Sandwich Chemiluminescent Immunoassay Research Articles

Enhancing Nanobody Immunoassays through Ferritin Fusion: Construction of a Salmonella-Specific Fenobody for Improved Avidity and Sensitivity.

Nanobodies (Nbs) serve as powerful tools in immunoassays. However, their small size and monovalent properties pose challenges for practical application. Multimerization emerges as a significant strategy to address these limitations, enhancing the utilization of nanobodies in immunoassays. Herein, we report the construction of a Salmonella-specific fenobody (Fb) through the fusion of a nanobody to ferritin, resulting in a self-assembled 24-valent nanocage-like structure. The fenobody exhibits a 35-fold increase in avidity compared to the conventional nanobody while retaining good thermostability and specificity. Leveraging this advancement, three ELISA modes were designed using Fb as the capture antibody, along with unmodified Nb422 (FbNb-ELISA), biotinylated Nb422 (FbBio-ELISA), and phage-displayed Nb422 (FbP-ELISA) as the detection antibody, respectively. Notably, the FbNb-ELISA demonstrates a detection limit (LOD) of 3.56 × 104 CFU/mL, which is 16-fold lower than that of FbBio-ELISA and similar to FbP-ELISA. Moreover, a fenobody and nanobody sandwich chemiluminescent enzyme immunoassay (FbNb-CLISA) was developed by replacing the TMB chromogenic substrate with luminal, resulting in a 12-fold reduction in the LOD. Overall, the ferritin-displayed technology represents a promising methodology for enhancing the detection performance of nanobody-based sandwich ELISAs, thereby expanding the applicability of Nbs in food detection and other fields requiring multivalent modification.

Development of Shark Single Domain Antibodies Specific for Human α-Fetoprotein and the Multimerization Strategy in Serum Detection.

Sensitive detection of cancer biomarkers can contribute to the timely diagnosis and treatment of diseases. In this study, the whitespotted bamboo sharks were immunized with human α-fetoprotein (AFP), and a phage-displayed variable new antigen receptor (VNAR) single domain antibody library was constructed. Then four unique VNARs (VNAR1, VNAR11, VNAR21, and VNAR25) against AFP were isolated from the library by biopanning for the first time. All of the sequences belong to type II of VNAR, and the VNAR11 was much different from the rest of the three sequences. Then VNAR1 and VNAR11 were selected to fuse with the C4-binding protein α chain (C4bpα) sequence and efficiently expressed in the Escherichia coli system. Furthermore, a VNAR-C4bpα-mediated sandwich chemiluminescence immunoassay (VSCLIA) was developed for the detection of AFP in human serum samples. After optimization, the VSCLIA showed a limit of detection of 0.74 ng/mL with good selectivity and accuracy. Moreover, the results of clinical serum samples detected by the VSCLIA were confirmed by an automatic immunoanalyzer in the hospital, indicating its practical application in actual samples. In conclusion, the novel antibody element VNAR exhibits great potential for immunodiagnosis, and this study also provides a new direction and experimental basis for AFP detection.

Evaluating peripheral blood inflammatory and metabolic biomarkers as predictors in diabetic retinopathy and diabetic macular edema.

To determine the correlation between serum inflammatory and metabolic biomarkers of patients with diabetic retinopathy (DR) and diabetic macular edema (DME). Serum samples were obtained from 100 diabetic patients. Patients were divided into three groups: group 1 (patients with no DR, n = 27), group 2 (DR with DME, n = 34), and group 3 (DR without DME, n = 39). Serum concentrations of C-reactive protein (CRP) and interleukin-6 (IL-6) were measured by quantitative turbidimetric immunoassay and sandwich chemiluminescence immunoassay, respectively. Metabolic parameters such as glycated hemoglobin (HbA1c), total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglyceride (TG), serum creatinine, and blood urea were determined by automated analyzer om-360 after standardization. The levels of IL-6 and CRP differed significantly in patients with DR and without DR (P < 0.001 and P = 0.045, respectively). We also found a positive correlation between IL-6 and CRP with the severity of DR. When DR patients with DME were compared to patients without DME, only IL-6 was observed to be significantly elevated (P < 0.001). None of the metabolic markers correlated significantly with DR and DME. Significantly raised levels of serum inflammatory biomarkers can be used to elucidate the significant role of inflammation in the pathogenesis of DR. Therefore, circulating biomarkers can serve as diagnostic and therapeutic predictors for monitoring the onset and progression of DR and DME.

