Fluorescence Enzyme Immunoassay Research Articles

Sensitivity and specificity of LDBio Aspergillus ICT lateral flow assay for diagnosing allergic bronchopulmonary aspergillosis in adult asthmatics.

Aspergillus fumigatus-specific IgG estimation is crucial for diagnosing allergic bronchopulmonary aspergillosis (ABPA). A point-of-care LDBio immunochromatographic lateral flow assay (LFA) had 0%-90% sensitivity to detect IgG/IgM antibodies against A. fumigatus. To assess the accuracy of LDBio-LFA in diagnosing ABPA, using the modified ISHAM-ABPA working group criteria as the reference standard. The secondary objective was to compare the diagnostic performance between LDBio-LFA and A. fumigatus-specific IgG (cut-offs, 27 and 40 mgA/L), using a multidisciplinary team (blinded to A. fumigatus-IgG and LDBio-LFA results) diagnosis of ABPA as the reference standard. We prospectively enrolled adult subjects with asthma and ABPA. We performed the LDBio-LFA per the manufacturer's recommendations. We used the commercially available automated fluorescent enzyme immunoassay for measuring serum A. fumigatus-specific IgG. We used the same serum sample to perform both index tests. The tests were performed by technicians blinded to the results of other tests and clinical diagnoses. We included 123 asthmatic and 166 ABPA subjects, with a mean ± SD age of 37.4 ± 14.4 years. Bronchiectasis and high-attenuation mucus were seen in 93.6% (146/156) and 24.3% (38/156) of the ABPA subjects. The sensitivity and specificity of LDBio-LFA in diagnosing ABPA were 84.9% and 82.9%, respectively. The sensitivity of serum A. fumigatus-specific IgG ≥27 mgA/L was 13% better than LDBio-LFA, with no difference in specificity. There was no significant difference in sensitivity and specificity between LDBio-LFA and serum A. fumigatus-IgG ≥40 mgA/L. LDBio-LFA is a valuable test for diagnosing ABPA. However, a negative test should be confirmed using an enzyme immunoassay.

Quantification of Antiphospholipid Antibodies: The Importance of Using an Appropriate Methodology for Each Clinical Profile.

The presence of antiphospholipid antibodies (aPLs) is associated with antiphospholipid syndrome (APS), characterized by thrombosis and obstetric morbidity. aPLs included in APS classification criteria are lupus anticoagulant, anti-cardiolipin and anti-beta-2-glycoprotein-I of IgG or IgM isotypes. Enzyme-linked immunosorbent assay is the most used diagnostic technique to determine aPLs. Recently, new automated technologies mainly based in antigen-coated beads have been developed. The aim is to compare a fluorescence enzyme immunoassay (M1) and an antigen-coated bead assay (M2) in obstetric and thrombotic APS patients. All samples from the first 1020 patients received in the Immune Service Laboratory (Hospital 12 de Octubre) during the recruitment period, without exclusions, were analysed for aPLs. The weighted kappa for both methods in all the patients was 0.39 (0.30-0.47). Agreement increased to 0.56 (0.38-0.73) in patients with autoimmune disease. Sensitivity and specificity obtained for M1 were 17.1% and 89.3%, respectively, and 12.7% and 91.4% for M2. The sensibility and specificity of IgG isotypes were higher than the IgM ones. Regarding obstetric patients, M1 obtained significant diagnostic performance and had more sensitivity 23.75 (14.95-34.58) compared to M2 12.50 (6.16-21.79). In conclusion, clinical suspicion-based method selection for aPLs should be considered. To identify obstetric APS patients, solid phase methods remain more preferable.

Prevalence and sociodemographic correlates of antinuclear antibody testing by indirect immunofluorescence or solid-phase assays in a Spanish population: the Camargo Cohort

Autoantibodies are the hallmark of autoimmunity, and specifically, antinuclear antibodies (ANA) are one of the most relevant antibodies present in systemic autoimmune diseases (AID). In the present study, we evaluate the relationship between ANA and sociodemographic and biobehavioral factors in a population with a low pre-test probability for systemic AID. ANA were determined in serum samples at baseline visit from 2997 participants from the Camargo Cohort using indirect immunofluorescence assay, and two solid phase assays (SPA), addressable laser bead immunoassay, and fluorescence enzyme immunoassay. Sociodemographic and biobehavioral features of the subjects were obtained at baseline visit using a structured questionnaire. The prevalence of ANA positive results was significantly higher when indirect immunofluorescence assay was used as screening method in comparison with SPAs, being higher in females, older subjects, and those with higher C-reactive protein levels. Considering biobehavioral features, the prevalence was higher in those individuals with a sedentary lifestyle, and in ex- and non-alcohol users. Moreover, considering the relevance of the antibody load using ANA Screen, the prevalence of the antibody load also increased with age, especially in females. In conclusion, the prevalence of ANA varies depending on sociodemographic and biobehavioral features of the subjects, which could be relevant specifically in a population with a low pre-test probability for systemic AIDs.

