correlation patterns with physical quality traits. Overall, cooking at 100°C caused more undesirable changes in YFC than broilers meat blocks. Therefore, it is suggested that cooking at 90°C could be a better temperature for YFC meat.

The effects of freezing temperature on the microstructure and moisture migration of beef were investigated, aiming to provide the potential theoretical basis for the beef storage. Drip loss, surface hydrophobicity, and secondary structure of myofibrillar proteins, ice crystal, and micro- and ultrastructure of meat were analyzed at 4°C, −1°C, −6°C, −9°C, −12°C, and −18°C, respectively. Results indicated that the drip loss and surface hydrophobicity of samples stored at −12°C were significantly lower than that stored at 4°C and −1°C (p0.05). Result from Fourier transform infrared spectroscopy suggested that protein denaturation occurred after storage. There was an increase in α-helices and decline in random coil at lower temperature (−12°C and −18°C). It was indicated that the samples stored at −12°C and −18°C could effectively restrain the denaturation of protein and maintain the stability of secondary structure. The analysis of the ice crystal and micro- and ultrastructure of the muscle indicated that the structure of samples stored at −12°C and −18°C had more integrity and was complete than that stored at 4°C and −1°C. The spaces (water “reservoir” and “channel”) where was the origination of drip were small. Furthermore, the results of low-field nuclear magnetic resonance and 1H magnetic relaxation image showed that the freezing at −12°C could inhibit the migration of immobilized water to free water.

During pregnancy, alterations take place in mother's immune system with the goal of maintaining a successful pregnancy, and delivering healthy offspring. Immune alterations include activation of the innate immune system and dampening of cell-mediated adaptive immunity. Due to these alterations, cell-mediated autoimmune diseases typically ameliorate during pregnancy. The objectives of this study were to evaluate whether C-reactive protein (CRP) concentration, a sensitive marker of systemic inflammation (1) is increased during MS pregnancy (2) predicts pregnancy-related co-morbidities associated with MS (3) predicts MS disease activity after delivery. CRP concentration was measured using a high sensitivity assay from seven prospectively collected serum samples of 41 MS patients and 19 controls during pregnancy and 6 months after delivery. Annualized relapse rates, EDSS, fatigue scores and obstetric details of the patients were recorded. Delivery-related CRP levels were significantly elevated both among MS patients and in controls. CRP levels were higher during pregnancy than during the postpartum period in both study groups. Delivery-related elevated CRP levels did not correlate with postpartum disease activity. MS patients with eventual gestational diabetes had a significantly higher median CRP in the beginning of pregnancy compared to non-diabetic MS patients (9.28 vs. 2.98 mg/l, p = 0.0025). MS patients reporting fatigue had a significantly higher CRP throughout pregnancy compared to patients without fatigue. Higher CRP values were associated with pregnancy-related co-morbidities but not with MS disease activity.

Exposure of the skin to UV radiation induces local inflammation. We hypothesized that inflammation induced by erythemal UV-B irradiation could elevate levels of serum C-reactive protein (CRP) and that suberythemal repeating doses of solar-simulating UV radiation (SSR) would produce photoadaptation to such inflammation. Separation-free high-sensitivity assays of CRP show an increase by 42% (P = 0.046) in CRP concentrations in healthy human subjects 24 h after a 3 minimal erythemal dose (MED) dose of UV-B delivered onto a 100 cm2 skin area. Preceding daily suberythemal doses of whole-body SSR for 10 or 30 consecutive days completely prevented the CRP increase. UV-B-induced skin erythema was partially attenuated by 30 preceding days of SSR only (P = 0.00066). After 10 daily SSR doses, the mean baseline CRP concentrations (0.24 +/- 0.21 mg/L) declined by 35% (P = 0.018). Using high-sensitivity analysis of serum CRP as the endpoint marker for cutaneous inflammation, we show that acute exposure of even a relatively small skin area to erythemal UV-B induces skin inflammation detectable also at the systemic level and that photoadaptation by preceding repeating suberythemal doses of SSR reduces signs of inflammation. Our data complement the view given by previous studies in that local photoadaptation also has systemic manifestations.