Correlation Between Quantitative Hepatitis B Surface Antigen and Hepatitis B Virus Deoxyribonucleic Acid Levels in Hepatitis B e Antigen-Positive and Hepatitis B e AntigenNegative Chronic Hepatitis B Patients.

This study aimed to analyze the relationship between quantitative hepatitis B surface antigen and hepatitis B virus deoxyribonucleic acid in hepatitis B e antigen-positive and hepatitis B e antigen-negative chronic hepatitis B patients and to determine the best cut-off value for quantitative hepatitis B surface antigen to predict high hepatitis B virus deoxyribonucleic acid levels (≥2000 IU/mL). Ninety-seven sera from chronic hepatitis B patients were collected in this study. Hepatitis B virus deoxyribonucleic acid levels were quantified by real-time polymerase chain reaction. Quantitative hepatitis B surface antigen and hepatitis B e antigen levels were determined by two-site sandwich chemiluminescence immunoassay. Alanine transaminase levels were measured by the International Federation of Clinical Chemistry-approved methods. A significant correlation between quantitative hepatitis B surface antigen and hepatitis B virus deoxyribonucleic acid levels was observed in hepatitis B e antigen-positive group (r = 0.453, P = .002), but not in hepatitis B e antigen-negative group (r = 0.117, P = .454). No significant correlation between quantitative hepatitis B surface antigen and alanine transaminase was found in the hepatitis B e antigen-positive group (r = 0.521, P = .241). However, a significant correlation was shown between quantitative hepatitis B surface antigen and alanine transaminase levels in the hepatitis B e antigen-negative group (r = 0.455, P = .001). The best cut-off value of quantitative hepatitis B surface antigen for predicting high hepatitis B virus deoxyribonucleic acid levels was 3.422 × 103 IU/mL. Correlation between quantitative hepatitis B surface antigen and hepatitis B virus deoxyribonucleic acid levels is significant in the hepatitis B e antigen-positive group. Quantitative hepatitis B surface antigen can be used to predict high hepatitis B virus deoxyribonucleic acid levels in the hepatitis B e antigen-positive group.

Establishment of Sensitive Sandwich-Type Chemiluminescence Immunoassay for Interleukin-18 in Urinary Samples.

Interleukin-18 (IL-18), a member of IL-1 cytokine superfamily, is deemed as an important indicator of the kidney disease. Herein a sandwich chemiluminescence immunoassay integrated with magnetic beads was conducted to detect IL-18 in kidney disease. The detection limit and linear range were 0.0044ng/mL and 0.01-2.7ng/mL, respectively. Satisfactory recoveries were ranged from 91.70 to 101.18% with the relative standard deviation below 10%; interference bias of most biomarkers were within allowable deviation range (± 15%). In summary, the whole study was successfully applied to detect IL-18 levels in urine samples for patients with kidney disease. The results showed that chemiluminescence immunoassay for IL-18 detection could be used in the clinical application.