Longitudinal study of patients with discrepant results in CLIFT and a solid-phase dsDNA antibody assay: does a gold standard dsDNA assay exist?

ObjectiveAntidouble-stranded DNA (dsDNA) antibodies are essential for diagnosis and follow-up of systemic lupus erythematous (SLE). To ensure the best diagnostic approach, most healthcare laboratories opt for a combination of highly...

B-055 Comparison of Sensitivity Between Tissue Anti-Transglutaminase and Anti-Endomysium IgA and IgG in Serological Screening for Celiac Disease in a Large Clinical Laboratory in Brazil

Abstract Background Celiac Disease (CD) is a genetic and autoimmune disorder characterized by permanent intolerance to gluten found in foods, resulting in small intestine injury in children and adults. Anti-tissue transglutaminase (anti-tTG) and anti-endomysium (anti-EM) antibodies are important serological markers of this disease. The aim was to analyze the analytical sensitivity of these markers in a large-scale Clinical Laboratory routine. Methods We employed a pure sample and a series of dilutions in the analysis of the functional sensitivity of each marker (anti-tTG IgA/IgG and anti-EM IgA/IgG) and a database survey of a large Clinical Laboratory in Sao Paulo/Brazil, from January to December 2021, analyzing 60 000 samples with concomitant results of anti-tTG IGA and anti-EM IgA and 7075 samples with results of anti-tTG IgG and anti-EM IgG, from patients with no defined clinic condition. The anti-tTG tests were performed by fluorescence enzyme immunoassay (FEIA) and the anti-EM tests by indirect immunofluorescence (IFI), with an initial dilution of 1:10, following the manufacturer's recommendations. Results The determination of functional sensitivity by checking the maximum dilution of antibody detection showed higher sensitivity of the anti-tTG IgA and IgG test (dilution factor 0.3 for both) compared to the anti-EM IgA and IgG (dilution factor 0.7 for both). Of the 60 000 samples analyzed for IgA markers, 59 083 (98.5%) were negative for both tests. Of the remaining 917 samples (1.5%), 609 (66.4%) were positive for both anti-tTG IgA and anti-EM IgA, 308 (33.6%) were positive only for anti-tTG IgA, and we did not observe any sample anti-tTG IgA negative with anti-EM IgA positive. Of the 7075 samples analyzed for IgG markers, 7032 (99.4%) were negative for both tests. Of the remaining 43 samples (0.6%), 18 (41.9%) were positive for anti-tTG IgG and anti-EM IgG, 14 (32.6%) were positive only for anti-tTG IgG, and 1 (2.3%) were positive only for anti-EM IgG. Conclusion The study showed greater sensitivity of anti-tTG compared to anti-EM for both IgA and IgG, in agreement with the literature data. The anti-tTG IgA test, in individuals without immunoglobulin A (IgA) deficiency, is recommended as the initial test in suspicion of celiac disease by current scientific guidelines on celiac disease. Based on our findings and literature data, in individuals with IgA deficiency, we suggest a screening using two automated IgG markers, tTG IgG and deamidated gliadin IgG (DG IgG). In addition to the lower sensitivity of anti-EM, indirect immunofluorescence (IFI), compared to the fully automated FEIA method, is a more subjective, observer-dependent, and labor-intensive methodology to be used as screening in large-scale Clinical Laboratory routines.

Reporting and Establishment of Reference Intervals for Antiphospholipid Antibody Immunoassays: A Survey of Participants in the College of American Pathologists Proficiency Testing Program.

