Commercial Enzyme Immunoassay Kit Research Articles

Dissociating Autoantibody Responses against Ro52 Antigen in Patients with Anti-Synthetase or Anti-MDA5 Antibodies.

We aimed to dissociate the autoantibody response against the Ro52 protein in patients with anti-synthetase or anti-melanoma differentiation-associated gene 5 (MDA5) antibodies to explore the potential roles of different anti-Ro52 autoantibody responses in disease subclassification. This study used a single-center, prospective myositis cohort involving 122 consecutive patients with anti-synthetase antibodies identified by RNA immunoprecipitation (RNA-IP) and 34 patients with anti-MDA5 antibodies detected using enzyme immunoassay (EIA). Anti-Ro52 antibodies were measured using commercial EIA kits, while anti-Ro/SSA antibodies were identified using RNA-IP. Clinical features and outcomes were stratified according to two different patterns of autoantibody responses against Ro52, including "isolated anti-Ro52", defined by positive anti-Ro52 and negative anti-Ro/SSA antibodies, and "anti-SSA-Ro52", defined by positive anti-Ro52 and anti-Ro/SSA antibodies. Isolated anti-Ro52 positivity was the most prevalent autoantibody response in patients with both anti-synthetase (40/122; 32.8%) and anti-MDA5 antibodies (8/34; 23.5%). Isolated anti-Ro52 or anti-SSA-Ro52 positivity was associated with Gottron's sign in patients with anti-synthetase antibodies, while in patients with anti-MDA5 antibodies, isolated anti-Ro52 positivity was associated with respiratory insufficiency at initial presentation and poor overall survival. Isolated anti-Ro52 positivity could be a potential biomarker for patient stratification; however, the clinical significance of dissociating isolated anti-Ro52 positivity from overall anti-Ro52 positivity was not evident.

Urinary C-peptide and total triiodothyronine as energetic biomarkers for studies of lemurs.

Measuring energy balance and energy metabolism can provide crucial information for understanding the ecological and behavioral drivers of an animal's energetic and physiological condition. Both urinary C-peptide (uCP) of insulin and urinary total triiodothyronine (uTT3) have been validated as noninvasive biomarkers of energy balance and metabolic activity in haplorrhine primates. This study attempts to validate uCP and uTT3 measures in strepsirrhines, a phylogenetically distinct primate clade, using the ruffed lemur (genus Varecia) as a model. We experimentally manipulated the diet of captive black-and-white (Varecia variegata) and red (Varecia rubra) ruffed lemurs at Duke Lemur Center across a 4-week period. We collected urine samples from subjects (n = 5) each day during 1 week of control diet, 2 weeks of calorie-restricted diet and 1 week of refeeding, designed to temporarily reduce energy balance and metabolism. We also tested the outcome of filter paper as a storage method by comparing to controls (frozen at -20°C) to assess its suitability for studies of wild populations. We successfully measured uCP and uTT3 levels in frozen urine samples using commercial enzyme immunoassay kits and found that both biomarkers were excreted at lower concentrations (C-peptide: 1.35 ng/mL, 54% reduction; TT3: 1.5 ng/mL, 37.5% reduction) during calorie-restricted periods compared to normal diet periods. Filter paper recovery for uCP was 19%, though values were significantly positively correlated with frozen control samples. uTT3 could not be recovered at measurable concentrations using filter paper. These methods enable noninvasive measurement of energetic conditions in wild strepsirrhines and subsequent assessment of relationships between energy balance and numerous socioecological drivers in primate populations.

