Development Of Enzyme Immunoassay Research Articles

Development of an enzyme linked immunosorbent assay using recombinant cathepsin B5 antigen for sero-surveillance of bovine tropical fasciolosis

Bovine tropical fasciolosis, caused by Fasciola gigantica, is a major parasitic disease in tropical countries responsible for significant production losses in animal husbandry practices. The disease is transmitted by the Radix sp. snails. In the early developmental stage of the parasite, the juveniles and immature flukes cause considerable damage to the liver parenchyma of the bovine host while migrating through the liver. The cathepsin (cat) B5 is a cysteine protease that is present in the excretory-secretory product of the fluke both in immature and adult stages. The early detection of fasciolosis is very critical in effective disease management. In this study, the cathepsin B5 gene from newly excysted juveniles were cloned, sequenced and analyzed. The phylogenetic analysis revealed existence of two distinct clades. The clade I includes cat B 1 to B3 whereas clade II consist of cat B4 to B7. Further, the present study was aimed to develop an enzyme linked immuno sorbent assay (ELISA) using recombinant cat B5 antigen. The developed enzyme immuno assay showed 95.3 % sensitivity and 92.4 % specificity with a cut-off of 60 % percent positive. It revealed weighted Kappa value as 0.768 (95 % CI 0.648–0.889) when compared with ELISA using native cathepsin protein. Hence, the developed assay can be exploited as a potent tool in the diagnosis and sero-surveillance of bovine tropical fasciolosis.

Development of a Competitive Enzyme Immunoassay Technique for the Detection of Peanut Traces in Gluten-free Products

The aim of this work was to develop a competititve enzymeimmunoassay technique, to detect the presence of traces of peanut in gluten-free products. Specific rabbit polyclonal antiserum against peanut was used as primary antibody. The optimal antigen concentration to be immobilized on the plate and the concentration of primary antibody to be used in competition was determined. The calibration curve was fitted using increasing concentrations of an extract of peanut product. The peanut product was extracted with Tris-HCl buffer 0.0625M with 3% sodium dodecylsulfate (SDS) and 2% sulphite (S) 0,1 M. All validation parameters studied were appropriate. Commercial samples of gluten-free products were analysed with this enzyme immunoassays and a commercial ELISA kit. Significant differences were observed in the quantitative results obtained with both methods; nevertheless the developed enzyme immunoassay could be used as screening method.

THE DYNAMICS OF HUMAN TOTAL AND ACTIVATED ANTI-MULLERIAN HORMONE SERUM LEVELS IN DIFFERENT LIFE PERIODS

Here, changes in the serum level of total anti-mullerian hormone (AMH) and its activated form in children of both sexes and women with different reproductive status are investigated. This TGFβ superfamily cytokine is known to provide the formation of the male-type reproductive system in mammalian embryogenesis, and regulate folliculogenesis, spermatogenesis and the balance of sex hormones after birth. The biologically active form of the hormone (aAMH) is formed as a result of limited proteolysis of the AMH molecule; it is not reliably known in which tissues and under the action of which enzyme it occurs. The serum level of aAMH seems to be a more informative clinical indicator than the content of total AMH (tAMH), but there are no ELISA systems at the world market that provide direct quantitative detection of aAMH. In this work, quantitative detection of total hormone (tAMH) and its biologically active form (aAMH) in serum was performed using specially developed enzyme immunoassay systems. We showed that in girls, the total serum AMH level, as well as the concentration ratio aAMH / tAMH, practically does not change with age, whereas in boys, there is not only a significant decrease in the total serum AMH level previously described in the literature (Pearson correlation coefficient R = - 0.86, p <0.001), but also in the ratios of the aAMH / tAMH level (R = -0.531, p <0.001). It was also found that in pregnant women, the amount of total AMH and the proportion of aAMH in serum was significantly higher (p <0.01 and p <0.001, respectively) than in the control group women. The obtained results are in good agreement with the available data on the total and activated AMH content in the blood serum of people of different sex and age and indicate a change in the ratio of aAMH / tAMH serum levels in pregnancy. These data may be important both for deepening the understanding of AMH biology and for interpreting the results obtained using AMH detection based diagnostics.