Misdiagnosis of antiphospholipid syndrome can occur owing to the wide diversity of antiphospholipid (aPL) assays and a lack of international calibrators and harmonized reference intervals. To assess laboratory practices regarding reporting and establishing reference intervals for immunoglobulin (Ig) G/IgM anti-cardiolipin (aCL) and anti-beta-2 glycoprotein I (anti-β2GPI) assays. Supplemental questions related to reporting and establishing reference ranges for aPL assays were sent as part of the Antiphospholipid Antibody (ACL)-B 2019 College of American Pathologists (CAP) proficiency testing survey. The response rate and methods assessment details were determined, as well as qualitative and quantitative results for 3 test samples. The number of participants reporting results for IgG aCL (n = 489), IgM aCL (n = 476), IgG anti-β2GPI (n = 354), and IgM anti-β2GPI (n = 331) varied by antibody type. The enzyme-linked immunosorbent assay (ELISA) (up to 58.6%, 260 of 444) was the most used method; others included multiplex (from 18.9% to 23.9%), fluorescence enzyme immunoassay (14.4%-17.6%), and chemiluminescence immunoassay (6.5%-9.0%). More respondents reported quantitative than qualitative results, and manufacturer cutoff ranges were used by 92.9% and 94.2% of respondents for aCL and anti-β2GPI, respectively. Despite variation in the use of semiquantitative ranges, qualitative negative/positive reporting of the test samples achieved almost 100% consensus. Qualitative consensus was met in contrast to the wide range of quantitative results obtained for each analyte across different kits. ELISA remains the most used method for detecting aPL antibodies, with most laboratories reporting quantitative results based on manufacturers' suggested reference ranges. The categorization of quantitative results as equivocal, weak positive, or positive for responders using kits from the same manufacturer was variable.

P-133 A study of the relationship between in vitro embryo development and maternal hypothyroidism

Abstract Study question Are there an association between the maternal thyroid condition and in vitro fertilization (IVF) outcomes? Summary answer Maternal hypothyroidism was found to be related to oocyte maturation and blastocyst development. What is known already Previous studies have shown that thyroid-stimulating hormone (TSH) levels affect ovarian stimulation in assisted reproductive technology (ART). In addition, changes in serum TSH levels have been linked to a sub-optimal environment for the implantation and development of the embryo. Thyroid-related receptors are present in granulosa cells, ovaries, and the endometrium. In addition, those receptors play a role in ovulation and folliculogenesis. This study aims to determine the association between maternal hypothyroidism and adverse implications for IVF outcomes in IVF/ICSI treated women. Study design, size, duration This retrospective study performed 333 cycles of 208 patients with TSH levels ranging from 0.043-16.627 µIU/mL, treated at the RMC National IVF Center between 2019 and 2022. The study population consisted of 1171 oocytes, which were divided into two groups based on their maternal basal TSH levels: normal TSH ≤ 2.5 µIU/mL (control group, n = 865, euthyroid) and higher TSH > 2.5 µIU/mL (case group, n = 306, hypothyroid). Participants/materials, setting, methods Maternal basal serum TSH levels were measured using Fluorescence Enzyme Immunoassay on the third day of the menstrual cycle. IVF outcome expressed oocyte maturation, fertilization, cleavage, and blastocyst formation rate. Oocytes were divided into mature (metaphase II) and immature (metaphase I and prophase I) groups. Blastocysts are graded by the Kato Ladies Clinic classification system and divided into high potential (A and B grades) and low potential (C, D, and E grades) groups. Main results and the role of chance In our study, there was no statistically significant difference in age and partner’s semen criteria between the two groups (p > 0.05). As regards oocyte maturation, MII oocytes retrieved significantly higher in the control group (82.2% vs. 75.8%). Logistic regression analysis found that TSH levels > 2.5 µIU/mL were related to statistically significantly increased risks of oocyte maturation stages like MI or GV (Age affect adjusted model: B = 0.456, p = 0.005; OR = 1.578, 95% CI = 1.146-2.175). The fertilization, cleavage, and blast formation rates have no differences between the groups. However, 91% (n = 324/356) of higher potential blastocysts in the control group were significantly higher than the case group (79.7%, n = 106/133). While, by the logistic regression analysis found that higher TSH levels ( > 2.5 µIU/mL) were related to statistically significantly increased risks of lower potential for implantation grades (B = 0.947, p = 0.001; OR = 2.579, 95%CI = 1.477-4.502). Limitations, reasons for caution The study relied on TSH as the primary indicator of thyroid function and excluded participants with abnormal prolactin levels. Other markers, such as FT4 and TSHR, should also be considered for a thorough evaluation of thyroid function. Wider implications of the findings Thyroid dysfunction can negatively impact IVF success, and screening and managing thyroid function in women undergoing IVF are essential. More research is needed to understand the mechanisms and develop strategies to improve oocyte and blastocyst quality. Trial registration number not applicable

Sympathetic innervation and endogenous catecholamines in neuromuscular preparations of muscles of different functional profiles

Influence of the sympathetic nervous system on the work of skeletal muscles contractile apparatus is now beyond doubt. However, until recently there was no evidence that the endings of sympathetic nerves can be located in close proximity to the neuromuscular synapses, and there is also no reliable data on how much endogenous adrenaline and noradrenaline can be contained near the synaptic contact in skeletal muscles. In this research, using fluorescent analysis, immunohistochemical and enzyme immunoassays the isolated neuromuscular preparations of three skeletal muscles of different functional profiles and containing different types of muscle fibers were examined. Close contact between the sympathetic and motor cholinergic nerve endings and the presence of tyrosine hydroxylase in this area were demonstrated. Concentrations of endogenous adrenaline and noradrenaline in the solution perfusing the neuromuscular preparation were determined under different modes of its functioning. The effects of α and β adrenoreceptor blockers on the processes of acetylcholine quantal secretion from the motor nerve endings were compared. The data obtained provide evidence for the presence of endogenous catecholamines in the neuromuscular junction region and their role in the modulation of the synaptic function.