JS-HR-9: A NEW ERA OF PROGRESS IN PRIMARY ALDOSTERONISM TREATMENT, A NEW ALDOSTERONE ASSAY, AND A CLINICAL PRACTICE GUIDELINE

Primary aldosteronism (PA), one of the mineralocorticoid receptor (MR)-associated hypertension, is a highly prevalent secondary hypertension that accounts for 5–15% of all hypertension and is a severe hypertension that is prone to cerebrovascular and cardiovascular disease. Therefore, it is expected that the pathophysiology and treatment strategies for PA will be elucidated. In recent years, the major changes in PA have been in treatment and diagnosis in Japan. Bilateral or non-surgical unilateral PA is treated with mineralocorticoid receptor antagonist (MRA). Esaxerenone, a non-steroidal MRA, is now widely used in Japan for hypertension including PA. It has been reported that add-on treatment using esaxerenone with maximal tolerable doses of a renin-angiotensin system inhibitor reduced the urinary albumin creatinine ratio in patients with type 2 diabetes mellitus (ESAX-DN), suggesting a renoprotective effect against diabetic nephropathy. Several clinical studies of another non-steroidal MRA, finerenone(FIDELIO-DKD, FIGARO-DKD, and FIDELITY), have shown its renoprotective effect against diabetic nephropathy as well as cardiovascular events, particularly hospitalization for heart failure in patients with type 2 diabetes and chronic kidney disease. Although there are no reports of clinical studies on finerenone for PA, finerenone may be used in the future for type 2 diabetes patients with PA, as obesity, glucose intolerance, and sleep apnea are common complications in patients with PA. Secondly, radiofrequency ablation of macroscopic adrenal tumors has been reported as an alternative treatment for PA to lower blood pressure and plasma aldosterone levels. This treatment has been covered by health insurance providers in Japan since April 2022, but long-term outcomes need to be validated. In diagnosis, serum and urine aldosterone has been measured in Japan since early 2021 by the two-step sandwich chemiluminescent enzyme immunoassay (CLEIA) method. This new CLEIA method correlates well with LC-MS/MS values and is expected to be more accurate. As CLEIA serum aldosterone levels are lower than those measured with a previous radioimmunoassay, the Japan Endocrine Society issued the 2021 Primary Aldosteronism Clinical Guidelines, which included a provisional positive value for the diagnosis of PA. More than ever, the diagnosis of PA needs to be carefully considered. This CLEIA method is currently available only in Japan. This presentation will cover the latest treatments, diagnostic procedures, and other hot topics in PA as described above.

317. Use cases for rapid antigen-detecting tests for COVID-19 screening and surveillance: a systematic review

Abstract Background Testing remains critical to controlling the COVID-19 pandemic. Antigen-detecting rapid diagnostic tests (Ag-RDTs), which can be used at the point of care, have the potential to increase access to COVID-19 testing, particularly in settings with limited laboratory capacity. This systematic review synthesized literature on specific use cases and performance of Ag-RDTs for detecting SARS-CoV-2, for the first comprehensive assessment of Ag-RDT use in real-world settings. Methods We searched three databases (PubMed, EMBASE and medRxiv) up to 12 April 2021 for publications on Ag-RDT use for large-scale screening and surveillance of COVID-19, excluding studies of only presumptive COVID-19 patients. We tabulated data on the study setting, populations, type of test, diagnostic performance, and operational findings. We assessed risk of bias using QUADAS-2 and an adapted tool for prevalence studies. Results From 4313 citations, 39 studies conducted in asymptomatic and symptomatic individuals were included. Of 39 studies, 37 (94.9%) investigated lateral flow Ag-RDTs and 2 (5.1%) investigated multiplex sandwich chemiluminescent enzyme immunoassay Ag-RDTs. Six categories of testing initiatives were identified: mass screening (n=13), targeted screening (n=11), healthcare entry testing (n=6), at-home testing (n=4), surveillance (n=4) and prevalence survey (n=1). Sensitivity and specificity values by testing category are shown in the table. Ag-RDTs were noted as convenient, easy-to-use, and low cost, with a rapid turnaround time and high user acceptability. Risk of bias was generally low or unclear across studies. Conclusion During the first year of the COVID-19 pandemic, Ag-RDTs were used across a wide range of real-world settings for screening and surveillance of COVID-19 in both symptomatic and asymptomatic individuals. Ag-RDTs were fast and simple to run, but due to their often low sensitivity, careful consideration must be given to their implementation and interpretation. Ag-RDTs have subsequently been rolled out more broadly and recommended for COVID-19 self-testing. Disclosures Talya Underwood, MPhil, Oncotherapeutics: Medical writing.