Sensitive and selective determination of butyl benzyl phthalate from environmental samples using an enzyme immunoassay

Increased awareness of phthalic acid esters (PAEs) toxicity has given rise to a dramatic increase in concern about the determination of these contaminations in the environment. In this paper, a sensitive, selective and rapid enzyme immunoassay of ELISA based on polyclonal antibody for detecting butyl benzyl phthalate (BBP) was developed and applied in the environmental water and soil samples. The hapten of BBP was synthesized, then applied to prepare artificial antigen and produce polyclonal antibody capable of specific recognizing BBP. From the optimal standard curve of ELISA for BBP, the values of LOD (limit of detection, IC10) and IC50 were 2.5 and 79.4 ng/mL, respectively. The ELISA showed high specificity, with the cross-reactivity toward BBP analogs < 9.6%. The satisfactory accuracy and precision were demonstrated by the recoveries of 76–116% and coefficient of variations (CVs) of 4.7–13.7%. Furthermore, BBP contamination was investigated at 3.1–25.2 ng/mL in real water samples and 4.2–76.4 ng/g in real soil samples (with the detection rate of 55% in 20 samples) by the developed ELISA, which also had shown a good correlation with that the results obtained by HPLC. All of this indicated that the developed enzyme immunoassay could be applied for sensitive and selective determination of BBP contamination in the environmental samples. Furthermore, the strategy of BBP hapten synthesis and an alternative method of BBP determination could be provided.

Prognostic Value of Serum Troponin T in Unstable Angina.

Abstract Background. Cardiac troponin T is a regulatory contractile protein not normally found in blood. Its detection in the circulation has been shown to be a sensitive and specific marker for myocardial cell damage. We used a newly developed enzyme immunoassay for troponin T to determine whether its presence in the serum of patients with unstable angina was a prognostic indicator. Methods. We screened 109 patients with unstable angina (25 with accelerated or subacute angina and 84 with acute angina at rest) for serum creatine kinase activity, creatine kinase isoenzyme MB activity, and troponin T every eight hours for two days after admission to the hospital. The outcomes of interest during the hospitalization were death and myocardial infarction. Results. Troponin T was detected (range, 0.20 to 3.64 μg per liter; mean, 0.78; median, 0.50) in the serum of 33 of the 84 patients (39 percent) with acute angina at rest. Only three of these patients had elevated creatine kinase MB activity (two were positive...

Detection of the tremorgenic mycotoxin paxilline and its desoxy analog in ergot of rye and barley: a new class of mycotoxins added to an old problem

A newly developed enzyme immunoassay (EIA) for the detection of the tremorgenic indole-diterpene alkaloid paxilline (PAX) and closely related analogs was used to analyze ergot sclerotia collected from rye and barley fields. The mean EIA standard curve detection limit was 0.47±0.14ng/mL; relative cross-reactivity of toxin standard solutions was found for 11-hydroxy-paspaline (terpendole E, 1.1%) but not for lolitrem B or ergot alkaloids. Sclerotia from all fields were positive in the PAX-EIA at concentration levels of 620±200 and 160±37μg/kg in ergot of rye and 130±47μg/kg in ergot of barley. Confirmatory analyses of sclerotia by liquid chromatography-tandem mass spectrometric detection identified PAX and its analog 13-desoxypaxilline. To the best of our knowledge, this is the first report on the natural occurrence of tremorgenic indole-diterpene alkaloid mycotoxins in ergot sclerotia from rye and barley. Along with details on the analytical methodology developed in this study, particularly PAX-antibody production, the relevance and implications of these findings for food and feed safety are discussed. Presence or absence of elevated levels of tremorgenic mycotoxins, along with the ergot alkaloids, would help in explaining the difference between the two distinct manifestations of historic ergotism, the convulsive and the gangrenous form. Further method development for paxilline and other tremorgenic mycotoxins in cereals used for food and feed is a prerequisite for a comprehensive risk assessment, which seems to be necessary in light of the findings reported here. Paxilline in ergot of rye.

