Immunoassay Results Research Articles

Concordance of Three Automated Procalcitonin Immunoassays at Medical Decision Points.

Procalcitonin (PCT) is a useful bacterial infection biomarker with the potential for guiding antibiotic therapy. We evaluated the concordance of three automated PCT immunoassays: Kryptor (BRAHMS GmbH, Hennigsdorf, Germany), Atellica IM 1600 (Siemens Healthcare Diagnostics, Munich, Germany), and Cobas e801 (Roche Diagnostics, Mannheim, Germany). In 119 serum samples with a PCT concentration <5.00 μg/L, Kryptor (reference assay) was compared with the other two immunoassays by Spearman’s rank correlation, regression analysis, and concordance at two antibiotic stewardship medical decision points: 0.25 and 0.50 μg/L. The Atellica IM 1600 and Cobas e801 results showed high correlations with those of Kryptor, with correlation coefficient (ρ) values of 0.97 and 0.99, respectively. However, negative biases were observed in both immunoassays (slope/y-intercept: 0.75/–0.00 for Atellica IM 1600; 0.88/–0.01 for Cobas e801). Atellica IM 1600 and Cobas e801 demonstrated excellent concordance with Kryptor at both medical decision points, with linearly weighted κ values of 0.90 and 0.92, respectively, despite discrepancies, which were more prominent at the 0.25 μg/L medical decision point. Based on these biases and discrepancies, the alternate use of different PCT immunoassays in repeat examinations is inadvisable. Standardization is required before comparing the results of different PCT immunoassays.

Dietary supplementation of Bacillus velezensis B8 enhances immune response and resistance against Aeromonas veronii in grass carp

The heavy use of prophylactic antibiotics in aquaculture leads to elevated antibiotic residues, posing a huge hidden danger in aquaculture products and other natural aquatic environments. Therefore, this study aims to isolate probiotics that can replace antibiotics from the gut of grass carp for disease control. Bacillus velezensis B8 was isolated from the gut of grass carp and showed broad-spectrum antimicrobial activity against several fish pathogenic bacteria, including Aeromonas hydrophilis, Aeromonas veronii, Vibrio parahaemolyticus, Escherichia coli, Edwardsiella tarda and Vibrio mimicus. The safety evaluation showed that the strain B8 was non-toxic to grass carp, had no hemolytic activity, and was sensitive to most antibiotics. In vitro study indicated that strain B8 was viable at pH 2–7, had weak tolerance to 0.1% (w/v) bile salt, and could grow at 10°C–40 °C. The grass carps were fed with diets containing 0 (control), 107, and 109 cfu/g of strain B8 for 4 weeks. Various immune parameters were measured at 1, 2, 3, and 4 weeks of post-feeding. The results of non-specific immunoassay showed that diets supplemented with B8 significantly increased alkaline phosphatase (AKP) and superoxide dismutase (SOD) activity in serum samples (p < 0.05). The expression levels of immune-related genes in the kidney and spleen of grass carp were measured. Among them, the expression levels of IgM and TNF-α both in spleen and kidney were significantly increased after 3 and 4 weeks of post-feeding (p < 0.05). The expression of IgD and MHCI in kidney was significantly upregulated in high-dose groups after 2 and 3 weeks of feeding, respectively (p < 0.05). In addition, after 7 days of challenging with A. veronii, the high-dose group and low-dose group had 48% and 53% survival compared to 25% survival for the control group. These results suggest that B. velezensis B8 has the potential to be developed into a microecological preparation for the alternatives of antibiotics in aquaculture.

Habitual khat chewing alters urinary inorganic profile in adult healthy males.

Khat (Catha edulis) is a stimulant plant, and it is abusive to induce euphoria, alertness and activity. Concomitant use of medications and khat chewing predisposes to the appearance of drug interactions result in treatment failure or toxicity. This study determined the changes in the urinary inorganic profile in adult healthy males who are chewing khat compared with non-khat chewer males. A total of 40 adult non-smoker healthy males (20 khat chewer and 20 non-khat chewer) aged 24-30 years were selected. Khat chewer samples were positive for cathinone and cathine and negative for other drug of abuse, while non-khat chewer samples were negative for drug of abuse include cathinone and cathine. Samples were selected according to their results in immunoassay and gas chromatography mass spectrometry (GCMS) analysis. Cathine and cathinone were confirmed using liquid chromatography mass spectrometry (LC-MSMS) analysis. Inorganic profile includes titanium (Ti), cobalt (Co), copper (Cu), zinc (Zn), cadmium (Cd), and lead (Pb) were determined by using inductively coupled plasma-mass spectrometry (ICP-MS). The levels of Ti, Co, Zn, Cd, and Pb in urine were significantly higher among the khat chewer group compared with non-khat chewer. Ti, Cd, Co, Pb and Zn urine levels were 0.5-, 1.5-, 1.15-, 5-, and 8.2-fold higher in the khat chewer group compared to non-khat chewer, respectively. We suggested that continuous khat chewing has a long term effect on metabolic pathway of therapeutic drugs that result in toxicity or failure of therapy.