We describe the use of fluorophore-doped nanoparticles as reporters in a recently developed ArcDia TPX bioaffinity assay technique. The ArcDia TPX technique is based on the use of polymer microspheres as solid-phase reaction carrier, fluorescent bioaffinity reagents, and detection of two-photon excited fluorescence. This new assay technique enables multiplexed, separation-free bioaffinity assays from microvolumes with high sensitivity. As a model analyte we chose C-reactive protein (CRP). The assay of CRP was optimized for assessment of CRP baseline levels using a nanoparticulate fluorescent reporter, 75 nm in diameter, and the assay performance was compared to that of CRP assay based on a molecular reporter of the same fluorophore core. The results show that using fluorescent nanoparticles as the reporter provides two orders of magnitude better sensitivity (87 fM) than using the molecular label, while no difference between precision profiles of the different assay types was found. The new assay method was applied for assessment of baseline levels of CRP in sera of apparently healthy individuals.

Microspheres provide a solid phase substrate for bioaffinity binding similar to the walls of traditional test tubes and the wells of microtiter plates. The coated microsphere concentrates analyte molecules in the reaction volume on its surface. When the bioaffinity binding reaction has reached an equilibrium, the local concentration of the analyte in close proximity of the microsphere is orders of magnitude higher than the concentration of the analyte in the total reaction volume. The preparation and quality control of microspheres coated with bioactive material is less costly and labour intensive when compared to test tube or microwell plate coating procedures. In addition, the cost for logistics and transportation of microsphere reagents is lower than that of coated tubes or plates. Moreover, microspheres can be easily used in miniaturised assay formats and several different detection schemes can be employed in the measurement of microsphere-based assays. Several different types of microspheres are commercially available. The microspheres can be manufactured in different sizes from many materials, such as polystyrene, acrylate, and glass. The surface of the microspheres can be activated to enable covalent binding of biomolecules. Further, the microspheres may contain internal fluorochrome or magnetic material, for identification or separation purposes. In this paper we review different assay formats for single-step measurement of bioaffinity assays employing microspheres. The term single-step is used to describe assays where all reagents and the sample are mixed, incubated and measured without separate washing steps.

Recent developments in infrared laser technology have enabled the design of a compact instrumentation for two-photon excitation microparticle fluorometry (TPX). The microparticles can be used in immunoassays as the antibody-coated solid phase to capture an antigen and then detect it with a fluorescently labeled tracer antibody. Unlike most other methods, TPX technology allows low-volume, homogeneous immunoassays with real-time measurements of assay particles in the presence of a moderate excess of fluorescent tracer. In this study, the TPX assay system was used for the reagent characterization and the measurement of C-reactive protein (CRP) in diluted plasma samples, targeting the assay range useful in infectious disease diagnosis. The pentameric structure of the CRP permitted the optimization of an assay with the lowest detectable concentration of 1 microg/L (7.5 pM) by using a single monoclonal antibody both for capture and as the tracer. With a 1:200 predilution of samples, the measurement range of the assay was 1-150 mg/L, but an additional 1:10 dilution was required for higher concentrations. The TPX method showed a good correlation with the reference result obtained in a routine hospital laboratory, demonstrating the feasibility of the technology for immunodiagnostic applications.

One-step immunocapture enzyme-immunoassay (EIA) was compared with time-resolved fluoroimmunoassay (TR-FIA) for rapid diagnosis of influenza A infection by antigen detection. The high-affinity monoclonal antibodies (MAbs) recognising two independent epitopes on the conservative nucleoprotein were used for capture (MAb 44) and detection (MAb 107L) of antigen by both assays. The detection limit for purified recombinant influenza A virus nucleoprotein was approximately 10 pg by EIA and 5 pg by TR-FIA. The performance of the methods was evaluated by testing 43 known positive and 50 negative clinical specimens (nasopharyngeal washes and aspirates). The sensitivity and specificity was 93% and 92% for EIA and 100% and 98% for TR-FIA, respectively, in comparison to the reference A3/A1 TR-FIA. The relationship of 44/107L immunoassays has been evaluated: in comparison to 44/107L TR-FIA (100%), EIA confirmed 93% of positive and 94% of negative samples. In conclusion, the capture-detector pair of MAbs 44 and 107L can be used for the sensitive detection of influenza A viral antigen in clinical samples by both immunocapture methods. Despite the slightly lower accuracy of the EIA, widespread availability and economy of the EIA methodology makes it an advantageous alternative for the laboratory diagnosis of influenza A virus infections.