Development of enzyme immunoassays (ELISA and Western blot) for the serological diagnosis of dermatophytosis in symptomatic and asymptomatic cats

Dermatophytosis is the most common fungal infection in cats worldwide and plays an important role in both animal and human health due to their high zoonotic potential. Effective screening is a strong preventive measure and the fungal culture is quite useful but requires full laboratorial experience and it takes a long time to obtain the result. A rapid and accurate screening test for dermatophytosis in cats is crucial for the effective control of disease outbreaks. The aim of this study was to develop and evaluate the diagnostic efficacy of enzyme immunoassays (ELISA and Western blot [WB]) for the rapid and precise diagnosis of dermatophytosis in cats. Seventy cats of various ages were divided into three groups: S (symptomatic, n = 20), AS (asymptomatic, n = 30), and N (negative, n = 20). All animals were submitted to fungal culture and blood samples for carrying out the serological tests. A significant difference (P < 0.05) was found between IgG-specific levels of sera of Microsporum canis positive and negative animals. There was no statistic difference between groups symptomatic and asymptomatic. The ELISA test showed sensitivity of 94% and specificity of 75%. Receiver operating characteristic (ROC) analysis also showed higher diagnostic accuracy (AUC 0.925). The WB technique detected 13 bands, and the 50kDa protein was considered the most immunogenic protein, observing reactivity in 83.3% in the symptomatic group and 66.6% in the asymptomatic group. The study concluded that ELISA and WB were useful tools to reliably detect cats that have been exposed to M. canis.

Development of a Competitive Enzyme Immunoassay Technique for the Detection of Soy Traces in Meat Products

The aim of this work was to develop a competitive enzyme immunoassay technique, to detect the presence of traces of soy in meat products. Specific rabbit polyclonal antiserum against soy protein was used as primary antibody. The optimal antigen concentration to be immobilized on the plate and the concentration of primary antibody to be used in competition was determined. The calibration curve was fitted using increasing concentrations of an extract of soy product. The soy product was extracted with Tris-HCl buffer 0.0625M with 3% sodium dodecylsulfate and 2% mercaptoethanol. The working range used in the enzyme immunoassay to detect soy was 9-280ppm SP with adequate linearity (R2: 0.9880). All validation parameters studied were appropriate. Commercial samples of meat products were analyzed with this enzyme immunoassays and a commercial ELISA kit. Significant differences were observed in the quantitative results obtained with both methods; nevertheless the developed enzyme immunoassay could be used as screening method.

Autoantibodies to Thrombopoietin and the Thrombopoietin Receptor in Patients with Immune Thrombocytopenia