Implementation of Experimental Design Techniques to Optimize Immunoglobulins Detection with Ultrasensitive Sandwich Immunoassays (Adv. Sensor Res. 1/2022)

Inside Front Cover Ultrasensitive Sandwich Immunoassays In article number 2200009, Eleonora Macchia and co-workers study a full factorial experimental design that is undertaken to optimize a sandwich chemiluminescent immunoassay, leading to a reduced experimental effort and increased quality of the information obtained with respect to one-variable-at-a-time approaches. The optimization leads to an immunoglobulin M limit-of-detection of 4 fM, reducing the involved antibody concentrations by one order of magnitude and dramatically reducing the assay costs.

Determination of the Association Between Serum Ferritin and C-Reactive Protein in Children Presented with Iron Deficiency Anemia in Combined Military Hospital Pathology Department Multan

Objective: To determine association between Serum ferritin and C-reactive protein (CRP) in children having iron scarcity anemia.&#x0D; Study Design: Cross sectional study.&#x0D; Place and Duration of Study: Pathology Department, Combined Military Hospital Multan Pakistan, from Jun to Dec 2018&#x0D; Methodology: One hundred and thirty-eight (138) children were consecutively selected as per inclusion and exclusion criteria. Each sample of blood was collected from children for serum ferritin, Blood complete picture (Blood CP) which include hemoglobin level and red blood cell indices and serum sample for C-reactive protein (CRP). Sandwich chemilumine-scence immunoassay technique was used to analyze serum ferritin, Blood CP was analyzed on sysmex and C-reactive protein was analyzed immuneturbidi-metric method.&#x0D; Result: A total of 138 children were included in our study. Mean age group of our population is 4.5 ± 0.65 years. Out of these 138, 67 (49%) were male children and 71 (51%) were female children. CRP was found negative in 50 (36%) and positive in 88 (64%) children. ANOVA and chi-square test showed association between CRP and anemia was statically significant.&#x0D; Conclusion: Increased ferritin level, secondary to inflammation may mask an underlying iron inadequacy in children. CRP in children with normal or high ferritin can be helpful in diagnosing iron deficiency. Serum ferritin was found to be positively correlated with CRP in children.

Post-vaccination Immunity Against Hepatitis B Among Mongolian Adolescents and Youths

Background: Mongolia introduced vaccination against hepatitis B (HepB) in 1991, leading to a significant decline in the number of infections and mortality associated with the liver disease among this generation. However, the prevalence of hepatitis B virus (HBV) infection and mortality rates among people born before the vaccination program have not declined. Although several studies have been conducted in Mongolia since the introduction of the HepB immunization program, long-term immunity has not been studied at the national level. Objectives: This study aimed to determine the prevalence of HBV infection in adolescents and young adults who received HepB vaccinations at 0, 2, and 8 months after birth and to assess their post-vaccination immunity against hepatitis B. Methods: A population-based cross-sectional study was conducted between December 2016 and December 2018 and included a sample aged 10 to 27 years in Mongolia who had received HepB vaccination according to the national program. A total of 3591 individuals were randomly selected, and data were collected using a structured questionnaire. Blood samples were collected, and serum titers of hepatitis B surface antigen (HBsAg), antibody to HBsAg (anti-HBs), and antibody to hepatitis B core antigen (anti-HBc) were determined by a two-step sandwich chemiluminescent enzyme immunoassay. The age-specific geometric mean of anti-HBs was also estimated. Results: Overall, 98.3% of participants were vaccinated against HepB as infants, according to the interview. The majority had an inadequate anti-HBs titer, while 17.9% had an anti-HBs level of &gt; 10 mIU/mL, of whom 5.7% had immunity induced by HBV infection. Up to 4% of children aged 10 - 19 years and an average of 8% of young adults were serologically positive for HBsAg. The geometric mean anti-HBs titer declined with age, from an average of 40.4 mIU/mL in 10-year-old children to 14.1 IU/mL in 27-year-old adults (P &lt; 0.001). Conclusions: In Mongolia, a small proportion of the population aged 10 - 27 years is immune to HBV, and the geometric mean titer of anti-HBS tended to decrease with age. In order to attain long-term protection against HBV, booster vaccination in adulthood may be necessary.