Adipomyokines in children with obesity

Adipomyokines are synthesized and secreted into the bloodstream by cells of both muscle and adipose tissue. They can have both a negative metabolic effect, acting as pro-inflammatory adipokines in obesity, and a positive one, increasing in response to physical exertion in the form of myokines. To study the features of adipocytokine secretion in children with constitutionally exogenous obesity. The study included 80 patients: 60 adolescents aged 15 [13; 16] years with constitutionally exogenous obesity SDS BMI: 3.0 [2.6; 3.3] and 20 control group children aged 16 [15; 17] years without excess weight SDS BMI: -0.3 [-1.25; 0.33]. Commercial enzyme immunoassay kits were used to determine the level of adipomyokines. The compositional composition of the body was evaluated by bioimpedance analysis (InBody 770 analyzer, South Korea) in the morning, on an empty stomach. Statistical processing was carried out using STATISTICA v.12.0 (StatSoft Inc., USA). The results are presented in the form of median (Me) and quartiles (Q1; Q3) corresponding to 25 and 75 percentiles. The critical significance level (p) was assumed to be <0.05. Levels of IL-6 and irisin are statistically significantly higher in obese adolescents compared to the control group: 0.55 [0.226; 1.35] pg/ml vs 0.202 [0.128; 0.652] pg/ml (p=0.041) and 11.16 [6.6; 22.76] mcg/ml vs 7.36 [6.48; 9.68] mcg/ml (p=0.043), respectively. Concentrations of IL-6, myostatin and decorin increase with an increase in the degree of obesity: grade I vs III: 0.226 [0.224; 0.398] vs 0.80 [0.36; 1.81] pg/ml (p=0,0197), 25,85 [21,53; 28,23] vs 31.41 [24.36; 35.06] ng/ml (p=0.03), 4065.3 [3244.9; 5245.5] vs 5322.5 [4199.8; 7702.4] pg/ml (p=0.0376), respectively. In obese children, IL-6 levels positively correlate with BMI, SDS BMI and the amount of adipose tissue, and myostatin - with BMI and SDS BMI. The concentration of irisin in the blood serum is significantly higher in obese girls than in obese boys and healthy girls. Obese patients, compared with lean peers, are characterized by a statistically significantly higher content of both fat and lean mass. With the progression of obesity, there is a statistically significant increase in the ratio of fat to lean mass (I degree - 0.66 [0.56; 0.7], III - 0.78 [0.68; 0.98] (p=0.0073). Patients with obesity and normal body weight have different levels of adipomyokines. An increase in the level of IL-6 with the progression of obesity is directly related to an increase in the content of adipose tissue. Further study of the features of adipocytokine secretion, their relationship with the features of the body composition and metabolic complications in obesity is required.

Cortisol Variations to Estimate the Physiological Stress Response in Horses at a Traditional Equestrian Event.

In many countries, horses remain involved in traditional equestrian events such as those celebrated in Menorca (Balearic Islands, Spain) every year since at least the 14th century. The present study aimed to evaluate the variations in salivary cortisol concentrations to estimate the physiological stress response in horses at the Menorca patronal festivals. Two different editions (years 2016 and 2018) of the festivals in honor of the Virgin of Grace in Maó (Menorca, Spain) were studied. Nineteen and seventeen Pure Breed Menorca stallions were included in the study, respectively. The stallions were aged between seven and twelve years. During celebrations, samples were collected before the start of the festivals between 8-9 a.m. and during the festivals at 8-9 p.m. On the second day of celebrations, the samples were collected at 8-9 a.m. and 3-4 p.m. Finally, on the day after the festivals, one sample was collected at 8-9 p.m. Additionally, a control group was sampled at 8-9 a.m., 3-4 p.m., and 8-9 p.m. Salivary cortisol concentrations were assessed by using a commercial enzyme immunoassay kit specially validated to quantify salivary cortisol in horses. Salivary cortisol concentrations did not show significant differences between sampling hours in the control group (p > 0.05). All the samples collected during festivals were significantly higher than samples of the control group (p < 0.05). Within the twenty-four hours after the end of the celebrations, cortisol concentrations returned to baseline levels and did not differ significantly from the control group (p > 0.05). Hence, the present study describes that the participation of the horses in these particular acts generate an acute and transitory stress response. Overall, the current work provides a reasonable basis for future research on the stress physiology and well-being of horses participating in traditional celebrations or similar events.