Background: Primary immune thrombocytopenia (ITP) is an autoimmune bleeding disorder caused by autoantibodies against platelet glycoproteins (GP). These autoantibodies are detected in only 40-60% of ITP patients even when sensitive techniques are used. Our understanding of the mechanisms of ITP is limited by the variability in clinical presentation, varied differences in treatment responses and the lack of reliable biomarkers. Several studies have exploited the possibilities of other immune-mediated mechanisms to account for low platelet counts in the absence of detectable antibodies. Thus, we hypothesized that other autoantibodies that target antigens involved in the platelet lifecycle can be important in ITP, such as thrombopoietin (TPO) and the thrombopoietin receptor (cMpl). The objective of this study was to evaluate the frequency and clinical significance of autoantibodies against TPO and cMpl in patients with ITP compared to patients with other thrombocytopenic disorders and healthy controls.Methods: We tested well-defined adult ITP patients with a platelet count less than 100 x109/L and ITP patients in remission (platelets > 100,000) all of whom had not received any immune-modulating treatments in the previous 3 months. We also tested patients with other immune-mediated platelet disorders, non-immune thrombocytopenia and healthy controls. Patients with immune-mediated platelet disorders had anti-phospholipid syndrome (APS); heparin induced thrombocytopenia (HIT); or thrombotic thrombocytopenic purpura (TTP). Patients with non-immune thrombocytopenia had hypersplenism with documented splenic enlargement; familial thrombocytopenia; or myelodysplastic syndrome. Samples were tested for circulating antibodies against TPO or cMpl using newly developed enzyme immunoassays (EIAs) and for antibodies against platelet glycoproteins (GPIIb/IIIa and GPIb/IX) using the antigen capture assay.Results: Among patients with active ITP, 36/42 (86%) had antibodies to TPO or c-Mpl: 4/42 (10%) had anti-TPO autoantibodies only and 5/42 (12%) had anti-cMpl autoantibodies only and 15/42 (36%) had both. Among patients with ITP in remission, 8/15 (53%) had autoantibodies to TPO or cMpl. Autoantibodies were not detected in healthy controls; however, all patients with non-immune thrombocytopenia had circulating autoantibodies to TPO or c-Mpl (10/10, 100%): 1/10 (10%) had anti-TPO autoantibodies only and 1/10 (10%) had anti-cMpl autoantibodies only and 8/10 (80%) had both. We also found antibodies against TPO and cMpl in patients with other immune-mediated platelet disorders. Among HIT patients (n=26), 73% had antibodies to TPO and 50% had antibodies to cMpl; among TTP patients (n=16), 31% had antibodies to TPO and 13% had antibodies to cMpl; and among APS patients (n=17), 29% had antibodies to TPO and 47% had antibodies to cMpl. Platelet bound antibodies to GPIIb/IIIa, GPIb/IX or both were detected in 18/42 (43%) active ITP samples; 6/15 (40%) remission ITP samples; and 2/10 (20%) patients with thrombocytopenia from non-immune causes. ITP patients with anti-TPO or anti-cMpl antibodies required fewer ITP therapies before remission was achieved compared with patients who had anti-GP autoantibodies.Conclusions: Testing the entire panel of autoantibodies that included anti-TPO, anti-cMpl and anti-GP, we were able to identify all patients with active ITP; however, we could not distinguish between patients with ITP and other thrombocytopenic syndromes. DisclosuresArnold:Novartis: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy; UCB: Consultancy; Amgen: Consultancy, Research Funding.

Is salivary estriol detectable in very early pregnancy?

Objective: Estriol (E3) is produced by the placenta and is important for early pregnancy maintenance. In blood, E3 can be detected from the 8th week of pregnancy. Under the influence of the hypothalamic-pituitary-adrenal (HPA) axis hormone ACTH, E3 levels increase sharply after the 10th week. Although E3 can be reliably analyzed in saliva, till now information about the concentrations during the first trimester is missing. The aim of this study was to verify whether the sensitivity of a newly developed enzyme immunoassay is sufficient for the determination of salivary E3 (sE3) in very early pregnancy.Methods: Saliva samples were collected at home, once weekly in 25 healthy pregnant women from the 6th week of gestation to the end of the first trimester.Results: sE3 was detectable from the beginning of the 6th pregnancy week (M = 3.17 pg/ml, SD = 2.13). A steep significant increase between the 7th and the 8th week (p = 0.029) and again between the 10th and the 11th week (p = 0.001) was apparent.Conclusion: Low concentrations of sE3 can be measured during very early pregnancy and may serve as a promising, easily assessable marker for future research on the mechanisms of healthy pregnancy.

PREVALENCE AND RISK FACTORS OF HEPATITIS C VIRUS INFECTION IN HAEMODIALYSIS PATIENTS: TESTING ANTIBODIES, RNA AND GENOTYPES