Evaluation of Five Commercial SARS-CoV-2 Antigen Tests in a Clinical Setting.

BackgroundPoint-of-care antigen tests (AgTs) for the detection of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) enable the rapid testing of infected individuals and are easy-to-use. However, there are few studies evaluating their clinical use.ObjectiveThe present study aimed to evaluate and compare the clinical performance characteristics of various commercial SARS-CoV-2 AgTs.DesignThe sensitivity of five AgTs, comprising four rapid antigen tests (RAT; AMP Rapid Test SARS-CoV-2 Ag, NADAL COVID-19 Antigen Rapid Test, CLINITEST Rapid COVID-19 Antigen Test, and Roche SARS-CoV-2 Rapid Antigen Test) and one sandwich chemiluminescence immunoassay (CLIA; LIAISON SARS-CoV-2 Assay), were evaluated in 300 nasopharyngeal (NP) swabs. Reverse transcriptase (RT) polymerase chain reaction (PCR) was used as a reference method.ParticipantsNP swabs were collected from patients admitted to hospital due to COVID-19.Key ResultsSensitivities of the AgTs ranged from 64.9 to 91.7% for samples with RT-PCR cycle threshold (Ct) values lower than 30 and were 100% for cycle threshold (Ct) values lower than 20. The highest sensitivity was observed for CLINITEST Rapid COVID-19 Antigen Test, and Roche SARS-CoV-2 rapid antigen test. Multivariate analysis using time from symptom onset and the Ct value for AgT sensitivity showed an inverse correlation. Further, the female sex was an independent factor of lower RAT sensitivity.ConclusionsAntigen tests from NP swab samples show high sensitivity in patients with a Ct value < 20. The best clinical sensitivity can be obtained using AgTs within the first 6 days after symptom onset.

Anterior Pituitary Hormones in Blood and Cerebrospinal Fluid of Patients in Neurocritical Care.

Background: Anterior pituitary hormones in blood follow a circadian rhythm, which may be influenced by various factors such as intracranial pathologies. In cerebrospinal fluid (CSF), pituitary hormones have been collected only selectively and circadian rhythm has not yet been investigated. This pilot study analysed diurnal variations of anterior pituitary hormones in patients in neurocritical care to determine whether circadian rhythmicity exists in these patients. Possible influences of intracranial pathologies were also investigated. Blood and CSF concentrations were assessed simultaneously to explore the value of blood concentrations as a surrogate parameter for CSF levels. Methods: Blood and CSF samples of 20 non-sedated patients were collected at 06:00, noon, 18:00 and midnight, and analysed for adrenocorticotropic hormone (ACTH), cortisol, thyroid-stimulating hormone (TSH) and insulin-like growth factor-1 (IGF-1) concentrations at each of the four time points. ACTH and IGF-1 were measured by sandwich chemiluminescence immunoassay. Cortisol and TSH were measured by electrochemiluminescence immunoassay. Results: Results showed inconsistent circadian rhythms. Less than 50% of the patients showed a circadian rhythmicity of ACTH, cortisol, TSH or IGF-1. Significance of diurnal variations was only present for blood concentrations of TSH. Correlations between blood and CSF concentrations were strong for cortisol and TSH. Conclusions: CSF concentrations were only in the measurable range in some of the patients. No clear circadian rhythmicity could be identified, except for TSH in blood. Absence of significant diurnal variations could be explained by the underlying pathologies or disturbing influences of the intensive care unit. Blood concentrations of cortisol and TSH may be suitable surrogate parameters for CSF.