Study of pulsed electrostatic field (PESF) in the perfusion of peripheral tissues: Microangiopathy, nutrition and quality of perceiver life.

Microangiopathy compromises the structural and functional integrity of organs and tissues in patients with type II diabetes mellitus (T2DM) negatively affecting the perceived quality of life. Nitric oxide (NO) is a multifunctional signalling molecule, acting as a vasodilator, neurotransmitter, and modulator of inflammatory processes. Patients with type II diabetes mellitus and chronic kidney disease, controlled from glycaemic status, were treated or not with pulsed electrostatic field (PESF) cycles to evaluate effect on the perfusion of peripheral tissues. Everyone was monitored for the metabolic profile, and we tested circulating NO with a commercial enzyme immunoassay kit. In addition, we tested the perceived quality of life of patients before/after a PESF cycle using a questionnaire. Patients treated with PESF were improved circulating NO levels, significant changes in systolic and diastolic blood pressure, heart rate and were more homogeneous for their metabolic profile. The questionnaire showed also a marked improvement in the perceived quality of life. The use of pulsed electrostatic fields has allowed us to observe an improvement in the metabolic, psychological, and clinical profile in patients with T2DM and chronic kidney disease whose pathological profile is strongly compromised.

MO735: Increased Serum Adipocyte Fatty Acid Binding Protein Level Associated with Endothelial Dysfunction in Patients Undergoing Hemodialysis

Abstract BACKGROUND AND AIMS Adipocyte fatty acid binding protein (A-FABP) is independently associated with atherosclerosis. Endothelial dysfunction is one of the causes of cardiovascular morbidity and mortality in patients with hemodialysis (HD). This study evaluated the correlation between serum A-FABP level and endothelial function measured by vascular reactivity index (VRI) in patients with HD. METHOD Fasting blood samples were obtained from 90 HD patients. A commercial enzyme immunoassay kit was used to measure A-FABP levels. The endothelial function and VRI were measured using a digital thermal monitoring (DTM) test (VENDYS). In this study, VRI &amp;lt; 1.0 was used as the poor vascular reactivity, 1.0 ≤ VRI &amp;lt; 2.0 was used as the intermediate vascular reactivity, and VRI ≥ 2.0 was used as the good vascular reactivity. RESULTS A total of 14 HD patients (15.6%) were categorized as poor vascular reactivity (VRI &amp;lt; 1.0), 38 HD patients (42.2%) were categorized as intermediate vascular reactivity (1.0 ≤ VRI &amp;lt; 2.0) and 38 HD patients had good vascular reactivity. Decreased pre-HD body weight (P = 0.016), post-HD body weight (P = 0.013), body mass index (P = 0.034), serum creatinine level (P = 0.010) and higher serum A-FABP level (P = 0.001) were associated with poor vascular reactivity. After multivariable forward stepwise linear regression analysis noted that serum creatinine level (β = 0.234, adjusted R2 change = 0.043; P = 0.019) was positively associated with, and serum A-FABP level (β = −0.347, adjusted R2 change = 0.152; P = 0.001) was negatively associated with VRI values in patients undergoing HD. CONCLUSION After adjusting for significant confounders, the serum A-FABP level was negatively correlated, and the serum creatinine level was positively correlated with VRI values in patients undergoing HD.

Association between Serum Adipocyte Fatty Acid Binding Protein Level and Endothelial Dysfunction in Chronic Hemodialysis Patients.