Hepatitis C virus (HCV) infection is an important global public health problem affecting approximately 180 million people. Multiple risk factors are associated with HCV transmission among haemodialysis (HD) patients leading to an increased risk for liver-related mortality. Patients undergoing HD may show a decreased humoral and cellular immunity, which lowers the sensitivity of the HCV antibodies (Abs) test resulting in false negative antibody test, thus requiring HCV RNA testing. Our study is to determine the prevalence of HCV markers (antibody RNA and genotype) and risk factors of HCV infection among patients in HD unit in Baghdad. A sample of 54 patients were interviewed. HCV Abs (anti-HCV) was tested using third generation enzyme immunoassay (EIA-3) and immunoblot assay (Lia-Tek III) as screening and confirmatory test respectively. Sera of 46 patients (irrespective to anti-HCV results) were subjected to molecular analysis, using the most developed RT-PCR and DNA Enzyme immunoassay (DEIA) method. Seropositive rate of anti-HCV and HCV-RNA were (66.6%) and (60.9%) respectively. Anti-HCV seropositive rate was significantly higher in males (77.1%), and history of blood transfusion (85%). Blood transfusion acts as a significant risk for acquiring HCV (OR 44.2, 95% CI 7.6-256.9). Genotype 4 was the most prevalent (33.3%), followed by genotype 1a (25.9%) and genotype 1b (22.2%). We concluded that, the prevalence of HCV among the haemodialysis patients is high. It is significantly related to gender, duration of dialysis and number of blood transfusion. Blood transfusion acts as a significant risk factor. Molecular test for detection for HCV RNA is necessary and proper nosocomial prevention program should be implemented to prevent HCV transmission.

Duration of hexobarbital sleep as a marker for resilience to postraumatic stress disoder (PTSD) in rats

tion about the concentrations before the 12th week of pregnancy is missing. Methods: The aim of this pilot study was to verify whether the sensitivity of a newly developed enzyme immunoassay of IBL International is sufficient for the determination of salivary estriol during very early pregnancy (N=25, 6th–12th week). Saliva samples were collected weekly between 10:00 am and 14.00 pm. Results: Salivary estriol was detectable from the beginning of the 6th week of pregnancy (M=3.17pg/ml, SD=2.13) and the concentrations ranged from .10 to 10.94 pg/ml. Salivary estriol concentrations showed a steep significant increase between the 10th and the 11th gestational week (t(11) =−3.847, p< .01). Conclusion: With the new enzyme immunoassay low concentrations of salivary estriol can be measured during very early pregnancy. Concordant with blood concentrations, salivary estriol starts to increase by the 10th week, when under stimulation of ACTH the placenta takes over the production of estriol from the corpus luteum. Salivary estriol may therefore serve as a promising marker for future research on the mechanisms and consequences of prenatal stress during very early pregnancy.

Higher HPA axis activity in healthy participants compared to depressed patients after MR blockade: Evidence for attenuated MR function in depression

tion about the concentrations before the 12th week of pregnancy is missing. Methods: The aim of this pilot study was to verify whether the sensitivity of a newly developed enzyme immunoassay of IBL International is sufficient for the determination of salivary estriol during very early pregnancy (N=25, 6th–12th week). Saliva samples were collected weekly between 10:00 am and 14.00 pm. Results: Salivary estriol was detectable from the beginning of the 6th week of pregnancy (M=3.17pg/ml, SD=2.13) and the concentrations ranged from .10 to 10.94 pg/ml. Salivary estriol concentrations showed a steep significant increase between the 10th and the 11th gestational week (t(11) =−3.847, p< .01). Conclusion: With the new enzyme immunoassay low concentrations of salivary estriol can be measured during very early pregnancy. Concordant with blood concentrations, salivary estriol starts to increase by the 10th week, when under stimulation of ACTH the placenta takes over the production of estriol from the corpus luteum. Salivary estriol may therefore serve as a promising marker for future research on the mechanisms and consequences of prenatal stress during very early pregnancy.

Is salivary estriol detectable in very early pregnancy?