Sympathetic Innervation and Endogenous Catecholamines in Neuromuscular Preparations of Muscles with Different Functional Profiles

Influence of the sympathetic nervous system on the work of skeletal muscles contractile apparatus is now beyond doubt. However, until recently there was no evidence that the endings of sympathetic nerves can be located in close proximity to the neuromuscular synapses, and there is also no reliable data on how much endogenous adrenaline and noradrenaline can be contained near the synaptic contact in skeletal muscles. In this research, using fluorescent analysis, immunohistochemical and enzyme immunoassays the isolated neuromuscular preparations of three skeletal muscles of different functional profiles and containing different types of muscle fibers were examined. Close contact between the sympathetic and motor cholinergic nerve endings and the presence of tyrosine hydroxylase in this area were demonstrated. Concentrations of endogenous adrenaline and noradrenaline in the solution perfusing the neuromuscular preparation were determined under different modes of its functioning. The effects of α and β adrenoreceptor blockers on the processes of acetylcholine quantal secretion from the motor nerve endings were compared. The data obtained provide evidence for the presence of endogenous catecholamines in the neuromuscular junction region and their role in modulation of the synaptic function.

Serum Immunoglobulin G Testing against Pigeon Egg in Stable Fibrotic Hypersensitivity Pneumonitis

Introduction: The accuracy of serum immunoglobulin (Ig) G testing for diagnosis of stable bird-related fibrotic hypersensitivity pneumonitis (HP) is controversial. Furthermore, avian serum, extracts, or feathers were employed as antigens in bird-related HP; however, the usage of egg whites has not been reported. We investigated the utility of IgG testing against pigeon egg whites in patients with stable bird-related fibrotic HP. Methods: Patients having a positive inhalation test for pigeon antigen and a histological investigation with diagnostic confidence of fibrotic HP greater than moderate confidence were included. The control group consisted of patients with interstitial lung diseases (ILDs) other than HP. To select patients in the stable phase, patients with fibrotic HP were excluded if they were clinically considered to be in the acute exacerbation or acute phase. The IgG testing against pigeon egg whites by enzyme-linked immunosorbent assay and the commercialized anti-pigeon IgG testing by fluorescence enzyme immunoassay were investigated. Results: In this study, 37 patients with stable bird-related fibrotic HP and 32 patients with ILDs other than HP participated. Serum IgG testing for pigeon egg whites revealed that the control group’s optical density was 0.147 and the group with bird-related fibrotic HP had a mean value of 0.207 (p = 0.011). IgG testing in bronchial alveolar lavage fluid was not significantly higher in the bird-related fibrotic HP group than in controls (p = 0.42). No significant difference in area under the curve between an IgG testing against pigeon egg whites and a commercialized anti-pigeon IgG testing was observed (p = 0.24). Test accuracy for stable bird-related fibrotic HP ranged from 62% to 76% sensitivity and 59–66% specificity. Conclusion: IgG testing to identify the inciting antigen in patients with stable bird-related fibrotic HP had relatively low accuracy.

Successful use of a benchtop fluorescent enzyme immunoassay analyzer to measure serum cortisol concentration as a screening test for hypoadrenocorticism in dogs.

To assess the diagnostic performance of a benchtop fluorescent enzyme immunoassay analyzer (AIA-360; Tosoh Bioscience Inc) for the measurement of serum cortisol concentration as a screening test for hypoadrenocorticism in dogs. 173 client-owned dogs (20 with hypoadrenocorticism and 153 with nonadrenal illness). Medical records of all dogs that underwent an ACTH stimulation test between June 2015 and October 2019 were reviewed retrospectively. Dogs were excluded if the ACTH stimulation test was performed on the basis of a suspicion of hypercortisolism, serum cortisol concentrations were measured using an analyzer other than the one assessed in the present study, or dogs had received medication known to affect the pituitary-adrenal axis in the 4 weeks1,2 preceding ACTH stimulation testing. The diagnostic performance of the benchtop analyzer was evaluated by calculating sensitivity, specificity, and likelihood ratios at various cutoff points. Serum resting cortisol cutoff point concentrations of 0.8 μg/dL (22 nmol/L), 1 μg/dL (28 nmol/L), and 2 μg/dL (55 nmol/L) had a sensitivity of 100%. An optimal serum resting cortisol cutoff point of 0.58 μg/dL (16 nmol/L) had a sensitivity, specificity, and positive and negative likelihood ratios of 100%, 97%, and 30.6 and 0.0, respectively. Findings indicated that previously derived cutoff points could be used with excellent sensitivity to exclude hypoadrenocorticism in this population of dogs when serum cortisol concentration was measured with the evaluated benchtop analyzer. An ACTH stimulation test may need to only be performed to diagnose hypoadrenocorticism if resting serum cortisol concentration is ≤ 0.58 μg/dL when measured with the evaluated benchtop analyzer.