Adipocyte fatty acid binding protein (A-FABP) is associated with atherosclerosis, and endothelial dysfunction is one of the reasons for adverse cardiovascular outcomes in patients undergoing hemodialysis (HD). This study investigated the correlation between serum A-FABP levels and endothelial function in HD patients. Fasting blood samples were collected from 90 HD patients. A-FABP levels were measured using a commercial enzyme immunoassay kit. Endothelial function was evaluated by a digital thermal monitoring test to measure vascular reactivity index (VRI). VRI < 1.0, 1.0 ≤ VRI < 2.0, and VRI ≥ 2.0 indicated poor, intermediate, and good vascular reactivity, respectively. In total, 14 (15.6%), 38 (42.2%), and 38 (42.2%) HD patients had poor, intermediate, and good VRI, respectively. Patients with poor VRI had lower pre-HD and post-HD body weight, body mass index, and serum creatinine level but higher serum A-FABP level (p = 0.001) than those with intermediate and good VRI. Log-transformed VRI (log-VRI) positively correlated with serum creatinine and negatively correlated with A-FABP by multivariate linear regression analysis. We concluded that A-FABP correlated with endothelial dysfunction in chronic HD patients.

Dual-readout fluorescence quenching immunochromatographic test strips for highly sensitive simultaneous detection of chloramphenicol and amantadine based on gold nanoparticle-triggered photoluminescent nanoswitch control

Herein, a novel fluorescence quenching immunochromatographic test strip (FQICTS) for simultaneous detection of chloramphenicol (CAP) and amantadine (AMD) was developed on the basis of inner filter effect (IFE), with the combination of gold nanoparticles (AuNPs) and highly luminescent green-emitting gold nanoclusters (AuNCs) as the IFE quencher/donor pair. The AuNPs could quench the excitation light and emission light of AuNCs and achieve a high IFE efficiency due to dual spectral overlapping. Under optimal conditions, the “turn-on” mode of the AuNCs-based dual-readout FQICTS showed good linearity for CAP detection in chicken samples from 0.05 ng/g to 10 ng/g, with a limit of detection (LOD) of 0.043 ng/g. The linear range of AMD is 0.5–50 ng/g, with LOD of 0.45 ng/g. The visual LODs of CAP and AMD in “turn-on” mode were 200 and 10 times lower than that in “turn-off” mode, respectively. The “turn-on” mode of FQICTS showed high recovery for detecting CAP (82.5–94.5%) and AMD (81.9–110.7%) spiked into chicken samples. The performance and practicability of the established method were verified with commercial enzyme-immunoassay kits, and good correlations were observed. Overall, the newly developed AuNCs-based dual-readout FQICTS is a promising on-site screening tool for rapid, high-sensitivity detection of multiple food contaminants in practical applications.

Plasma calcitonin gene-related peptide (CGRP) in migraine and endometriosis during the menstrual cycle.

ObjectiveMigraine, endometriosis, and the comorbidity of both are frequent pain disorders of special relevance for women. The neuropeptide calcitonin gene‐related peptide (CGRP) is critically involved in migraine, and circumstantial evidence suggests a role in endometriosis. We assessed CGRP levels at different times of menstrual cycle in four groups: healthy women, women with migraine or endometriosis and with the comorbidity of both.MethodsWomen with episodic migraine and women with a histologically confirmed endometriosis were recruited from specialized centers. For CGRP determination with a commercial enzyme immunoassay kit, cubital vein blood samples were collected on menstrual cycle day 2 ± 2 (during menstruation) and on day 15 ± 2 (periovulatory period). The primary endpoint of the study was the absolute difference of CGRP plasma levels between the menstrual and the periovulatory phase of all study groups. Groups were compared using nonparametric test procedures.ResultsA total of 124 women were included in the study. The change of CGRP plasma levels between menstruation and the periovulatory period was different between groups (p = 0.007). Women with comorbid migraine and endometriosis showed an increase of CGRP in the menstrual phase of +6.32 (interquartile range, IQR −3.64–13.60) compared to the periovulatory time, while healthy controls had a decrease of −10.14 (−22.54–0.91, p = 0.004). CGRP levels were different in the periovulatory phase among groups (p = 0.008), with highest values in healthy controls.InterpretationCGRP levels change significantly during the menstrual cycle. Different patterns in women with the comorbidity point to a deviant regulation of CGRP release.