An ultrasensitive sandwich chemiluminescent enzyme immunoassay based on phage-mediated double-nanobody for detection of Salmonella Typhimurium in food

In this study, we demonstrated the first report of a phage-mediated double-nanobody sandwich chemiluminescent enzyme immmunoassay (P-CLISA) for Salmonella Typhimurium (S. Typhimurium) determination. Epitope mapping was first conducted for pairing nanobodies, and then soluble and phage-displayed nanobodies were used as detection antibodies in Nb-ELISA and P-ELISA, respectively. Compared with Nb-ELISA, P-ELISA has a 100-fold improvement in sensitivity given the signal amplification mediated by phage. Moreover, a chemiluminescence reaction was applied to replace the traditional chromogenic reaction in the assay, which achieved the detection of S. Typhimurium with an LOD of 3.63 × 103 CFU/mL in a linear range from 5.1 × 103 to 1.2 × 106 CFU/mL. The P-CLISA was successfully applied in actual sample analysis and able to detect fewer than 10 S. Typhimurium cells within 6–8 h of incubation. This study provides guidelines to develop reliable and sensitive double nanobody sandwich immunoassays, which also have great potential for other large molecule monitoring in food samples.

Development of a Sandwich Chemiluminescence Immunoassay for the Detection of Intact Procollagen Type I N Propeptide with Magnetic Nanosphere Carrier Technology.

The metabolic product of type I collagen synthesis, intact procollagen type I N propeptide (intact PINP), is a potential marker of bone formation and osteoporosis, which is not affected by kidney function. We sought to establish a chemiluminescent immunoassay method for the detection of serum intact PINP with previously prepared paired monoclonal antibodies and to evaluate the diagnostic value of the assay in osteoporosis. Using the capture molecule and monoclonal antibody as detection molecule, a diagnostic reagent was developed to detect intact PINP in serum with magnetic nanosphere carriers by the chemiluminescence method, and its analytical performance in the laboratory was evaluated. Serum intact PINP was measured in 142 healthy people and 115 osteoporosis patients. Results were matched with results of a similar test kit, Roche total PINP Elecsys Chemiluminescent Immunoassay Assay. Compared with the performance of the Roche PINP assay product, our method had higher sensitivity (0.02 ng/mL), wider linear range (0.02-1500 ng/mL), and anti-interference. Serum intact PINP values in osteoporosis patients were significantly higher than in healthy subjects (p < 0.001). Our method had good consistency compared with the Roche PINP assay (r = 0.9794). This chemiluminescence method for detecting serum intact PINP (CLIA-intact PINP) with magnetic nanosphere carrier technology meets the requirements of a clinical testing reagent and is expected to have clinical application after further evaluation and can compete with expensive imported kits on the market.

BRAIN NATRIURETIC PEPTIDE (BNP) SEBAGAI BIOMARKER GAGAL JANTUNG KONGESTIF

Brain Natriuretic Peptide (BNP) is mainly secreted by the heart ventricles and acts as an antagonist to Renin-Angiotensin-Aldosterone. BNP is secreted as pre-proBNP which is broken down into BNP and proBNP in circulation. The BNP examination is done by measuring the levels of BNP or N-Terminal-proBNP depending on the method and the manufacturer. Increased levels of BNP and NT-proBNP indicate heart failure so that BNP and NT-proBNP are considered markers of heart failure. This examination is also indicated to help establish the diagnosis, monitoring, and prognosis of heart failure. Natriuretic peptide examination can be done by several methods such as radioimmunoassay (RIA), Enzyme immunoassay (EIA), Fluorescence immunoassay (FIA), and Sandwich Electro Chemiluminescence Immuno Assay (Sandwich ECLIA).

