Detection of Specific IgE against Molds Involved in Allergic Bronchopulmonary Mycoses in Patients with Cystic Fibrosis.

CONTEXT: Allergic bronchopulmonary mycoses (ABPM) can be due to molds other than Aspergillus fumigatus in patients with cystic fibrosis (pwCF). We aimed to develop immunoassays for the detection of specific IgE (sIgE) directed against five fungal species involved in ABPM: Aspergillus terreus, Scedosporium apiospermum, Lomentospora prolificans, Rasamsonia argillacea, and Exophiala dermatitidis. MATERIALS AND METHODS: Serum samples (n = 356) from 238 pwCF, collected in eight CF care centers in France, Germany, and Italy, were analyzed by dissociated enhanced lanthanide fluorescent immunoassay (DELFIA®) to assess levels of sIgE directed against antigenic extracts of each fungus. Clinical, biological, and radiological data were collected for each episode. One hundred serum samples from healthy blood donors were used as controls. Sera were classified into four groups depending on the level of sIgE according to the quartile repartition calculated for the pwCF population. A score of 4 for values above the 3rd quartile corresponds to an elevated level of sIgE. RESULTS: PwCF showed higher levels of sIgE than controls. Based on criteria from the ABPA-ISHAM working group, with an additional criterion of "a sIgE score of 4 for at least one non-A. fumigatus mold", we were able to diagnose six cases of ABPM. CONCLUSIONS: Using 417 IU/mL as the threshold for total IgE and the same additional criterion, we identified seven additional pwCF with "putative ABPM". Detection of sIgE by DELFIA® showed good analytical performance and supports the role played by non-A. fumigatus molds in ABPM. However, commercially available kits usable in routine practice are needed to improve the diagnosis of ABPM.

Development of a Method for Detection of SARS-CoV-2 Nucleocapsid Antibodies on Dried Blood Spot by DELFIA Immunoassay.

Antibodies against the SARS-CoV-2 nucleocapsid protein are produced by the immune system in response to SARS-CoV-2 infection, but most available vaccines developed to fight the pandemic spread target the SARS-CoV-2 spike protein. The aim of this study was to improve the detection of antibodies against the SARS-CoV-2 nucleocapsid by providing a simple and robust method applicable to a large population. For this purpose, we developed a DELFIA immunoassay on dried blood spots (DBSs) by converting a commercially available IVD ELISA assay. A total of forty-seven paired plasma and dried blood spots were collected from vaccinated and/or previously SARS-CoV-2-infected subjects. The DBS-DELFIA resulted in a wider dynamic range and higher sensitivity for detecting antibodies against the SARS-CoV-2 nucleocapsid. Moreover, the DBS-DELFIA showed a good total intra-assay coefficient of variability of 14.6%. Finally, a strong correlation was found between SARS-CoV-2 nucleocapsid antibodies detected by the DBS-DELFIA and ELISA immunoassays (r = 0.9). Therefore, the association of dried blood sampling with DELFIA technology may provide an easier, minimally invasive, and accurate measurement of SARS-CoV-2 nucleocapsid antibodies in previously SARS-CoV-2-infected subjects. In conclusion, these results justify further research to develop a certified IVD DBS-DELFIA assay for detecting SARS-CoV-2 nucleocapsid antibodies useful for diagnostics as well as for serosurveillance studies.

NK Cell Isolation and Cytotoxicity by Radioactive Chromium Release Assay and DELFIA-EuTDA Cytotoxicity Assay.

Cytotoxicity assays are important in vitro tools to measure the lysis of desired target cells via an effector immune cell of choice. Specific lysis of the target cells can be determined by labeling the target cells with a radioactive isotope or fluorescent molecule, co-incubating it with an effector cell, then measuring the release of the labeled molecule in the supernatant. Here, we describe and compare different cell cytotoxicity assays using a chromium-51 (51Cr) release and DELFIA EuTDA fluorescent assay using K562 as the target cells and peripheral blood mononuclear cell (PBMC) derived natural killer (NK) cells as the effector cells.