AbstractObjective: Estriol (E3) is produced by the placenta and is important for early pregnancy maintenance. In blood, E3 can be detected from the 8th week of pregnancy. Under the influence of the hypothalamic-pituitary-adrenal (HPA) axis hormone ACTH, E3 levels increase sharply after the 10th week. Although E3 can be reliably analyzed in saliva, till now information about the concentrations during the first trimester is missing. The aim of this study was to verify whether the sensitivity of a newly developed enzyme immunoassay is sufficient for the determination of salivary E3 (sE3) in very early pregnancy.Methods: Saliva samples were collected at home, once weekly in 25 healthy pregnant women from the 6th week of gestation to the end of the first trimester.Results: sE3 was detectable from the beginning of the 6th pregnancy week (M = 3.17 pg/ml, SD = 2.13). A steep significant increase between the 7th and the 8th week (p = 0.029) and again between the 10th and the 11th week (p = 0.001) was apparent.Conc...

A New Enzyme Immunoassay for the Quantitative Determination of Classical Autotaxins (ATXα, ATXβ, and ATXγ) and Novel Autotaxins (ATXδ and ATXε).

BackgroundAutotaxin (ATX) is a secreted enzyme that converts lysophosphatidylcholine to lysophosphatidic acid, a potent bioactive lipid mediator, through its lysophospholipase D activity. Although five alternative splicing isoforms of ATX have been identified as ATXα, ATXβ, ATXγ, ATXδ, and ATXε and the expression patterns of each isoform differ among several tissues, the clinical significance of each isoform remains to be elucidated.MethodsAnti-ATXβ and anti-ATXδ monoclonal antibodies were produced by immunization with recombinant human ATXβ and ATXδ expressed using a baculovirus system, respectively. We then developed enzyme immunoassays to measure the serum concentrations of “classical ATX” (ATXα, ATXβ, and ATXγ) and “novel ATX” (ATXδ and ATXε) antigens and evaluated the usefulness of these assays using human serum samples.ResultsThe with-run and between-run precision, interference, detection limit, and linearity studies for the present assay were well validated. In healthy subjects, the serum concentrations of classical ATX and novel ATX were significantly (P < 0.01) higher in women than in men, while the ratios of classical ATX or novel ATX to total ATX were not different between women and men. The concentrations of both classical ATX and novel ATX in normal pregnant subjects and patients with chronic liver diseases or follicular lymphoma were significantly higher than those in healthy subjects, while the ratio of both ATX isoforms to total ATX did not vary among these groups.ConclusionsWe have developed a new enzyme immunoassay to determine the concentrations of classical ATX and novel ATX in human serum. These assays may be helpful for elucidating the distinct functional roles of each ATX isoform, which are largely unknown at present.

Validation of an enzyme immunoassay for the measurement of faecal glucocorticoid metabolites in Eurasian (Lynx lynx) and Iberian lynx (Lynx pardinus)

Stress hormone levels are important indicator of an animal’s well-being, as stress has harmful effects on reproduction, growth and immune function. The development of enzyme immunoassays (EIA) to monitor faecal glucocorticoid metabolites (fGM) contributes a powerful tool to assess an animal’s adrenal status non-invasively. We aimed to identify a suitable EIA for monitoring fGM by assessing the suitability of six different EIAs for detecting quantitative changes in fGM concentrations in response to an ACTH challenge test in Eurasian lynx. FGM were characterised in a male Eurasian lynx that received an injection of 3H-cortisol. Using HPLC analyses radiolabeled metabolites were compared with immunoreactive metabolites. The second aim was to biologically validate the established EIA for monitoring adrenocortical activity of captive Iberian lynxes after a translocation to new enclosures in relation to behaviour. Additionally faecal samples of ten pregnant Iberian lynxes from the peripartal period were analysed. The ACTH challenge revealed an 11β-hydroxyetiocholanolone EIA as the most sensitive assay to reflect acute fGM elevations in the Eurasian lynx. HPLC immunograms demonstrated that the 11β-hydroxyetiocholanolone EIA measured significant amounts of immunoreactivities corresponding to radiolabeled metabolites with strong similarities across both lynx species. Additionally, HPLC and GC–MS analyses confirmed the presence of 11β-hydroxyetiocholanolone in faeces of both, the Eurasian and the Iberian lynx. Longitudinal fGM profiles of Iberian lynx revealed increases in concentrations associated with management events. During the peripartal period, however, fGM concentrations were not significantly elevated. Our results show that the 11β-hydroxyetiocholanolone EIA is a reliable tool to assess fGM in both lynx species.