Comparison of current methods for anti-dsDNA antibody detection and reshaping diagnostic strategies.

Anti-double-stranded DNA antibodies (anti-dsDNA) are considered a specific marker for systemic lupus erythematosus (SLE). Though the Farr technique was once the reference method for their detection, it has been almost entirely replaced by more recently developed assays. However, there is still no solid evidence of the commutability of these methods in terms of diagnostic accuracy and their correlation with the Crithidia luciliae immunofluorescence test (CLIFT). Anti-dsDNA antibody levels were measured in 80 subjects: 24 patients with SLE, 36 disease controls drawn from different autoimmune rheumatic diseases (14 systemic sclerosis, 10 Sjögren's syndrome, nine autoimmune myositis, three mixed connective tissue disease), 10 inflammatory arthritis and 10 apparently healthy blood donors by eight different methods: fluorescence enzyme immunoassay, microdot array, chemiluminescent immunoassay (two assays), multiplex flow immunoassay, particle multi-analyte technology immunoassay and two CLIFT. At the recommended manufacturer cut-off, the sensitivity varied from 67% to 92%, while the specificity ranged from 84% to 98%. Positive agreement among CLIFT and the other assays was higher than negative agreement. Mean agreement among methods assessed by the Cohen's kappa was 0.715, ranging from moderate (0.588) to almost perfect (0.888). Evaluation of the concordance among quantitative values by regression analysis showed a poor correlation index (mean r2, 0.66). The present study shows that current technologies for anti-dsDNA antibody detection are not fully comparable. In particular, their different correlation with CLIFT influences their positioning in the diagnostic algorithm for SLE (either in association or sequentially). Considering the high intermethod variability, harmonization and commutability of anti-dsDNA antibody testing remains an unachieved goal.

Analytical and clinical performance of a fluorescence enzyme immunoassay for progesterone and determination of ovulation day in bitches.

We evaluated the performance of a third-generation fluorescence enzyme immunoassay kit (FEIA; Tosoh Bioscience) for progesterone (P4) measurement in canine serum to identify the day of luteinizing hormone (LH) peak and ovulation in bitches. We conducted P4 assays on 54 serum samples using a FEIA and a chemiluminescence immunoassay (CLIA; Siemens). For the FEIA kit, the linearity test, recovery test, inter- and intra-assay CVs, and total error observed (TEo) were calculated. Serum samples from 28 bitches were used to evaluate the association of P4 concentration with the day of LH peak and the day of ovulation based on P4 thresholds (P4 ≥ 2 ng/mL, and doubling at 4-8 ng/mL in the following 48 h), and with pregnancy length. Linearity was 75-97% and 86-94% for high (37.0 ng/mL) and medium (3.8 ng/mL) pool serum samples, respectively. Recovery was 86.4-119%. Intra-assay CVs were 2.6%, 3.3%, and 5.2% for low (0.23 ng/mL), medium (6.24 ng/mL), and high (38.3 ng/mL) pool concentrations. Inter-assay CVs were 2.22% and 2.53% for P4 concentrations of 2.70 ng/mL and 8.2 ng/mL, respectively. TEo was 8.72% and 22.7% for P4 concentrations of 2 and 8 ng/mL, respectively. The mean pregnancy length from the day of LH peak and ovulation were 64.6 ± 1.7 and 63.3 ± 1.1 d, respectively. The third-generation FEIA kit that we tested was highly reliable.

Antiphospholipid antibodies are persistently positive at high titers. Additive value of platelet-bound C4d.