Habitual khat chewing alters urinary inorganic profile in adult healthy males.

Khat (Catha edulis) is a stimulant plant, and it is abusive to induce euphoria, alertness and activity. Concomitant use of medications and khat chewing predisposes to the appearance of drug interactions result in treatment failure or toxicity. This study determined the changes in the urinary inorganic profile in adult healthy males who are chewing khat compared with non-khat chewer males. A total of 40 adult non-smoker healthy males (20 khat chewer and 20 non-khat chewer) aged 24-30 years were selected. Khat chewer samples were positive for cathinone and cathine and negative for other drug of abuse, while non-khat chewer samples were negative for drug of abuse include cathinone and cathine. Samples were selected according to their results in immunoassay and gas chromatography mass spectrometry (GCMS) analysis. Cathine and cathinone were confirmed using liquid chromatography mass spectrometry (LC-MSMS) analysis. Inorganic profile includes titanium (Ti), cobalt (Co), copper (Cu), zinc (Zn), cadmium (Cd), and lead (Pb) were determined by using inductively coupled plasma-mass spectrometry (ICP-MS). The levels of Ti, Co, Zn, Cd, and Pb in urine were significantly higher among the khat chewer group compared with non-khat chewer. Ti, Cd, Co, Pb and Zn urine levels were 0.5-, 1.5-, 1.15-, 5-, and 8.2-fold higher in the khat chewer group compared to non-khat chewer, respectively. We suggested that continuous khat chewing has a long term effect on metabolic pathway of therapeutic drugs that result in toxicity or failure of therapy.

Rheumatoid factor and falsely elevated results in commercial immunoassays: data from an early arthritis cohort

The aim of the study was to assess RF cross-reactivity to animal antibodies used in immunoassays, and to test if selected commercial immunoassays are vulnerable to interference from RF, causing false test results. Our study included samples from patients with RF-positive rheumatoid arthritis (RA) and controls (patients with RF-negative RA and psoriatic arthritis), included in an early arthritis-cohort. Reactivity to mouse IgG1, mouse IgG2a, rabbit IgG, bovine IgG, sheep/goat IgG and human IgG was analysed using in-house interference assays. RF-positive sera with strong reactivity to mouse IgG1 were analysed in three commercial immunoassays. To reveal interference, results before and after addition of blocking aggregated murine IgG1 were compared. Samples from 124 RF-positive RA patients and 66 controls were tested. We found considerably stronger reactivity toward animal antibodies, particularly mouse IgG1 (73% vs. 12%) and rabbit IgG (81% vs. 6%), in sera from RF-positive RA-patients compared to controls (p < 0.001). After selecting samples for testing in commercial assays, interference was revealed in 6/30 sera in the Architect β-hCG assay, 7/10 sera in the 27-plex cytokine assays, and in 2/33 samples in the Elecsys Soluble Transferrin Receptor assay. Our study revealed considerable RF reactivity to animal antibodies used in immunoassays and RF was associated with falsely elevated results in immunoassays used in clinical care and research. Clinicians, laboratorians, researchers and assay manufacturers must be alert to the risk of falsely elevated test results in RF-positive RA patients.

Optimizing immunoassay-based selection of donors for COVID-19 convalescent plasma

Abstract COVID-19 convalescent plasma (CCP) is currently administered to hospitalized COVID-19 patients under an EAU by the FDA. CCP units are selected by commercially available ELISA quantification of anti-SARS-CoV-2 (SARS2) antibodies under the rationale that higher levels of anti-SARS2 antibodies correlate with higher neutralizing capabilities and thus greater efficacy. The validity of this selection process has yet to be investigated. We analyzed serum samples from 60 convalesced donors with PCR-confirmed COVID-19. Samples were analyzed using commercial SARS2 ELISAs (DiaSorin, Euroimmun, Ortho, and Roche) and a cell-based enzyme-linked immunosorbent assay (cbELISA) that detects antibodies against the native conformation of the SARS2 spike glycoprotein (GP). Sera were also tested for neutralizing activity against wild-type SARS2 and a pseudovirus expressing the SARS2 GP. Immunoassay and neutralization values were compared to determine the precision of each immunoassay to predict differing levels of neutralizing activity, and ability of each immunoassay to identify high neutralizing activity. Results of immunoassays that apply SARS2 GP as the antigen (DiaSorin, Euroimmun, Ortho, and cbELISA) correlated well with neutralization titers measured by wild-type SARS2 or SARS2 GP-pseudotyped virus as compared with assays using other SARS2 antigens (Roche; N protein). Differences in precision of these assays were evident when samples within the top quintile or decile of immunoassay values were analyzed, with DiaSorin and Ortho assays showing superior performance in predicting neutralizing capability. We hope this approach will assist in the selection of highly neutralizing CCP units that confer greater clinical efficacy.