Development of enzyme immunoassay of amphetamines in human biological fluids

Enzyme-linked immunosorbent assay (ELISA) for detection of amphetamines in urine was developed. The conditions for ELISA using an antigen obtained from the derivative of amphetamine and ovalbumin were optimized. It was shown that the specificity of the proposed method when used in clinical conditions corresponds to that of gas chromatography with mass spectrometric detection.

Serologically verified human bocavirus pneumonia in children

Human bocavirus (HBoV) is a newly identified parvovirus frequently found in children suffering from acute respiratory and intestinal infections. The aim of the present study was to evaluate, by using a newly developed antibody assay, the role of HBoV in pediatric community-acquired pneumonia (CAP) and the seropositivity rate to HBoV in a prospective study in North-Italian children. During a 15-month study period, 124 children were admitted due to presumptive pneumonia, and in 101 of them, pneumonia was radiologically confirmed. The etiology of CAP was studied by antibody assays to 16 microbes, including the newly developed enzyme immunoassay for HBoV. Serological evidence of acute HBoV infection was found in 12 (12%) children, being single in 7 and mixed in 5 cases (4 with other viruses and 3 with bacteria). IgM was positive in 11 cases. A diagnostic rise in IgG antibodies between paired sera was observed in six cases. HBoV was the second most common virus next to respiratory syncytial virus (17%). The seropositivity rate to HBoV increased with age, reaching nearly 100% before school age. The present results show that HBoV is able to induce significant antibody responses and suggest that HBoV may be a fairly common cause of pneumonia in children. Seroconversion to HBoV in most children takes place in early childhood.

Urinary hormone analysis assists reproductive monitoring and sex identification of bell frogs ( Litoria raniformis)

With the world currently facing a global amphibian extinction crisis, the development of techniques to help meet the needs of conservation managers and researchers studying the reproductive biology of amphibians is needed. Here, we developed enzyme immunoassays to measure estrone, testosterone, and progesterone hormone metabolites in the urine of Litoria raniformis, the southern bell frog. Concentrations of urinary estrone, testosterone, and progesterone increased during the breeding season for females (P < 0.05). Concentrations of urinary testosterone and progesterone increased for males during the breeding season compared with that for months where no reproductive behaviors were observed (P < 0.05). Furthermore, urinary estrone concentrations proved to be a reliable sexing tool for adult frogs, with no overlap between the sexes in 98% of cases, regardless of season. There was no difference in estrone (P = 0.204) or testosterone (P = 0.485) metabolite concentrations between samples taken immediately upon capture and those taken 12 to 24 h later from the same individual. Progesterone metabolite concentrations were lower on Day 2 than upon collection (P = 0.004). This is the first study to show that urinary hormone analysis can be a useful technique for reproductive monitoring in an amphibian. Additionally, hormone metabolite measures offer promise as sex identification tools for monomorphic species and for those whose secondary sex characteristics are visible only during the breeding season.

Short-Term Highly Intense Physiological Stress Causes an Increase in the Expression of Heat Shock Protein in Human Leukocytes

Extracellular heat shock protein with molecular weight of 70 kDa is a signal molecule of the immune system. It is secreted by the peripheral blood, liver and muscle cells in response to physiological, thermal, and mental stresses. The main goal of our study was to compare the levels of expression of heat shock protein (70 kDa) matrix ribonucleic acid in leukocytes and serum concentrations of the protein before and after physiological stress. In order to solve this problem, we developed enzyme immunoassay of serum heat shock (70 kDa) protein concentration and a method for evaluating the expression of matrix ribonucleic acid of this protein in leukocytes by the real time PCR. The concentration of 70 kDa heat shock protein in the serum increased 1.7 times as a result of even a short-term highly intense physiological stress, while the expression of its matrix ribonucleic acid in leukocytes increased 1.5 times. The individual features determine the response to physiological stress. Probable sources of 70 kDa heat shock protein are discussed.