Diluted Serum Samples Research Articles

Optimisation and standardisation of a multiplex immunoassay of diverse Plasmodium falciparum antigens to assess changes in malaria transmission using sero-epidemiology

Background: Antibody responses have been used to characterise transmission and exposure history in malaria-endemic settings for over a decade. Such studies have typically been conducted on well-standardised enzyme-linked immunosorbent assays (ELISAs). However, recently developed quantitative suspension array technologies (qSAT) are now capable of high-throughput and multiplexed screening of up to hundreds of analytes at a time. This study presents a customised protocol for the Luminex MAGPIX © qSAT using a diverse set of malaria antigens. The aim is to develop a standardised assay for routine serological surveillance that is implementable across laboratories and epidemiological settings. Methods: A panel of eight Plasmodiumfalciparumrecombinant antigens, associated with long- and short-lived antibody responses, was designed for the Luminex MAGPIX © platform. The assay was optimised for key steps in the protocol: antigen-bead coupling concentration, buffer composition, serum sample dilution, and bead storage conditions. Quality control procedures and data normalisation methods were developed to address high-throughput assay processing. Antigen-specific limits of quantification (LOQs) were also estimated using both in-house and WHO reference serum as positive controls. Results: Antigen-specific bead coupling was optimised across five serum dilutions and two positive controls, resulting in concentrations operational within stable analytical ranges. Coupled beads were stable after storage at room temperature (22⁰C) for up to eight weeks. High sensitivity and specificity for distinguishing positive and negative controls at serum sample dilutions of 1:500 (AUC 0.94 95%CI 0.91-0.96) and 1:1000 (AUC 0.96 95%CI 0.94-0.98) were observed. LOQs were also successfully estimated for all analytes but varied by antigen and positive control. Conclusions: This study demonstrates that developing a standardised malaria-specific qSAT protocol for a diverse set of antigens is achievable, though further optimisations may be required. Quality control and data standardisation methods may also be useful for future analysis of large sero-epidemiological surveys.

Nanoscale fluorescent metal–organic framework composites as a logic platform for potential diagnosis of asthma

Asthma is a common chronic disorder, and the decreased hydrogen sulfide (H2S) production in the lung has been considered as an early detection biomarker for asthma. However, the detection of H2S in biological systems remains a challenge; because it requires the designed sensors to have the following features: nanoscale size, good biocompatibility, real-time detection, high selectivity/sensitivity, and good water stability. Here, we propose the potential of using nanoscale fluorescent metal–organic framework (MOF) composites Eu3+/Ag+@UiO-66-(COOH)2 (hereafter denoted as EAUC) as a logic platform for tentative diagnosis of asthma by detecting the biomarker H2S. This INHIBIT logic gate based on Eu3+@UiO-66-(COOH)2 (EUC) can be produced by choosing Ag+ and H2S as inputs and by monitoring the fluorescent signal (I615) as an output. Our fluorescent studies indicate that the EAUC exhibits excellent selectivity, extreme sensitivity (limit of detection: 23.53 μM), and real-time in situ detection of H2S. Further, MTT analysis in PC12 cells shows that the EAUC possesses low cytotoxicity and favourable biocompatibility that are suitable for the detection of biomarker H2S in vivo, as demonstrated by the successful detection of spiked H2S in the diluted serum samples. This work represents the possibility of using MOF-based logic platform for tentative diagnosis of asthma in clinical medicine.

Electrochemical Studies on the Binding between Surface-Tethered DNA Aptamers and Lysozyme

Lysozyme is an enzyme that is responsible for the catalysis of β - 1,4 bond breakage in peptidoglycan residues of Gram-positive bacterium cell walls between N-acetylmuramic acid and N-acetylglucosamine. Lysozyme is commonly applied in food industry and its elevated concentration might indicate a development of leukemia or kidney failure, hence it is necessary to elaborate fast and sensitive tools for its detection.Herein, we present the studies on the elaboration of electrochemical aptasensor for lysozyme determination. For that purpose thiolated aptamer probes were immobilized on gold electrode surface that was subsequently modified with 6-mercapto-1-hexanol as blocking agent. It was found that 2h incubation period is sufficient to form monolayers that efficiently bind to target analyte. Methylene blue at 2 μM concentration was chosen as redox indicator as it provided the highest biosensor response in comparison to other tested electroactive molecules. 5 min. incubation time enabled lysozyme detection within the range from 0.1 fM to 10 nM. The developed biosensor exhibited good selectivity toward target analyte as well as minor response toward a reference DNA aptamer specific for HER2 biomarker. The proposed sensor was also successfully used for discrimination of lysozyme in diluted serum samples.