We evaluated a commercial immunoglobulin M (IgM)-capture immunoassay for the detection of Mycoplasma pneumoniae infections in 278 pediatric patients with community-acquired, radiographically defined pneumonia. Acute- and convalescent-phase serum samples were collected from all patients and were tested for M. pneumoniae-specific IgM and IgG antibodies by Platelia enzyme immunoassays (Sanofi Diagnostica Pasteur, Marnes la Coquette, France). Nasopharyngeal aspirates (NPAs) were collected at the time of admission to the hospital. A total of 227 NPAs were subjected to the detection of M. pneumoniae DNA by PCR, and 191 NPAs were cultured by using the Pneumofast kit (International Mycoplasma, Signeswere, France). Southern hybridization of PCR products and the IgM test with solid-phase antigen (Serion Immunodiagnostica, Würzburg, Germany) were used for additional confirmation of a positive result, which required agreement of at least two different methods. A total of 24 (9%) confirmed diagnoses of mycoplasma infection were made, 5 (21%) of which were in children <5 years of age. Of the positive children, 24 of 24 (sensitivity, 100%) were positive by the IgM-capture test with convalescent-phase serum, 19 of 24 (79%) were positive by the IgM-capture test with acute-phase serum, 19 of 24 (79%) were positive by IgG serology, 10 of 20 (50%) were positive by PCR, and 8 of 17 (47%) were positive by culture. An additional 5 (of 254) children were positive by the Platelia IgM test alone (specificity, 98%). When the PCR with Southern hybridization result was combined with the IgM-capture test result with the acute-phase sera, the sensitivity of rapid laboratory diagnosis increased to 95%. In conclusion, the IgM serology test was the single most valuable tool for the diagnosis of M. pneumoniae pneumonia in children of any age.

To investigate enhanced disease associated with a formalin-inactivated (FI) respiratory syncytial virus (RSV) vaccine, we studied the pulmonary inflammatory response to RSV in BALB/c mice immunized with live RSV, FI-RSV, or combinations of the two. After RSV challenge, the number of granular cells, the ratio of CD4+/CD8+ lymphocytes, and the level of Th2-like cytokine mRNAs in the bronchoalveolar lavage specimens in mice immunized first with live RSV and then with FI-RSV were lower than that in FI-RSV-immunized mice and close to that in live RSV-immunized mice. These data suggest that prior live RSV infection prevents most of the enhanced inflammatory response seen in FI-RSV-immunized mice and might explain lack of enhanced disease in older FI-RSV-immunized children. A live RSV vaccine might similarly decrease the risk of enhanced disease with non-live RSV vaccines.

Vaccination with formalin-inactivated respiratory syncytial virus (FI-RSV) caused excessive disease in infants upon subsequent natural infection with RSV. Recent studies with BALB/c mice have suggested that T cells are important contributors to lung immunopathology during RSV infection. In this study, we investigated vaccine-induced enhanced disease by immunizing BALB/c mice with live RSV intranasally or with FI-RSV intramuscularly. The mice were challenged with RSV 6 weeks later, and the pulmonary inflammatory response was studied by analyzing cells obtained by bronchoalveolar lavage 4 and 8 days after challenge. FI-RSV-immunized mice had an increased number of total cells, granulocytes, eosinophils, and CD4+ cells but a decreased number of CD8+ cells. The immunized mice also had a marked increase in the expression of mRNA for the Th2-type cytokines interleukin-5 (IL-5) and IL-13 as well as some increase in the expression of IL-10 (a Th2-type cytokine) mRNA and some decrease in the expression of IL-12 (a Th1-type cytokine) mRNA. The clear difference in the pulmonary inflammatory response to RSV between FI-RSV- and live-RSV-immunized mice suggests that this model can be used to evaluate the disease-enhancing potential of candidate RSV vaccines and better understand enhanced disease.