Development of in-house ELISAs for the detection of anti-SARS‑CoV‑2 RBD and N IgG and IgM antibodies in biological samples

By the end of year 2019, the new virus SARS-CoV-2 appeared, causing the Coronavirus Disease 2019 (COVID-19), and spread very fast globally. A continuing need for diagnostic tools is a must to contain its spread. Till now, the gold standard method, the reverse transcription polymerase chain reaction (RT-PCR), is the precise procedure to detect the virus. However, SARS-CoV-2 may escape RT-PCR detection for several reasons. The development of well-designed, specific and sensitive serological test like enzyme immunoassay (EIA) is needed. This EIA can stand alone or work side by side with RT-PCR.In this study, we developed several EIAs including plates that are coated with either specially designed SARS-CoV-2 nucleocapsid or surface recombinant proteins. Each protein type can separately detect anti-SARS-CoV-2 IgM or IgG antibodies. For each EIAs, the cut-off value, specificity and sensitivity were determined utilizing RT-PCR confirmed Covid-19 and pre-pandemic healthy and other viruses-infected sera. Also, the receiver operator characteristic (ROC) analysis was performed to define the specificities and sensitivities of the optimized assay. The in-house EIAs were validated by comparing against commercial EIA kits. All in-house EIAs showed high specificity (98–99%) and sensitivity (97.8–98.9%) for the detection of IgG/IgM against RBD and N proteins of SARS-CoV-2.From these results, the developed Anti-RBD and anti-N IgG and IgM antibodies EIAs can be used as a specific and sensitive tool to detect SARS-CoV-2 infection, calculate the burden of disease and case fatality rates.

Characterization of antibodies in human immunoglobulin products from different regions worldwide

AimThe antibody levels against a broad spectrum of pathogens were assessed in commercial intravenous immunoglobulin (IVIG) manufactured from pooled plasma obtained from different global regions. MethodsTwenty-four IVIG commercial lots from eight manufacturers corresponding to 12 brands were analyzed. The plasma was collected in 10 countries/regions. Depending on each pathogen, antibody levels were measured using specific commercial IgG-specific enzyme immunoassay kits or by cell culture neutralization test and guinea pig skin neutralization test. A principal component analysis was performed. ResultsFor polio and diphtheria (reference markers of the US authorities), all IVIGs had relevant titers in accordance with reference levels. IVIGs from Canada, Australia, and the USA were positive for titers against globally distributed pathogens or those under vaccination programs in the developed world (parainfluenza, Epstein–Barr, varicella-zoster, influenza B, parvovirus B19, and measles viruses). IVIG from Taiwan and Hong Kong showed low antibody titers for these pathogens but high titers for Pseudomonas aeruginosa. IVIG from India had high titers for pathogens frequently found in developing countries (West Nile, dengue, chikungunya, and hepatitis E viruses and Streptococcus pneumoniae). IVIGs from Argentina, Spain, Israel, and Czechia showed intermediate antibody concentrations. ConclusionThe antibody profile in IVIG was greatly influenced by regional characteristics including climate, vaccination programs, and the prevalence of pathogens in the different countries and regions.

Soroepidemiological survey of chlamydia abortus infection in bovine in the State of Pernambuco, Brazil

The objective of this study was to conduct a seroepidemiological survey of Chlamydia abortus infection in dairy cattle herds. A total of 303 blood serum samples were collected from 24 property in Vale do Ipanema microregion in the state of Pernambuco, Brazil. For the diagnosis of C. abortus infection, a commercial enzyme immunoassay kit (ELISA) was used. A prevalence of 34.0% (103/303; 95% CI: 28.7%-39.7%) of infected animals was identified. In 79.8% (19/24) of the properties, at least one infected animal was detected. The risk factors identified were: semi-intensive system (OR = 3.47, p ≤ 0.000), extensive system (OR = 8.14; p ≤ 0.000), supply of water in troughs and directly at the fountain (OR = 2.29, p = 0.002), pasture rent (OR = 1.72, p = 0.041), use of artificial insemination (AI) (OR = 3.07, p = 0.002), and use of AI associated with natural mount (OR = 2.22, p = 0.003). The occurrence of C. abortus infection in dairy cattle in the state of Pernambuco, Brazil, was recorded for the first time. It is concluded that the infection by this agent is present in the analyzed herds and that hygienic and sanitary management measures based on the identified risk factors should be implemented to avoid reproductive losses and losses to the producers.