Accurate measurement of extended half-life and unmodified factor VIII low levels with one-stage FVIII assays is dependent on the matrix of calibration curves.

The monitoring of factor VIII (FVIII) replacement therapy relies on the accurate measurement of FVIII activity over a large concentration range. However, unexplained overestimation of low FVIII levels has recently been reported with extended half-life recombinant FVIIIs. The objective of this study was to confirm previous publications indicating that the reagents used to generate the calibration curves determine the accuracy of the measurement of low FVIII levels. We generated FVIII calibration curves with FVIII-deficient plasmas or a commercial diluent buffer. We then measured FVIII levels in FVIII-deficient plasma spiked with plasma FVIII, a full-length recombinant FVIII (Advate® , Shire, Brussels, Belgium) and two extended half-life recombinant FVIIIs (Elocta® , Swedish Orphan Biovitrum, Woluwe Saint-Lambert, Belgium and Afstyla® , CSL Behring, Mechelen, Belgium). FVIII levels were also analyzed in spiked samples prediluted two- and fourfold, either in diluent buffer or in FVIII-deficient plasma to evaluate parallelism. Coagulation times of calibration curves generated with diluent buffer were longer than those with FVIII-deficient plasmas. This resulted in an overestimation of FVIII levels lower than 25IU/dL in spiked samples and in detection of FVIII activity (≥1IU/dL) in FVIII-deficient plasma. Predilution of samples with diluent buffer rather than with FVIII-deficient plasma also led to discordant results. Our data confirm that the generation of calibration curves by dilution in FVIII-deficient plasma is crucial for the accurate measurement of low FVIII levels. When serial dilutions of samples are analyzed, predilution in FVIII-deficient plasma is required to respect the parallelism criteria. These methods should be more generally implemented for coagulation instruments.

Staining Traditional Colloidal Gold Test Strips with Pt Nanoshell Enables Quantitative Point-of-Care Testing with Simple and Portable Pressure Meter Readout.

Traditional immunochromatographic test strips based on colloidal gold are effective devices for portable and low-cost point-of-care (POC) testing. Nevertheless, they still suffer from the limitation of qualitative or semiquantitative tests via naked-eye detection. Replacement of gold with other signal entities, such as magnetic particles or fluorescent particles, requires professional instrumentation to obtain quantitative results. A pressure-based assay with platinum nanoparticles (PtNPs) can provide quantitative results using a portable pressure meter but is also hampered by the long-term instability of PtNPs. Consequently, we developed a Pt-staining method based on test strips to create platinum nanoshells on the surface of colloidal gold. This method not only preserves the original advantages of colloidal gold with easy synthesis and decoration but also introduces PtNPs with excellent catalytic activity as signal labels to achieve sensitive quantitative detection. Myoglobin was tested as a model target, and the limit of detection was 5.47 ng/mL in 20% diluted serum samples, which satisfies the requirements for clinical monitoring of acute myocardial infarction. In addition, the two most common colloidal gold strips available in the marketplace were applied to demonstrate the compatibility of Pt-staining. Taking advantage of low cost, user-friendliness, compatibility, simplicity, and stability, colloidal gold test strips with Pt-staining are expected to satisfy the need for quantitative POC testing of biomarkers, especially in resource-limited regions.