BRAIN NATRIURETIC PEPTIDE (BNP) SEBAGAI BIOMARKER GAGAL JANTUNG KONGESTIF

Brain Natriuretic Peptide (BNP) is mainly secreted by the heart ventricles and acts as antagonist to Renin-Angiotensin-Aldosterone. BNP is secreted as pre-proBNP which is broken down into BNP and proBNP in circulation. The BNP examination is done by measuring the levels of BNP or N-Terminal-proBNP depending on the method and the manufacturer. Increased levels of BNP and NT-proBNP indicate heart failure so that BNP and NT-proBNP are considered markers of heart failure. This examination is also indicated to help establish the diagnosis, monitoring, and prognosis of heart failure. Natriuretic peptide examination can be done by several methods such as radioimmunoassay (RIA), Enzyme immunoassay (EIA), Fluorescence immunoassay (FIA), and Sandwich Electro Chemiluminescence Immuno Assay (Sandwich ECLIA).

P1185LUNG COMETS AND EVALUATION OF HYDRATION STATUS IN PERITONEAL DIALYSIS PATIENTS

Abstract Background and Aims Assessment of optimal hydration status in patients with end-stage renal disease (ESRD) on peritoneal dialysis (PD) is a challenge. Multiple diagnostic options to determine hydration status in PD patients are available. Multifrequency bioimpedance spectroscopy (MBIS) is a cheap, simple and non-invasive method of estimating body composition, including estimates of total body water (TBW), extracellular water (ECW), intracellular water (ICW) and the ratio between both spaces (ECW/ICW). Lung ultrasonography (LUS) and lung B-lines (lung comets) can be used for the evaluation of extravascular lung water. Ultrasound evaluation of inferior vena cava (UIVC) provides rapid, non-invasive assessment of a patient's hemodynamic and volume status. N-terminal pro-brain natriuretic peptide (NT-proBNP) is related to fluid status and fluid distribution. The aim of our study was to assess fluid status in PD patients comparing four different methods: MBIS, LUS, UIVC and NT-proBNP. Method We performed a single-centre cohort study in 19 PD patients. The body composition was measured using the portable whole-body MBIS device, Body Composition Monitor-BCM(®) (Fresenius Medical Care, Bad Homburg, Germany), LUS with portable US device (VScan, General Electrics Corporate), UIVC index with SonoSite US device. NT-proBNP was measured in a one-step sandwich chemiluminescent immunoassay (Dimension Vista® System 1500, Siemens Healthcare Diagnostics Inc., Newark, NJ, USA). Results The mean age of patients was 54 ± 10 years, mean dialysis vintage 53 (10-194) months, 63% were men. Thirteen (68.4%) patients had fluid overload (FO) &amp;gt; 1.1 L. Data of patients are presented in table 1. We found a statistically significant correlation between the number of lung comets and ECW/ICW ratio (r = 0.496, P = 0.031) and NT-proBNP (r = 0.759, P &amp;lt; 0.0001). In contrast, there was no significant correlation between the number of lung comets and UIVC (r = 0.221, P &amp;lt; 0.364). Conclusion According to our results, LUS with lung comets, MBIS with ECW/ICW ratio and NT-proBNP are useful and complementary methods for evaluation of fluid status in PD patients.