HEV prevalence and potential risk factors in a large multi-ethnic youth cohort in China

BackgroundThis cohort study was designed to investigate the prevalence of and potential risk factors of HEV infection in a large multi-ethnic youth cohort in China.MethodsBlood samples were collected from participants (n = 6269) and serum was isolated. All serum samples were tested for anti-HEV IgG, anti-HEV IgM antibodies using commercial enzyme immunoassay kits (Wantai Biological Pharmacy Enterprise, Beijing, China).ResultsThe overall rate of anti-HEV IgG and anti-HEV IgM prevalence was 4.78% and 0.14%, 0.03% were positive for both anti-HEV IgG and anti-HEV IgM antibodies. Anti-HEV IgG positivity is significantly higher in females (5.27%) compared to males (4.14%) (P = 0.028). Anti-HEV IgG prevalence is significantly (P = 0.0001) higher in Dong (17.57%), Miao (12.23%), Yi (11.04%), Gelao (9.76%), and Bai (10.00%) compared to other ethnic groups. It is significantly higher in Guizhou (11.4%), Sichuan (10.1%), Yunnan (9.3%), and Guangxi (6.9%) than that other province. We found that ethnicity and provincial background are significantly associated with HEV infection in this cohort.ConclusionThis study provides comprehensive information on HEV prevalence in multi-ethnic populations in China. However, our study only focused on a youth population from different provinces of China. Future studies are recommended to investigate HEV prevalence in other age groups of the ethnic populations.

Changing trends in rotavirus detection, genotypes and clinical severity among children

Background: Rotavirus is a major cause of acute gastroenteritis in developing countries like India. Rotavirus causes approximately 11.37 million episodes of acute gastroenteritis (AGE), 872,000 inpatient admissions and 78,000 deaths in children <5 years annually in India. This study aims to detect rotavirus among children <5 years admitted with acute gastroenteritis and to determine the common circulating genotypes and assess their clinical severity. Methods and materials: A total of 772 stool samples were collected from children <5years who were admitted for acute gastroenteritis (AGE) during February 2014 to October 2019. Rotavirus antigen was detected using a commercial enzyme immunoassay kit, Rotaclone®). RNA was extracted from stool samples using viral RNA mini kit (Qiagen, Germany). The extracted RNA was converted into cDNA by MMLV-RT enzyme. The cDNA is used for genotyping by a semi-nested RT-PCR targeting VP7 and VP4. Modified Vesikari scale was used to analyse the severity of acute gastroenteritis and Modified Kuppuswamy scale was used to assess the socio-economic class. Results: Rotavirus was detected in 23.6% of children with AGE. The year-wise detection rate of rotavirus was 33.8% (2014), 29.8% (2015), 26.1% (2016), 19.7% (2017), 17.7% (2018) and 20.9% (2019). Based on Vesikari score rotavirus G2P [4] infected children had more vomiting and episodes (56%) than those infected with G1P [8] (29%) and G3P [8] (32%). Children aged 12–24 months had a higher detection rate of 27.9%. Rotavirus was detected significantly higher among lower-middle class 26.7% in contrast to upper-middle class 17.4% (p = 0.006). G1P [8] (42%) and G3P [8] (32%) were the predominant circulating genotypes. G1P [8] was initially the predominant genotype (2014–2016) and it has been replaced by G3P [8] (2017–2019). Conclusion: Rotavirus shows a declining trend since 2017 and this could probably because of the introduction of rotavirus vaccine in National immunisation schedule. There is a change in genotype distribution of rotavirus G3P [8] replaced G1P [8] as the predominant circulating genotype in the past 3 years however less circulating G2P [4] genotype was associated with longer duration of vomiting and more episodes leads to severe gastroenteritis.