Alkaline phosphatase ınterference in an unconjugated estriol assay causing a false positive Down syndrome screening result

Abstract Objective Decreased unconjugated estriol (uE3) concentrations increase calculated risk of Down syndrome. Therefore, falsely low uE3, due to assay interference, have the potential to cause false-positive screening results. Here we present a 35 years old woman with a pregnancy of 17+2 weeks. Materials and methods Second-trimester screening test was performed on the UniCelDxI 800 (Beckman Coulter, Brea, CA, USA) analyzer and her uE3 level was 0.21 ng/mL (0.21 MoM). Risk calculated for DS was 1/8. Measurements were repeated on IMMULITE 2000 XPi (Siemens Healthcare Diagnostics Inc., USA). uE3 result was 0.614 ng/mL (0.97 MoM). The risk for DS was negative with this system. There was no sign of fetal anomaly on three-dimensional ultrasound examination and cell-free fetal DNA screening test. We suspected assay interference for uE3. Results Serial dilutions of serum samples revealed nonlinearity. 36.3% increase was observed with heterophile antibody blocking tubes. The post-polyethylene glycol treatment resulted approximately the same uE3 levels as IMMULITE system. Addition of alkaline phosphatase Scavenger to serum increased the result by 90% showing that falsely low E3 result was due to an interferent reacting on assay medium. Conclusion Laboratories should be aware that falsely low uE3 results due to interference may be obtained.

Colorimetric determination of glucose in solution and via the use of a paper strip by exploiting the peroxidase and oxidase mimicking activity of bimetallic Cu-Pd nanoparticles deposited on reduced graphene oxide, graphitic carbon nitride, or MoS2 nanosheets.

This work describes the preparation of bimetallic Cu-Pd nanoparticles (NPs) on supports like reduced graphene oxide (rGO), graphitic carbon nitride (g-C3N4) and MoS2 sheets with a size of <10nm. rGO is found to be the best support for synthesizing Cu-Pd NPs with controlled shape, size and oxidation state. The Cu-Pd/rGO nanocomposite also demonstrated the best peroxidase and oxidase mimicking activity compared to Cu-Pd/g-C3N4 and Cu-Pd/MoS2 nanocomposites. The peroxidase mimicking activity of Cu-Pd/rGO was investigated in more detail, and a glucose oxidase (GOx) based glucose sensor was constructed that is based on the enzymatic formation of H2O2 and the Cu-Pd NPs-assisted oxidation of tetramethylbenzidine by H2O2 to give a blue-green coloration with absorption maxima at 652nm. The assay has a 0.29μM detection limit and a detection range that extends from 0.2 to 50μM. The method was applied to the determination of glucose in diluted serum samples, and results compared well to those acquired with a clinical analyzer. The method alsowas applied in a colorimetric paper-based test stripe that can detect glucose within 10min. Graphical abstract Schematic representation of a sensitive colorimetric glucoseassay based on bimetallic Cu-Pd nanoparticles supported on 2D nanosheets, and construction of a paper based device for rapid glucose detection.

Contamination by Pathogenic Microorganisms in Water used for Agricultural Irrigation on the Sabana de Bogota, Colombia

The aim of this study was to evaluate the microbiological quality of water used to irrigate food crops, which can lead to the spread of certain microorganism, such as <em>Escherichia coli</em>, which poses a high risk of contamination for food, especially food consumed as fresh vegetables and milk, making it important to monitor the hygienic state of water based on the detection of 'indicator' bacteria, which are normally only present in human and animal intestines, such as coliforms, which have been accepted worldwide as a unfailing parameter. To evaluate the water quality, the coliforms present in the water used for agricultural irrigation on the Sabana de Bogota were quantified in the La Ramada irrigation district. Water samples were taken at ten agricultural irrigation points in municipalities around Bogota. The methodology for the analysis included the technique that uses serial dilutions of samples in a specific culture media. In the different sampled sites where the water quality used for irrigation was evaluated, the total and fecal coliforms values were higher in CFU than the established limits (Decree 1594 of 1984) and values were found in some samples that were higher than $1×10^6 \>CFU \>100/mL$ for total coliforms and $1×10^5 \>CFU \>100/mL$ for fecal coliforms. This study indicated that, with the current conditions of some water sources on the Sabana de Bogotá, there is an inherent source of contamination, creating an obstacle for agricultural irrigation and a microbiological risk for consumers.

PH-Based Detection of Target Analytes in Diluted Serum Samples Using Surface Plasmon Resonance Immunosensor.