Seroprevalence and correlates of SARS-CoV-2 neutralizing antibodies from a population-based study in Bonn, Germany

To estimate the seroprevalence and temporal course of SARS-CoV-2 neutralizing antibodies, we embedded a multi-tiered seroprevalence survey within an ongoing community-based cohort study in Bonn, Germany. We first assessed anti-SARS-CoV-2 immunoglobulin G levels with an immunoassay, followed by confirmatory testing of borderline and positive test results with a recombinant spike-based immunofluorescence assay and a plaque reduction neutralization test (PRNT). Those with a borderline or positive immunoassay result were retested after 4 to 5 months. At baseline, 4771 persons participated (88% response rate). Between April 24th and June 30th, 2020, seroprevalence was 0.97% (95% CI: 0.72−1.30) by immunoassay and 0.36% (95% CI: 0.21−0.61) when considering only those with two additional positive confirmatory tests. Importantly, about 20% of PRNT+ individuals lost their neutralizing antibodies within five months. Here, we show that neutralizing antibodies are detectable in only one third of those with a positive immunoassay result, and wane relatively quickly.

Seasonality in testing and positive respiratory bacterial infections in the Australian Capital Territory, 1997-2007.

Chlamydia pneumoniae (Cp) and Mycoplasma pneumoniae (Myco) bacteria are atypical pathogens that can cause pneumonia and exacerbate underlying conditions such as asthma and chronic obstructive pulmonary disease. In the Australian Capital Territory, there is limited information on how seasonal patterns for positive infections and testing may vary, a gap that has implications for control strategies. We examined seasonal patterns of immunoassay results of patients from Canberra Hospital, Australia, who were tested for Cp and/or Myco. Pathology data, collected from August 1997 to March 2007 from 7,275 patients, were analysed with time series additive decomposition and time series regression. The proportion of positive Cp infections was highest in March and April (autumn) and lowest in June and August (winter). The proportion of positive Myco infections was highest in December and January (summer) and lowest in August (winter), even though testing for the pathogen peaked in winter with a low in summer. Models with a long-term trend and a variable for month were a better fit for the data than the null models for both infections. We found differences in seasonal patterns of testing and in the proportion of positive infections. These findings suggest that preventative measures for common infections need to account for seasonal testing practices so as to build an accurate picture of temporal changes in these infections.

Affordable mobile microfluidic diagnostics: minimum requirements for smartphones and digital imaging for colorimetric and fluorometric anti-dengue and anti-SARS-CoV-2 antibody detection.

Background: Miniaturised bioassays permit diagnostic testing near the patient, and the results can be recorded digitally using inexpensive cameras including smartphone and mobile phone cameras. Although digital cameras are now inexpensive and portable, the minimum performance required for microfluidic diagnostic bioassays has not been defined. We present a systematic comparison of a wide range of different digital cameras for capturing and measuring results of microfluidic bioassays and describe a framework to specify performance requirements to quantify immunoassays. Methods: A set of 200 µm diameter microchannels was filled with a range of concentrations of dyes used in colorimetric and fluorometric enzyme immunoassays. These were imaged in parallel using cameras of varying cost and performance ranging from <£30 to >£500. Results: Higher resolution imaging allowed larger numbers of microdevices to be resolved and analysed in a single image. In contrast, low quality cameras were still able to quantify results but for fewer samples. In some cases, an additional macro lens was added to focus closely. If image resolution was sufficient to identify individual microfluidic channels as separate lines, all cameras were able to quantify a similar range of concentrations of both colorimetric and fluorometric dyes. However, the mid-range cameras performed better, with the lowest cost cameras only allowing one or two samples to be quantified per image. Consistent with these findings, we demonstrate that quantitation (to determine endpoint titre) of antibodies against dengue and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses is possible using a wide range of digital imaging devices including the mid-range smartphone iPhone 6S and a budget Android smartphone costing <£50. Conclusions: In conclusion, while more expensive and higher quality cameras allow larger numbers of devices to be simultaneously imaged, even the lowest resolution and cheapest cameras were sufficient to record and quantify immunoassay results.