Development of a DELFIA method to detect oncofetal antigen ROR1-positive exosomes.

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is highly expressed in a wide variety of hematological and solid cancers, but is low or absent in adult tissues. Here, we show that ROR1 is released with exosomes from ROR1-positive cancer cells. We also developed a simple dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) to detect cancer-derived ROR1-positive exosomes, which are captured by two anti-ROR1 antibodies and detected by the fluorescence of free chelating europium. This new DELFIA method can detect cancer-derived ROR1-positive exosomes in the cell supernatant and serum with a wide range and rapidly compared with the conventional Western blot assay. This method may be useful as a companion diagnostics for ROR1-positive cancers.

Diagnostic evaluation of heel prick newborn screening of thyroid stimulating hormone on dissociation-enhanced lanthanide fluorescence immunoassay with the establishment of reference value in Pakistani neonates.

OBJECTIVE: To develop and validate a method on Dissociation Enhanced Lanthanide Fluorescence Immunoassay for neonatal heel prick blood human thyroid stimulating hormone and the establishment of its reference value in the local population. METHODS: The multi-centre cross-sectional validation study was conducted from September 2016 to September 2018 at Zahra Beau Naqvi Foundation Welfare Trust laboratory, Islamabad, Pakistan, and comprised samples related to newborns aged 1 month or less taken from neonatal units of 39 hospitals based in Punjab, Khyber Pakhtunkhuwa, Gilgit-Baltistan and Azad Jammu and Kashmir. Samples were collected after 24 hours of birth using the heel prick test. The samples were dried and sent to the laboratory for assessment where Dissociation Enhanced Lanthanide Fluorescent Immunoassay was used to estimate thyroid stimulating hormone levels. Data recorded included age, gender, and birth detail, like gestational age, mode of delivery etc. Data was analysed using SPSS 21. Method validation and reference value were manually calculated. RESULTS: Of the 14,147 samples received, 8,207(58%) related to boys and 5,940(42%) to girls. Most samples 4903(34.6%) came from Peshawar. The overall mean age of the newborns was 5.6±4.8 days. Thyroid stimulating hormone data was divided into three groups; positive with median value 27.8±36.6 uIU/ml, negative with median 1.42±1.60 uIU/ml, and borderline with median 11.4±4.12uIU/ml. Prevalence of congenital hypothyroidism in high-risk population in the positive group was 39(0.3%), negative 14,012(99.0%) and borderline 96(0.7%). Reference cut-off was calculated as 7.06uIU/ml for screening of healthy and positive cases of congenital hypothyroidism. Method Validation results showed limit of detection -0.5uU/ml, limit of quantitation LOQ 0.8uU/ml, accuracy 100±5%, precision coefficient of variation at each level of calibrators -4, 8.8, 1.2, 11.3, 7.2 and 4.3% respectively, and linearity from to 0.8uU/ml to 254.1uU/ml. CONCLUSIONS: Neonatal human thyroid stimulating hormone by heel prick blood was found to be an affordable and highly sensitive method of screening for congenital hypothyroidism.

Forced Self-Modification Assays as a Strategy to Screen MonoPARP Enzymes.

Mono(ADP-ribosylation) (MARylation) and poly(ADP-ribosylation) (PARylation) are posttranslational modifications found on multiple amino acids. There are 12 enzymatically active mono(ADP-ribose) polymerase (monoPARP) enzymes and 4 enzymatically active poly(ADP-ribose) polymerase (polyPARP) enzymes that use nicotinamide adenine dinucleotide (NAD+) as the ADP-ribose donating substrate to generate these modifications. While there are approved drugs and clinical trials ongoing for the enzymes that perform PARylation, MARylation is gaining recognition for its role in immune function, inflammation, and cancer. However, there is a lack of chemical probes to study the function of monoPARPs in cells and in vivo. An important first step to generating chemical probes for monoPARPs is to develop biochemical assays to enable hit finding, and determination of the potency and selectivity of inhibitors. Complicating the development of enzymatic assays is that it is poorly understood how monoPARPs engage their substrates. To overcome this, we have developed a family-wide approach to developing robust high-throughput monoPARP assays where the enzymes are immobilized and forced to self-modify using biotinylated-NAD+, which is detected using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) readout. Herein we describe the development of assays for 12 monoPARPs and 3 polyPARPs and apply them to understand the potency and selectivity of a focused library of inhibitors across this family.