Detection of minute quantities of target antigens in serum samples (consisting of a mixture of proteins/biomolecules) can be achieved by enhancement of the capture efficiencies of heterogeneous immunosensors. An important process parameter which affects the capture of target analytes in such immunosensors is the pH of the solution as the target proteins present in the serum samples are charged molecules. Here, we investigated the capture of prostate-specific antigens (PSAs), first in a mixed-analyte system wherein the solution contained two other non-specific proteins along with the target analyte, using the surface plasmon resonance spectroscopy. There are no reports on the detection of antigens in a mixed system based on the optimization of the pH values of the carrier fluid, and this is the motivation of the present work. Further, we studied interference effects caused by the presence of these non-specific proteins in the mixed-analyte systems by artificially increasing the ratio of the interfering proteins to that of the target protein. Eventually PSA spiked into the rabbit serum samples was captured through the optimization of the pH of the solution. We could detect PSA in the serum samples when diluted to 100 times or more, where the amounts of other interfering proteins were ~ 66 times that of the amount of PSA. This study proposes a heterogeneous immunosensor to detect the target analytes in the diluted serum samples by tuning pH the of solution mixture, which can be utilized to detect disease biomarkers in serum samples.

Impedimetric immunosensor for dengue diagnosis using graphite screen-printed electrodes coated with poly(4-aminophenylacetic acid).

Dengue is an infectious viral disease transmitted by the Aedes aegypti mosquito, the control of which is complex. In addition, the clinical diagnosis is difficult to perform since it resembles other febrile infections; thus, the development of more effective methods to detect dengue virus (DV) has drawn increasing attention. The present study aimed to develop an impedimetric immunosensor for dengue diagnosis using a screen-printed electrode (SPE) functionalized with polymer films derived from 4-aminophenylacetic acid (4-APA). Data obtained from scanning electron microscopy (SEM) showed the deposition of a uniformly distributed material over the electrode surface. The immunosensor was based on the specific interaction between dengue antigen, NS1 protein, and anti-NS1 antibodies, IgG and IgM. In a characterization study using cyclic voltammetry (CV), the polymer film showed two oxidation peaks at +0.17 and + 0.35V in 0.50M sulfuric acid solution, indicating its adsorption and electroactivity at the SPE surface. Electrochemical impedance spectroscopy (EIS) measurements showed a higher charge transfer resistance (Rct) to the polymer film-modified SPE as compared with the bare SPE, corroborating a previous study. The best rNS1 concentration for immobilization was 1.00ng/mL, and the immunoreaction time between the antigen (Ag) and the antibody (Ab) was 20min. Dilutions of positive and negative clinical serum samples were evaluated by EIE, from which it was possible to elucidate, for the positive serum, that the more diluted the serum the greater the Rct. Negative serum also showed an analytical signal, probably due to the presence of non-specific antibodies; however, the generated signal presented values closer to the rNS1 signal, indicating good selectivity of the proposed platform. The experiments were repeated using bare SPE to verify the importance of the polymer film in biosensor construction. No significant difference was observed between these results. Graphical abstract Proposed schematic for the genosensor development.

Establishment of mouse neutralizing antibody detection method as potency assay for acellular pertussis vaccines

Objective To establish a functional antibody detection method for acellular pertussis vaccines in order to conveniently and effectively evaluate the production consistency and potency of acellular pertussis vaccine bulks and final products. Methods Chinese hamster ovary (CHO) cell clustering assay was optimized and used to measure titers of neutralizing antibodies against pertussis toxin in mouse immune serum samples. Results Vaccine samples were determined to be immunized intraperitoneally with 1/5 the human dose to ten female NIH mice (20-24 g, 5-week-old). Four weeks after immunization, blood samples were collected to isolate serum. Serially diluted serum samples were used to neutralize 0.1 IU/ml of pertussis toxin national reference product for 2 hours. Results of clustering were determined after 48 hours of incubation in pre-cultured CHO cell wells. The geometric mean of the serum dilution of the final unclustered wells was the neutralizing antibody titer of vaccine sample. There were significant differences in the titers of neutralizing antibodies elicited by acellular pertussis vaccines prepared with different manufacturing processes. Vaccine samples succeed or failed the modified intracerebral challenge assay (MICA) were easily distinguishable by neutralizing antibodies. Conclusion The method of detecting neutralizing antibodies to pertussis toxin greatly reduces the amount of animals used in research. CHO cell clustering assay that has better repeatability and precision can be used for monitoring and initial evaluation of the consistency and potency of the bulks and final products of pertussis vaccines prepared with different manufacturing processes. Key words: Pertussis toxin; Acellular pertussis vaccine; Neutralizing antibody; CHO cell; Potency assay