Development of sandwich chemiluminescent immunoassay based on an anti-staphylococcal enterotoxin B Nanobody–Alkaline phosphatase fusion protein for detection of staphylococcal enterotoxin B

In this study, sandwich chemiluminescent immunoassay (CLIA) for the detection of Staphylococcal enterotoxin B (SEB) was developed using nanobody–alkaline phosphatase (Nb–ALP) fusion protein. The SEB-binding nanobodies were obtained from a naïve phage-display library and the Nb–ALP fusion protein was constructed and obtained as a thermally stable and potentially effective substance for detecting antibodies in CLIA. The working range of the sandwich CLIA based on anti-SEB monoclonal antibodies (mAbs) and our fusion protein, Nb37–ALP, was 3.12–50.0 ng mL−1 with SC50 = 8.59 ± 0.37 ng mL−1. The limit of detection was 1.44 ng mL−1 according to the blank value plus 3 standard deviations. In order to understand the interaction of SEB and Nb37 in depth, the 3D structure of the SEB–Nb37 complex was constructed and verified by molecular modeling and the docking method. The results showed that the complementary-determining region 3 (CDR3) of Nb37 embedded itself in the opening generated by the major histocompatibility complex (MHC) and T-cell receptor- (TcR) binding sites of SEB, indicating that Nb37 may affect the recognition of SEB by MHC class Ⅱ molecules and the TcR. The arginine residue (Arg) 101, Arg102 and phenylalanine residue (Phe)103 of CDR3 in Nb37 may have contributed to specific binding to form six salt-bridges between these and SEB. In conclusion, in terms of their specificity and sensitivity, the obtained anti-SEB Nb–ALP appears to have the potential to replace chemically labeled probes for the detection of SEB.

Determination of Prothrombin Time, Activated Partial Thromboplastin Time and D-Dimer Levels among Malaria Infected Patients in Sinnar State, Sudan

Malaria is one of the major causes of disease for people living in tropical and subtropical areas. Blood coagulation activation is frequently found in patients with malaria. The aim of the current study is to assess prothrombin time, activated partial thromboplastin time and D-dimer in patient suffering from malaria. This study includes 50 patients who have malaria parasite and 50 persons as control. Sandwich chemiluminescence immunoassay uses ABEI to label an anti-D-dimer monoclonal antibody and uses another D-dimer monoclonal antibody to coat magnetic microbeads. Prothrombin Time (PT) and Partial Thromboplastin Time (PTT) were measured for all case and control and SPSS software was used to evaluate the correlation. Results of D-dimer in all patient in mean were 1.92 ± 1.09 ng/ml; the D-dimer level was significantly higher among patients (p value = 0.00) compared to control (0.41 ± 0.10 ng/ml). PT in patient in mean was 16.34 ± 2.67 seconds and the mean of PT result in control was 14.29 ± 0.92 seconds; the mean of INR results in patients was 1.22 and the mean of INR results in control was 1.19. The results of PTT in patients in mean were (30.40 ± 4.73 second) and the mean of PTT results in control were (27.82 ± 1.49 seconds). Also, our result shows no statistically significant in PT, APTT and D-dimer between male and female. In conclusion, our study revealed higher D-dimer levels, PT and APTT among patient with Malaria, while the gender showed no effect on the level of D-dimer levels, PT and APTT.

Phage-displayed nanobody based double antibody sandwich chemiluminescent immunoassay for the detection of Cry2A toxin in cereals

ABSTRACT In this study, we developed a double antibody sandwich chemiluminescent immunoassay (DAS-CLIA) based on phage-displayed nanobodies for the determination of Cry2A toxin. Nine phage-displayed nanobodies specifically to Cry2A were obtained from a naive nanobody library after four rounds of biopanning. The phage-displayed nanobody P2 with high affinity binding was selected as the detection antibody for the sensitive DAS-CLIA development. The proposed method exhibited high sensitivity with a limit of detection of 0.09 ng/mL, has a wide linear range for Cry2A toxin detection, in the range of 0.1–1000 ng/mL and negligible cross-reactivities to the other Bt toxins. The recoveries of Cry2A toxin spiked in cereal samples (rice, wheat, and corn) ranged from 82.7% to 118%, with a coefficient of variation of less than 10%. This study demonstrated that the DAS-CLIA procedure based on phage-displayed nanobodies may provide an alternative sensitive method for the detection of Bt toxins in agri-products.