BackgroundClassification criteria for antiphospholipid syndrome (APS) require that antiphospholipid antibody (aPL) positivity is confirmed after at least 12 weeks. We tested the hypothesis that aPL at high titers remain positive while low titers fluctuate over time. As both platelet-bound C4d (PC4d) and aPL are associated with thrombosis in systemic lupus erythematosus (SLE), we also evaluated whether PC4d can aid in APS diagnosis.MethodsData from serum or plasma sent to Exagen’s laboratory for routine aPL testing were analyzed. Anti-cardiolipin (aCL) and anti-beta2 glycoprotein-1 antibodies (aB2GP1) were measured by chemiluminescence or ELiA fluorescence enzyme immunoassay; anti-phosphatidylserine/prothrombin complex antibodies (aPS/PT) by ELISA; PC4d by flow cytometry. Statistical analysis included descriptive statistics, logistic regression, and Pearson correlation.ResultsMore than 80% of positive samples with aCL and aB2GP1 at high titers - but not low titers - were positive at a retest. Non-criteria aPL (aPS/PT) followed a similar trend. aCL and aB2GP1 measured with two different technologies were highly correlated. PC4d and IgG of the three aPL were at best moderately correlated even when only positive aPL samples were analyzed (coefficient: 0.1917 to 0.2649).ConclusionsHigh titers aPL are often persistently positive, allowing an earlier diagnosis and risk assessment at the time of the initial screening. Conversely, a retest may be necessary for low titers. The high correlation between two methodologies suggests that these findings are independent of assay platform. The low to moderate correlation between PC4d and aPL might suggest a possible additive value to evaluate association with thrombosis in autoimmune diseases.

Differences in Linear Epitopes of Ara h 9 Recognition in Peanut Allergic and Tolerant, Peach Allergic Patients.

BackgroundPeanut-allergic patients from the Mediterranean region are predominantly sensitized to the lipid transfer protein (LTP) Ara h 9, and the peach LTP Pru p 3 seems to be the primary sensitizer. However, LTP sensitization in peanut allergy is not a predictive marker for clinically relevant symptoms.ObjectiveWe aimed to identify sequential epitopes of IgE and IgG4 from Pru p 3 and Ara h 9 in peach-allergic patients sensitized to peanuts. We also sought to determine the differences in IgE and IgG4 binding between patients who had developed peanut allergy and those tolerating peanuts.MethodsA total of 46 peach-allergic patients sensitized to peanuts were selected. A total of 35 patients were allergic to peanuts (peanut-allergic group) and 11 were tolerant to peanuts (peanut-tolerant group). We measured sIgE and sIgG4 in peanut, peach, and their recombinant allergen (Ara h 1, Ara h 2, Ara h 3, Ara h 8, and Ara h 9) with fluorescence enzyme immunoassay. We examined the IgE and IgG4 binding to sequential epitopes using a peptide microarray corresponding to linear sequences of the LTPs Ara h 9 and Pru p 3 with a library of overlapping peptides with a length of 20 amino acids (aa) and an offset of 3 aa.ResultsThe frequency and the intensity of IgE recognition of Ara h 9 and Pru p 3 peptides were higher in the peanut-tolerant group than in the peanut-allergic group. We found four Ara h 9 peptides (p4, p14, p21, and p25) and four Pru p 3 peptides (p1, p3, p21, and p24) with a significantly elevated IgE recognition in peanut-tolerant patients. Only one peptide of Ara h 9 (p4) recognized by IgG4 was significantly elevated in the peanut-tolerant group. The IgG4/IgE ratio of Ara h 9 peptide 4 was significantly higher in peanut-tolerant patients than in peanut-allergic patients, while no significant differences were observed in the IgG4/IgE ratio of this peptide in Pru p 3.ConclusionAlthough we found significant differences in IgE and IgG4 recognition of Ara h 9 and Pru p 3 between peanut-tolerant and peanut-allergic patients (all of whom were allergic to peach), polyclonal IgE peptide recognition of both LTPs was observed in peach-allergic patients tolerating peanuts. However, the IgG4 blocking antibodies against Ara h 9 peptide 4 could provide an explanation for the absence of clinical reactivity in peanut-tolerant peach-allergic patients. Further studies are needed to validate the usefulness of IgG4 antibodies against Ara h 9 peptide 4 for peanut allergy diagnosis.