Stringent thresholds in SARS-CoV-2 IgG assays lead to under-detection of mild infections

BackgroundThresholds for SARS-CoV-2 antibody assays have typically been determined using samples from symptomatic, often hospitalised, patients. In this setting the sensitivity and specificity of the best performing assays can both exceed 98%. However, antibody assay performance following mild infection is less clear.MethodsWe assessed quantitative IgG responses in a cohort of healthcare workers in Oxford, UK, with a high pre-test probability of Covid-19, in particular the 991/11,475(8.6%) who reported loss of smell/taste. We use anosmia/ageusia and other risk factors as probes for Covid-19 infection potentially undiagnosed by immunoassays by investigating their relationship with antibody readings either side of assay thresholds.ResultsThe proportion of healthcare workers reporting anosmia/ageusia increased at antibody readings below diagnostic thresholds using an in-house ELISA (n = 9324) and the Abbott Architect chemiluminescent microparticle immunoassay (CMIA; n = 11,324): 426/906 (47%) reported anosmia/ageusia with a positive ELISA, 59/449 (13.1%) with high-negative and 326/7969 (4.1%) with low-negative readings. Similarly, by CMIA, 518/1093 (47.4%) with a positive result reported anosmia/ageusia, 106/686 (15.5%) with a high-negative and 358/9563 (3.7%) with a low-negative result. Adjusting for the proportion of staff reporting anosmia/ageusia suggests the sensitivity of both assays in mild infection is lower than previously reported: Oxford ELISA 89.8% (95%CI 86.6–92.8%) and Abbott CMIA 79.3% (75.9–82.7%).ConclusionFollowing mild SARS-CoV-2 infection 10–30% of individuals may have negative immunoassay results. While lowered diagnostic thresholds may result in unacceptable specificity, our findings have implications for epidemiological analyses and result interpretation in individuals with a high pre-test probability. Samples from mild PCR-confirmed infections should be included in SARS-CoV-2 immunoassay evaluations.

Evaluation of the Effect of Hemolysis on Quantitative Chemiluminescent Immunoassay Results for 10 Analytes.

The aim is to evaluate the effect of hemolysis on the quantitative chemiluminescent immunoassay results of 10 analytes and to provide a basis for formulating specific sample rejection criteria and reviewing report results. Hemolysis based on the clinical hemolysis index, hemolysis 1+, 2+, and 3+ samples and matched normal samples were collected. The quantitative chemiluminescent immunoassay results of 10 analytes from the two samples (hemolysis and normal) were determined and differences between the results obtained from samples with different degrees of hemolysis and those obtained from normal samples were evaluated. A total of 34 pairs of samples were collected, including 10 pairs of 1+ hemolysis samples, 10 pairs of 2+ hemolysis samples, and 14 pairs of 3+ hemolysis samples. The quantitative chemiluminescence immunoassay detection results for the 10 analytes showed that regardless of the degree of hemolysis, the differences in alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen (CA19-9), luteinizing hormone (LH), folli-cle-stimulating hormone (FSH), and ferritin (FER) between the hemolysis and normal samples were all lower than the total allowable error (TEa) based on biological variation; there were no statistically significant differences between the samples. However, the results for insulin (INS) began to decrease significantly at a hemolytic index of 1+, folic acid (FOL) showed an increase at a hemolytic index of 2+, and there was a significant difference at a he-molytic index of 3+. This research identified the analytes that are susceptible to hemolysis interference in chemiluminescent immunoassays. The influence of hemolysis on hemolytic clinical laboratory tests was closely related to the assay system used; thus, laboratories should evaluate the effect of hemolysis on their own analysis systems and define assay-specific hemolysis warning indices.

Isolation and characterization of extracellular vesicles in saliva of children with asthma.