Target-programmed and autonomous proximity binding aptasensor for amplified electronic detection of thrombin

The development of sensitive and simple approaches capable of monitoring trace amounts of protein biomarkers is appealing for disease diagnosis and treatment. Towards this end, we have developed an electrochemical sensing platform for sensitive and simple detection of protein biomarkers by using thrombin as the model target molecules via a target-programmed proximity binding amplification approach. The binding of thrombin to the aptamer sequences in the partial dsDNA duplex probes induces the release of the ssDNA trigger strands, which catalyze subsequent assembly formation of many methylene blue (MB)-tagged proximate DNA motifs with the presence of the DNA fuel strands through cascaded toehold-mediated strand displacement reactions. Due to the proximity-binding effect, these MB-tagged proximate DNA motifs anneal with the capture probes on the sensor surface with significantly enhanced stability against the corresponding single component counterpart, thereby pulling the MB tags close to the sensor surface and generating substantially amplified signal responses for sensitive determination of thrombin down to 23.6 pM. In addition, such aptasensor can specifically discriminate thrombin from other interference proteins, and can also be utilized to monitor thrombin in diluted serum samples, demonstrating its great potential for sensitive determination of proteins for early disease diagnosis.

A nonfouling voltammetric immunosensor for the carcinoembryonic antigen based on the use of polyaniline nanowires wrapped with hyaluronic acid.

A non-fouling electrochemical immunosensor is described for determination of the tumor biomarker carcinoembryonic antigen (CEA). It is based on the use of composite wires made by chemical grafting of hyaluronic acid onto polyaniline nanowires. The modified nanowires possess excellent antifouling property both in single protein solutions and in dilute serum samples. The current of immunoelectrode exhibits a linear response in the 0.01pgmL-1 to 10,000pgmL-1 CEA concentration range and 0.0075pgmL-1 detection limit. This work demonstrates that coating an electrode with hyaluronic acid can largely reduce unspecific adsorption of proteins on the electrode surface. Graphical abstract Schematic of a nonfouling electrochemical immunosensor for the carcinoembryonic antigen. It is based on novel composite wires made through the chemical grafting of easily available hyaluronic acid (HA) onto polyaniline (PANI) nanowires. The HA/PANI demonstrated excellent antifouling property both in single protein solutions and human serum samples.

Protein-Induced Fluorescence Enhancement Based Detection of Plasmodium falciparum Glutamate Dehydrogenase Using Carbon Dot Coupled Specific Aptamer.

A novel 90-mer long ssDNA aptamer (NG3) covering a 40-mer random region targeting Plasmodium falciparum glutamate dehydrogenase ( PfGDH) developed through systematic evolution of ligands by exponential enrichment (SELEX) technique. The binding affinity of the aptamer to PfGDH discerned by circular dichroism (CD) was 0.5 ± 0.04 μM. The specificity of the aptamer toward the target was confirmed by gel electrophoresis and CD studies. The presence of two quadruplex forming regions, two big and four small stem loop structures with a δG of -7.99 kcal mol-1 for NG3 were deduced by computational studies. The spherical carbon dots (Cdots) of size 2-4 nm, synthesized by pyrolysis method using l-glutamate as a substrate were covalently linked to the amine modified aptamer. The Cdot with a band gap of 2.8 eV and a quantum yield of 34% produced fluorescence at ∼ λ410nm when excited at λ320nm. The quantum yield of Cdot-aptamer assembly was increased up to 40% in the presence of the PfGDH in solution. A linear relationship with a dynamic range of 0.5 nM to 25 nM (R2 = 0.98) and a limit of detection (LOD) of 0.48 nM was observed between the fluorescence intensity of the Cdots-aptamer conjugate and the concentration of PfGDH. The method could detect PfGDH with an LOD of 2.85 nM in diluted serum sample. This novel simple, sensitive and specific protein induced fluorescence enhancement based detection of PfGDH has a great potential to develop as a method for malaria detection.