PlGF isoform 3 in maternal serum and placental tissue.

OBJECTIVES: Four isoforms originating from alternative splicing of PGF gene have been reported for placental growth factor (PlGF). Main PlGF isoforms 1 and 2 have been associated with screening and diagnosis of pre-eclampsia (PE). Despite of the vast amount of research around PlGF in PE, protein levels of isoforms PlGF-3 and -4 have not been reported in human serum samples. STUDY DESIGN: In this study a PlGF-3 specific DELFIA research immunoassay based on a custom recombinant Fab binder was developed and characterized. Serum levels of a third PlGF isoform during pregnancy were determined and screening performance of PlGF-3 for PE and small for gestational age (SGA) was investigated. MAIN OUTCOME MEASURES: Levels of serum and placental tissue PlGF 3 and predictive power of PlGF-3 for Pre-eclampsia and SGA. RESULTS: PlGF-3 was below the detection limit of 1.6 pg/mL in most of the serum samples collected during pregnancy. Detected protein levels of PlGF-3 were not associated to be predictive for PE or SGA. However, measurable, and relatively higher amounts of PlGF-3 was extracted from placental tissue samples. CONCLUSION: Data obtained indicates that very low amounts of PlGF-3 is present in blood but significantly higher amounts of protein is present in placental tissue where it is prominently associated with cellular membranes.

Comparison of three commercially available placental growth factor-based tests in women with suspected preterm pre-eclampsia: the COMPARE study.

OBJECTIVE: To compare the performance of three placental growth factor (PlGF)-based tests in predicting delivery within 14 days from testing in women with suspected preterm pre-eclampsia before 35 weeks' gestation. METHODS: This was a retrospective analysis of samples collected from three prospective pregnancy cohort studies. Participants were pregnant women with suspected preterm pre-eclampsia recruited in tertiary maternity units in the UK and Ireland. Samples were analyzed simultaneously according to the manufacturers' directions. The tests compared were the DELFIA Xpress PlGF 1-2-3 test, the Triage PlGF test and the Elecsys immunoassay soluble fms-like tyrosine kinase-1 (sFlt-1)/PlGF ratio. Areas under receiver-operating characteristics curves (AUCs) were compared. The main outcome measure was detection of a difference of 0.05 in AUC between tests for delivery within 14 days of testing. RESULTS: Plasma samples from 396 women and serum samples from 244 women were assayed. In predicting delivery within 14 days secondary to suspected pre-eclampsia prior to 35 weeks' gestation, no significant differences were observed in AUCs (P = 0.795), sensitivities (P = 0.249), positive predictive values (P = 0.765) or negative predictive values (P = 0.920) between the three tests. The specificity of the Elecsys sFlt-1/PlGF ratio test was higher than that of the other two tests (P < 0.001). CONCLUSIONS: The tests perform similarly in their prediction of need for delivery within 14 days in women with suspected pre-eclampsia. The high negative predictive values support the role of PlGF-based tests as 'rule-out' tests for pre-eclampsia. © 2018 Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Development of a sensitive potency assay to measure the anti-proliferation effect of an anti-HER2 antibody.