Post-vaccine rotavirus genotype distribution in Nairobi County, Kenya

BackgroundRotaviruses are primary etiological agents of gastroenteritis in young children. In Kenya, G1P8 monovalent vaccine (Rotarix) was introduced in July 2014 for mandatory vaccination of all newborns at 6 and 10 weeks of age. Since then, no studies have been done to identify the rotavirus genotypes circulating in Nairobi County, Kenya, following the vaccine introduction, hence the post-vaccine genotype distribution is not known. ObjectivesThe aim of this study was to determine the post-vaccine occurrence of rotavirus genotypes in children <5 years of age in Nairobi County, Kenya. MethodsStool samples were collected from children presenting with diarrhea for whom the vaccination status was card-confirmed. Fecal samples were analyzed for rotavirus antigen using a commercial enzyme immunoassay (EIA) kit, followed by characterization by polyacrylamide gel electrophoresis, RT-PCR, and nested PCR genotyping, targeting the most medically important genotypes. ResultsThe strains observed included G1P[8] (38.8%), G9P[8] (20.4%), G2P[4] (12.2%), G3[P4] (6.1%), G2P[6] (4.1%), and G9P[6] (4.1%). Mixed genotype constellations G3P[4][8] were also detected (4.1%). Remarkably, an increased prevalence of G2 genotypes was observed, revealing a change in genetic diversity of rotavirus strains. While the dominance of G1P[8] decreased after vaccination, an upsurge in G2P[4] (12.2%) and G9P[8] (20.4%) was observed. Additionally, G3[P4] (6.1%) and G2P[6] (4.1%) prevalence increased over the 3 years of study. ConclusionsThe results inform the need for robust longitudinal surveillance and epidemiological studies to assess the long-term interaction between rotavirus vaccine and strain ecology.

Detection of Staphylococcal Enterotoxins in Rice Using Enzyme Immunoassay Kits

Four commercial enzyme immunoassay kits specific for the detection of staphylococcal enterotoxins (SE) were used to examine Stayhylococcus aureus strains isolated from samples of food poisoning outbreaks. Results showed that 89.5% and 10.5% of the strains tested could produce SEA and SEC, respectively. No SEB or SED producing strains were observed. A S.aureus strain which produces SEA was inoculated into some sterilized cooked rice at a concentration of 1.7x103 CFU/g. After 6 h incubation at 37℃,cell concentration reached 2.5x105 CFU/g and SEA reached O.11 ng toxin/g. When the rice sample was kept at 4℃ for 48 h, cell growth and toxin production was not detected. Using eight strains of non-S.aureus bacteria which may be commonly found in foods, was used as control to test the detection specificity of the four commercial available enzyme immunoassay kits. Results showed that no cross-reaction had occurred indicating that the kits are highly specific for enterotoxin detection.

Lateral flow immunoassay for rapid qualitative and quantitative control of the veterinary drug bacitracin in milk

The lateral flow method was first developed for the rapid control of the presence and determination of the amount of polypeptide antibiotic bacitracin. The method is based on the competitive interaction of (a) the bacitracin potentially contained in the sample and (b) the bacitracin-protein conjugate immobilized on the membrane for binding with (c) gold nanoparticles labeled by the anti-bacitracin antibodies during the lateral flow of the reagents and the sample along the test strip. The qualitative assessment of the assay results consists of visual control of the presence or absence of bound colored gold nanoparticles in the zone with the immobilized bacitracin-protein conjugate. The quantitative assay uses a photometric measurement of the intensity of this zone coloration. The thresholds for visual and instrumental detection of bacitracin using the developed assay are 100 and 1.0 ng/mL, and the working range of quantitatively measured concentrations is 3 to 80 ng/mL. The average error of measurement is in the range of 1.5%–7.0%. The analysis time is 10 min. The specificity of the assay has been confirmed in relation to other antibiotics. The test system obtains reliable information on the amount of bacitracin in milk without sample preparation. Testing the milk samples (n = 90) showed the coincidence of the results for the developed lateral flow assay and for a commercial enzyme immunoassay kit. The recovery of bacitracin in milk by the proposed lateral flow method ranged from 75 to 140%.