Relation between lower respiratory tract microbiota and type of immune response against tuberculosis

In this study, the interaction between the microbiota of the lower respiratory tract and the type of immune response against Mycobacterium tuberculosis were studied. Bronchoalveolar lavage (BAL) samples of 10 tuberculosis (TB) patients and 5 cases suspected of lung cancer as control were obtained. Clinical symptoms were recorded for the TB patients. Serial dilutions of samples were prepared and cultured on a selective medium in order to count Streptococcus spp., Neisseria spp., Haemophilus spp. and Veillonella in the lung. To determine the type of immune response of Th1/Th2, Real Time-PCR method was used. The prevalence of Streptococcus spp. in the lungs of patients with TB increased when compared with the control group and the Th1-response in this group may be influenced by Neisseria and Haemophilus. However, reducing the number of Streptococcus and Neisseria can be involved in the development of Th1-response in the control group. Prevalence of Neisseria and Veillonella of the lung microbiota in this group may be associated with fever. The chest x-ray influenced both Th1 and Th2-responses in the lung, but only Th1-response was involved in reducing the weight of patients. The relationship between each of the clinical symptoms with immune response and with each genus of microbiota were reviewed separately, and these data are the new information on TB disease and can be the beginning of the study on the impact of genus, different species and strains of microbiota on the clinical signs of disease.

Accelerating the Peroxidase-Like Activity of Gold Nanoclusters at Neutral pH for Colorimetric Detection of Heparin and Heparinase Activity.

The peroxidase-like catalytic activity of gold nanoclusters (Au-NCs) is quite low around physiological pH, which greatly limits their biological applications. Herein, we found heparin can greatly accelerate the peroxidase-like activity of Au-NCs at neutral pH. The catalytic activity of Au-NCs toward the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) oxidation by H2O2 was 25-fold increased in the presence of heparin at pH 7. The addition of heparin not only accelerated the initial catalytic rate of Au-NCs but also prevented the Au-NCs from catalyst deactivation. This allows the sensitive colorimetric detection of heparin at neutral pH. In the presence of heparinase, heparin was hydrolyzed into small fragments, weakening the enhancement effect of catalytic activity. On the basis of this phenomenon, the colorimetric determination of heparinase in the range from 0.1 to 3 μg·mL-1 was developed with a detection limit of 0.06 μg·mL-1. Finally, the detection of heparin and heparinase activity in diluted serum samples was also demonstrated.

Improved quantification of a commercial enzyme-linked immunosorbent assay kit for measuring anti-MDA5 antibody

Objectives: To compare the quantitative performance for measuring anti-MDA5 antibody titer of two enzyme-linked immunosorbent assay (ELISA) systems: an in-house ELISA and the commercial MESACUPTM anti-MDA5 test.Methods: Anti-MDA5 antibody titer was measured in sera from 70 patients with dermatomyositis using an in-house ELISA and the MESACUPTM anti-MDA5 test side-by-side. For the commercial ELISA kit, serum samples diluted 1:101 were used according to the manufacturer’s protocol, but serial dilutions of sera were also examined to identify the optimal serum dilution for quantification.Results: The anti-MDA5 antibody titers measured by the in-house and commercial ELISAs were positively correlated with each other (r = 0.53, p = .0001), but the antibody titer measured by the commercial ELISA was less sensitive to change after medical treatment, and 37 (80%) of 46 anti-MDA5-positive sera had antibody titer exceeding the quantification range specified by the manufacturer (≥150 index). Experiments using diluted serum samples revealed that diluting the sera 1:5050 improved the quantitative performance of the MESACUPTM anti-MDA5 test, including a better correlation with the in-house ELISA results and an increased sensitivity to change.Conclusion: We improved the ability of the commercial ELISA kit to quantify anti-MDA5 antibody titer by altering its protocol.

Preanalytical validation of an in-house radioimmunoassay for measuring calprotectin in feline specimens.