Improved performance of confirmatory assays for detecting dense fine speckled (DFS) 70 antibodies

The presence of monospecific dense fine speckled 70 (DFS70) pattern in indirect immunofluorescence assay (IFA) without concomitant systemic autoimmune related diseases (SARD)-associated antibodies could be an exclusion biomarker for SARD. Since interpretation of DFS pattern on IFA is subjective, an assay for confirming the presence of anti-DFS70 is required. We evaluated the performance of two commercial assays [fluorescence enzyme immunoassay (FEIA) and line immunoassay (LIA)] for detecting anti-DFS70. Sera with monospecific DFS (n=176) and without DFS (n=179) pattern from referred patients for ANA testing, in two independent laboratories and healthy donors, were investigated for anti-DFS70 by FEIA (Phadia EliA) and LIA (Euroimmun). The assay performance including sensitivity and specificity at different cut-offs was evaluated, and optimal cut-offs were determined. The newly established optimal cut-offs (2.7 U/mL on EliA, band intensity of 28 on LIA) showed significantly better assay performance in detecting anti-DFS70 and confirming monospecific DFS pattern. A relative sensitivity of 93.7% with relative specificity of 100% on EliA and a relative sensitivity of 96.6% with relative specificity of 95.3% on LIA were achieved. Superior Cohen's Kappa agreements with IFA were also achieved for both assays (0.936 for EliA and 0.906 for LIA). Application of an optimal cut-off can significantly increase the assay performance for anti-DFS70 and enhance the accuracy in reporting DFS pattern by IFA.

Liraglutide increases islet Ca2+ oscillation frequency and insulin secretion by activating hyperpolarization-activated cyclic nucleotide-gated channels.

To determine whether hyperpolarization-activated cyclic nucleotide-gated (HCN) channels impact glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) modulation of islet Ca2+ handling and insulin secretion. The impact of liraglutide (GLP-1 analogue) on islet Ca2+ handling, HCN currents and insulin secretion was monitored with fluorescence microscopy, electrophysiology and enzyme immunoassays, respectively. Furthermore, liraglutide-mediated β-to-δ-cell cross-communication was assessed following selective ablation of either mouse islet δ or β cells. Liraglutide increased β-cell Ca2+ oscillation frequency in mouse and human islets under stimulatory glucose conditions. This was dependent in part on liraglutide activation of HCN channels, which also enhanced insulin secretion. Similarly, liraglutide activation of HCN channels also increased β-cell Ca2+ oscillation frequency in islets from rodents exposed to a diabetogenic diet. Interestingly, liraglutide accelerated Ca2+ oscillations in a majority of islet δ cells, which showed synchronized Ca2+ oscillations equivalent to β cells; therefore, we assessed if either cell type was driving this liraglutide-mediated islet Ca2+ response. Although δ-cell loss did not impact liraglutide-mediated increase in β-cell Ca2+ oscillation frequency, β-cell ablation attenuated liraglutide-facilitated acceleration of δ-cell Ca2+ oscillations. The data presented here show that liraglutide-induced stimulation of islet HCN channels augments Ca2+ oscillation frequency. As insulin secretion oscillates with β-cell Ca2+ , these findings have important implications for pulsatile insulin secretion that is probably enhanced by liraglutide activation of HCN channels and therapeutics that target GLP-1Rs for treating diabetes. Furthermore, these studies suggest that liraglutide as well as GLP-1-based therapies enhance δ-cell Ca2+ oscillation frequency and somatostatin secretion kinetics in a β-cell-dependent manner.

Microarray Analysis Confirms ImmunoCAP-Fluorescence Enzyme Immunoassay Results on Specific IgE in Patients with Atopic Dermatitis and Suspected Birch Pollen-Related Food Allergy

Background: Previous studies demonstrated that birch pollen-related foods can cause late eczematous responses in birch pollen-sensitized patients with atopic dermatitis (AD). However, suitable markers to predict birch pollen-related food allergy in patients with AD are still lacking. Objective: We evaluated the correlation of the results from ImmunoCAP® fluorescence enzyme immunoassay (FEIA) singleplex and ImmunoCAP® immuno solid-phase allergen chip (ISAC) multiplex system in AD patients and investigated the diagnostic validity of allergen microarray analysis, measuring specific IgE (sIgE) with ImmunoCAP® ISAC to predict birch pollen-related food allergy in patients with AD. Methods: A total of 19 children and adults with AD, existing IgE-mediated birch pollen sensitization, and suspected birch pollen-related food allergy underwent a double-blind placebo-controlled food challenge (DBPCFC) in the clinical routine. Total and sIgE levels to birch pollen, Bet v 1, Bet v 2, and birch pollen-related foods (apple, carrot, celery, and hazelnut) were determined prior to the DBPCFC by ImmunoCAP®-FEIA. Additionally, allergen microarray ImmunoCAP® ISAC analysis was performed. Data were analyzed retrospectively. Results: Twelve out of 19 patients (63% responders) experienced an allergic reaction upon DBPCFC. Overall, 7 patients (37%) developed a significant deterioration of AD with a median increase of 12.4 points in the scoring of atopic dermatitis (SCORAD) index (range 10.0–15.7). Oral allergy syndrome was the predominant immediate-type symptom (n = 11/12 responders). There were no differences in sensitization frequencies regarding allergens of the pathogenesis-related protein family 10 between responders and non-responders. In all patients, correlation of IgE levels determined with ImmunoCAP® ISAC and ImmunoCAP®-FEIA, respectively, was significant with high correlation coefficients regarding birch pollen allergen extract, rBet v 1, and rBet v 2 (r_s > 0.8, p < 0.001) and lower but also significant correlation coefficients regarding food allergens (r_s < 0.8, p < 0.05–<0.001). Conclusion: ImmunoCAP® ISAC microarray allows displaying a differentiated sensitization profile in birch pollen-sensitized patients with AD. However, IgE-mediated sensitization against birch pollen-related allergens revealed by the allergen multiplex system does not predict late eczematous reactions upon DBPCFC with birch pollen-related foods.