EXPRESS CONTROL OF ANTIBIOTIC BACITRICIN IN MILK: DEVELOPMENT AND TESTING OF AN IMMUNOCHROMATOGRAPHIC TEST SYSTEM

The immunochromatographic test system was developed for rapid control of polypeptide antibiotic bacitracin in milk. Its analytical parameters were determined. Visual and instrumental detection limits are 100 and 1.0 ng/ml, working range of quantitative measurements – 3–80 ng/ml, time of the assay – 10 min. The test system allows obtaining reliable information about the content of bacitracin in milk without sample preparation. The test system was validated for 90 samples of milk; good correlation with the results obtained using a commercial enzyme immunoassay kit was shown. The degree of bacitracin revealimg in milk ranged from 75 to 140%.

Neurokinin-1 Receptor Antagonism Ameliorates Dry Eye Disease by Inhibiting Antigen-Presenting Cell Maturation and T Helper 17 Cell Activation

Neuroinflammation plays an important role in the pathogenesis of ocular surface disease, including dry eye disease (DED), but little is known about the contribution of substance P (SP) to DED. In this study, we investigated the expression of SP at the ocular surface and evaluated its effect on maturation of antigen-presenting cells (APCs), the key cell component involved in the induction of type 17 helper T-cell (Th17) response in DED. The effect of topical blockade of SP signaling was further investigated using neurokinin-1 receptor (NK1R) inhibitors on APC maturation, Th17 cell activation, and disease severity in a mouse model of DED. The results demonstrate that SP is constitutively expressed at the ocular surface, and trigeminal ganglion neurons are the major source of SP in DED. SP derived from trigeminal ganglion enhanced the expression of major histocompatibility complex class II maturation marker by bone marrow-derived dendritic cells, an effect that is abrogated by blockade of SP signaling using NK1R antagonist spantide. Finally, using a well-established murine model of DED, topical treatment of DED mice with NK1R antagonists CP-99,994 and L-733,060 suppressed APC acquisition of major histocompatibility complex class II, reduced Th17 cell activity, and ameliorated DED severity. These findings are of translational value, as they suggest that antagonizing NK1R-mediated SP signaling may be an effective strategy in suppressing Th17-mediated ocular surface disease.

Is EIPH a misnomer and could it be hampering our attempts to understand the etiology of the condition?

Salivary cortisol provides information about free plasma cortisol concentration, and salivary sampling is a noninvasive well-tolerated procedure. The aim of this study was to validate a commercial enzyme immunoassay for the determination of salivary cortisol in donkeys. Saliva samples were collected in four donkey stallions on 13 nonconsecutive days at 8:30 AM to avoid circadian variation. Animals were already accustomed to be handled. Saliva was collected using a swab inserted at the angle of the lips, placed onto the tongue for 1 minute and returned into a polypropylene tube. Tubes were centrifuged, and at least 1 mL of saliva was aspirated from each sample and frozen at −20°C until analysis. A commercial enzyme immunoassay kit without extraction was used for determination of cortisol in saliva. Median cortisol concentrations with minimum and maximum value were calculated. Recovery of cortisol standard in donkey saliva was between 97.3% and 99.7%, and serial dilution of donkey saliva samples with assay buffer resulted in changes in optical density parallel to the standard curve. Cross-reactivity of the antiserum was 10.4% with 11-deoxycortisol, 5.2% with corticosterone, 0.4% with 11-deoxycorticosterone, 0.2% with cortisone, and <0.1% with testosterone, progesterone, and estradiol. The intra-assay coefficient of variation was 10.7%, the interassay variation was 8.0%, and the minimal detectable concentration was 0.01 ng/mL. The results of the present study demonstrate the validity of a commercial kit to determine the concentration of cortisol in donkey saliva as already reported in other species.