For therapeutic antibodies that inhibit the growth of cancer cells, proliferation assays that measure cell number changes after the antibody treatment are often used to determine the potency of the antibody. Two of the most commonly used non-radioactive readout systems for proliferation assays, the ATP bioluminescence assay and the fluorescent dye Alamar Blue assay, were initially tested as potency assays an anti-HER2 antibody. Due to the slow growth of the target cells, these assays only produced less than 3-fold difference after 5 days of antibody treatment. BrdU incorporation-based proliferation assay, which differentiates proliferating cells from arrested cells, was developed, and showed superior sign-to-background ratio. Colorimetric, chemiluminescent, and DELFIA readouts were compared for BrdU incorporation assays, and DELFIA-based assay was further optimized using a Design of Experiment (DoE) approach. The final DELFIA-based BrdU incorporation assay demonstrated superior signal-to-background ratio, robustness, accuracy, and precision, and represented significant improvement over traditional proliferation assays.

Mimicry epitope from Ehrlichia canis for interphotoreceptor retinoid-binding protein 201-216 prevents autoimmune uveoretinitis by acting as altered peptide ligand.

We report here identification of novel mimicry epitopes for interphotoreceptor retinoid-binding protein (IRBP) 201-216, a candidate ocular antigen that causes experimental autoimmune uveoretinitis (EAU) in A/J mice. One mimicry epitope from Ehrlichia canis (EHC), designated EHC 44-59, induced cross-reactive T cells for IRBP 201-216 capable of producing T helper (Th)1 and Th17 cytokines, but failed to induce EAU in A/J mice. In addition, animals first primed with suboptimal doses of IRBP 201-216 and subsequently immunized with EHC 44-59 did not develop EAU; rather, the mimicry epitope prevented the disease induced by IRBP 201-216. However, alteration in the composition of EHC 44-59 by substituting alanine with valine at position 49, similar to the composition of IRBP 201-216, enabled the mimicry epitope to acquire uveitogenicity. The data provide new insights as to how microbes containing mimicry sequences for retinal antigens can prevent ocular inflammation by acting as naturally occurring altered peptide ligands.

Application of serum PSA, fPSA detections and fPSA/PSA evaluation for diagnosis and therapy of prostatic diseases with DELFIA technique / 吉林大学学报(医学版)

Objective To investigate the application of PSA, fPSA concentrations and fPSA/PSA value for diagnosis and therapy of prostatic diseases detected with dissociation enhanced lauthanide fluoroimmunoassay (DELFIA) technique . Methods Thirty-four cases of normal individuals were used as control group, 46 patients with prostatitis as prostatitis group, 123 patients with prostatic hyperplasia as BPH group and 39 patients with prostate cancer as prostate cancer group. PSA, fPSA concentrations and fPSA/PSA value were detected with DELFIA technique. Results In prostate cancer group, the PSA and fPSA concentrations were significantly higher than those in other 3 groups (P0.05). Conclusion DELFI A technique is an effective method to improve the diagnosis and differential diagnosis of prostatic diseases, especially for prostate cancer.

[Comparative analysis of the determination of serum testosterobe levels by different methods].

The serum concentration of total testosterone were measured in 100 male patients aged 19-65 years who had visited the clinic and polyclinic of the Endocrinology Research Center, Russian Academy of Medical Sciences for complaints about different dysfunctions of the endocrine glands. Testosterone was measured by the well-known currently available technologies: automatic Vitros ECi, Architect, and Access multianalyzers, manual Delfia and DRS enzyme immunoassay kits, and radioimmunoassay (RIA) that had been developed within the framework of the WHO Human Reproduction Programme and adjusted by the reference assay (gas chromatography/mass spectroscopy). The closest results were obtained when determining the level of testosterone by RIA and by means of the Vitros ECi multianalyzer (13.1 vs 13.0 nmol/l, medians). All other technologies demonstrated a low correlation with RIS and had positive biases, especially with the use of the Delfia kits (as high as 60 and, at low concentrations, 100%). The positive bias tends to decrease in patients with higher testosterone levels ( > 10 nmol/l) for all studied methods.