LDBio Aspergillus immunochromatographic test lateral flow assay for IgG/IgM antibody detection in chronic pulmonary aspergillosis: Single-centre evaluation and meta-analysis

PurposeChronic pulmonary aspergillosis (CPA) is an infection of the lung usually caused by Aspergillus fumigatus in patients with pre-existing pulmonary diseases. Its diagnosis hinges on demonstrating IgG antibodies against A. fumigatus. Herein, we evaluated the performance of a newly introduced point of care test (POCT) kit, the LDBio Aspergillus IgG/IgM lateral flow assay (LFA) in India with the standard ImmunoCAP kit for diagnosing CPA. MethodsA total of 60 serum samples (30 CPA cases and 30 controls) were evaluated by the Aspergillus immunochromatographic test (ICT) IgG/IgM LFA. Fluorescent-enzyme immunoassay was used to determine specific A. fumigatus-IgG concentrations (positive >27 mgA/L). Further, a systematic review and meta-analysis of studies (up to August 26, 2021) reporting the performance of LDBio ICT for the diagnosis of CPA was performed. ResultA sensitivity of 86.7%, specificity of 90%, negative predictive value of 87.1%, positive predictive value of 89.7%, negative likelihood ratio of 0.15, positive likelihood ratio of 8.67, and was observed for the LDBio IC. There was good agreement between LDBio ICT and ImmunoCAP (88.3%) with a Cohen's Kappa score of 0.77. Our systematic review identified four studies and the pooled sensitivity of 90%, specificity of 91%, area under the curve of 0.94 and diagnostic odds ratio of 57.2, for CPA diagnosis by LDBio ICT. ConclusionAspergillus LDBio ICT assay exhibits good sensitivity and can be used to screen CPA cases.

Antinuclear antibody pattern and autoantibody profiling of systemic sclerosis patients in a tertiary referral center in North India.

Antinuclear antibody (ANA) pattern and autoantibody (autoAb) profiling of 150 adult systemic sclerosis (SSc) patients concerning their clinical association and diagnostic significance were analyzed by indirect immunofluorescence (IIF), immunoblot, and fluorescence enzyme immunoassay. One hundred and forty-three (95.3%) patients had positive ANA: DNA topoisomerase I (topo I)-like pattern-84(56%); speckled pattern-44(29.3%);centromere pattern-7(4.6%); and nucleolar pattern-4(2.6%). Three distinct topo I-like immunofluorescence patterns were detected at 1:40 dilution. Topo I-like pattern (32/75-limited cutaneous systemic sclerosis (lcSSc) vs. 52/75-diffuse cutaneous systemic sclerosis (dcSSc); p < 0.001) was found to be associated with dcSSc subset and speckled pattern (lcSSc 28/75 vs. dcSSc 16/75; p < 0.03) with lcSSc subset. One hundred and thirty-eight(92%) patients were positive for SSc-associated autoAbs. The frequency distribution of autoAbs to topo I, centromere A (CENP A) and centromereB (CENP B), RNA polymerase III (RP11, RP155), fibrillarin (U3RNP), nucleolus organizer region (NOR)-90, Th/To, PM-Scl75, PM-Scl100, Ku, platelet-derived growth factor receptor (PDGFR) and Ro-52, were 87(58%), 9(6%), 8(5.3%), 6(4%), 9(6%), 0, 6(4%), 6(4%), 8(5.3%), 5(3.3%), 11(7.3%),0 and 46(30.6%), respectively. Topo I autoAb was strongly associated with dcSSc (35/75 lcSSc vs. 52/75 dcSSc; p < 0.004), Raynaud's (p < 0.003), interstitial lung disease (ILD) (p < 0.001) and pulmonary arterial hypertension (PAH) (p < 0.04). This study helps in defining SSc clinical subset, prognostic markers of disease severity, characterization of the topo I-like ANA pattern, and provides a definite association between the ANA patterns and corresponding autoAb.