Calprotectin is a marker of inflammatory disorders in people, and serum and fecal calprotectin were shown to be increased in dogs with gastrointestinal inflammation. Biomarkers of gastrointestinal inflammation are currently lacking in cats. The purpose of the study was to analytically validate the canine calprotectin radioimmunoassay for quantification of calprotectin in feline specimens. The immunoassay was analytically validated by determining assay working range, dilutional parallelism, spiking recovery, and intra- and inter-assay variability. Reference intervals for fecal calprotectin were established from healthy cats, and the influence of age, sex, and housing condition on fecal calprotectin was determined. The working range of the assay was 1.5-346.2μg/g of feces and 11.2-8654.4μg/L of serum. Observed-to-expected ratios (O/E) for serial dilutions of fecal extracts ranged from 77.3% to 112.0% (mean: 99.2%) and from 95.7% to 161.4% (mean: 118.5%) for spiking recovery. Intra- and inter-assay coefficients of variation for fecal samples were ≤11.0% and ≤12.8%, respectively. Fecal calprotectin concentrations ranged 1.5-66.5μg/g (3-day sample mean) and 1.5-126.1μg/g (3-day sample maximum). Housing conditions, sex, or age did not affect fecal calprotectin (all P>.05). For serial dilutions of serum samples, O/E ranged from 96.0% to 152.0% (mean: 115.7%). Serum calprotectin concentrations in healthy cats ranged from 108.8 to 255.3μg/L (median: 158.2μg/L). The canine radioimmunoassay for the measurement of calprotectin is analytically sensitive, linear, reproducible, accurate, and sufficiently precise (CVA ≤43.2%) for use with feline feces (with a loss of accuracy at high calprotectin concentrations). The RIs for feline fecal calprotectin are comparable to those established for dogs. Independence of fecal calprotectin from age and sex agrees with findings in dogs.

Direct Determination of Selenium in Serum Matrix by Electrothermal Atomic Absorption Spectrometry: Application on Healthy Individuals from Algeria

Selenium is a trace element implicated significantly in oxidative stress in biological systems. In this work, a direct method for selenium determination in serum samples by electrothermal atomic absorption spectrometry with Zeeman background correction is proposed.Serum samples were five-fold diluted in a 0.14% HNO3 and 0.2% Triton X-100 solution. Aliquots of 20 µL of the diluted serum samples were directly introduced into transversely heated graphite tubes. A total of 5 µg Pd and 3 µg Mg(NO3)2 was used as chemical modifier. Optimization of heating program was conducted by varying pyrolysis and atomization characteristic values. The optimal conditions were found to be 1400 and 2000 °C for pyrolysis and atomization temperatures respectively and 11 seconds for pyrolysis hold time.The standard additions method was employed for calibration. Intra-day and inter-day validation using quality control samples at each point of the addition calibration curve were performed. Good accuracy, precision and recovery were achieved with the proposed method (less than 5% for accuracy and precision). A characteristic mass of 92.550 pg, a limit of detection of 4.010 ppb and a limit of quantification of 13.375 ppb in undiluted serum samples were obtained.Several reference values of serum selenium concentrations were reported. As an application of the present method, selenium levels were determined in serum samples collected from 20 healthy individuals from Northern-Center of Algeria. Selenium concentrations ranged from 86.883 to 133.955 ppb with a mean value of 110.824±11.898 ppb.

Secondary Syphilis in Man: Prozone Effect

Introduction: Syphilis consists in a systemic infect contagious pathology with a chronic character. The etiological agent consists of an anaerobic spirochete bacterium, Treponema pallidum. Laboratory diagnosis can be made through a direct investigation of the etiologic agent or non-treponemal (VDRL) and treponemal (FTA-Abs) serological tests. False-negative results are rare. Objectives: The present report presents a rare case of a man with a secondary syphilis syndrome who was initially undiagnosed due to the prozone effect, since he presented VDRL in low titers, and was later confirmed with a new diluted serum sample and VDRL and FTA-Abs, which were positive. Conclusion: A close look should be given to patients who, although the clinical condition is compatible, complementary exams may not be, and clinical cases should be carefully evaluated so that the patient is not treated late.