Aim:To confirm the presence of extracellular vesicles (EVs) in cell-free saliva (CFS) of children with asthma and describe the isolated EV population.Methods:A pooled sample of CFS EVs isolated from 180 participants using ExoQuick-TC was examined in downstream analyses. Transmission electron microscopy (TEM) was used to confirm the presence of EVs. Nanoparticle tracking analysis (NTA) and single particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence were used for sizing, counting, and phenotyping of EVs. Capillary immunoassays were used for protein quantitation.Results:TEM confirmed the presence of EVs of diverse sizes, indicating the prep contained a heterogeneous population of EVs. Capillary immunoassays confirmed the presence of EV-associated proteins (CD9, CD63, CD81, ICAM-1, and ANXA5) and indicated limited cellular contamination. As others have also reported, there were discrepancies in the EV sizing and enumeration across platforms. Fluorescent NTA detected particles with a mode diameter of ~90 nm, whereas SP-IRIS reported sizes of ~55–60 nm that more closely approximated the TEM results. Consistent with protein immunoassay results, SP-IRIS with fluorescence showed that the majority of these EVs were CD9- and CD63-positive, with little expression of CD81.Conclusion:EVs from CFS can be isolated using a high-throughput method that can be scaled to large epidemiological studies. To our knowledge, we are the first to characterize CFS EVs from patients with asthma. The use of CFS EVs as potential novel biomarkers in asthma warrants further investigation and opens a new avenue of research for future studies.

Predicting various forms of endometriosis using artificial neural networks

Introduction. Endometriosis is a difficult-to-diagnose pathology due to the diversity of clinical manifestations and the lack of high-precision markers necessary for rapid noninvasive diagnosis and timely administration of pathogenetically justified treatment.The aim of this work was to develop a computer system that allows us to assess the probability of endometriosis with various localizations in women, based on artificial neural networks.Material and Methods. The neural network mathematical models were constructed and tested based on data from 110 patients with morphologically pre-confirmed endometriosis. Patients were divided into training and test samples. The models were built based on anamnestic data and results of proteomic and enzyme immunoassays in blood plasma samples.Results and Discussion. In the course of the study, four mathematical models of neural networks were constructed to predict the presence or absence of endometriosis in a woman and its localization if present. Based on these mathematical models, a computer system “Differential diagnosis of endometriosis” was developed. This system allowed to assess the probability and localization of endometriosis in a patient based on parameters obtained as a result of neural network training.Conclusion. The developed computer diagnostic system allowed predicting the presence of endometriosis and its localization with a probability over 80%, depending on the predicted localization, based on data about the patient and the results of her examination. This system may be used for differential diagnosis of endometriosis from other diseases of the female reproductive system, as well as for differential diagnosis of various endometriosis localizations.

Hapten-Branched Polyethylenimine as a New Antigen Affinity Ligand to Purify Antibodies with High Efficiency and Specificity

Purification of antibodies has become a critical factor in antibody production. A high-purity specific antibody against antigens, especially small molecules, seems to be difficult to obtain, even with the help of a protein A affinity column, which is a conventional and broadly used ligand for the separation of antibody and non-antibody proteins. Therefore, it is urgent to develop a cheap, simple, efficient, and stable method to separate the specific antibody from other antibodies. In this study, to improve the sensitivity and accuracy of immunoassay results, enrofloxacin (ENR) was grafted onto polyethylenimine (PEI) by the abundant amino groups and then the whole ligand (ENR-PEI) was conjugated to CNBr-Sepharose 4B to prepare the affinity column for the purification of the specific antibody against ENR from polyclonal antibodies. Scanning electron microscopy and Fourier transform infrared spectroscopy verification showed that Sepharose 4B was successfully modified by ENR-PEI with excellent uniformity. The capacity of the prepared column could reach to 6.15 mg of specific antibody with high purity per milliliter resin due to the high coupling ratio (49.3:1) of ENR on PEI, and the IC50 value of the antibody after purification was 47.58 ng/mL with a lowest limit of detection (IC10) of 1.099 ng/mL-18 times lower than those of the antibody purified through the protein A column. All the results showed that this new kind of resin could be used as the potential ligand in the purification of the trace-specific antibody against antigens in complex mixtures with high efficiency and specificity.

Sociodemographic and sanitary profile of chikungunya virus infection in medium-sized municipality in Mato Grosso, from January to March 2018, Brasil.

Introduction: The chikungunya virus has already been identified in more than 60 countries in Asia, Africa, Europe and the Americas, and chronicity after the disease impacts the lives of those affected by the virus, as well as society, the economy and public health. The objective was to characterize, through epidemiological survey, the profile of chikungunya infection in a mid-sized municipality in Mato Grosso according to sociodemographic and sanitary factors. Methods: The serum-epidemiological survey was conducted with 596 adults aged ≥ 18 years selected by cluster sampling process, with application of questionnaires and biological material collection. Positive cases of chikungunya fever were those with positive results in the anti-chikungunya virus enzyme immunoassay (ELISA). The statistical analyses used descriptive and inferential techniques with confidence intervals of 95% and a significance level of 5%. Results: The overall prevalence of chikungunya fever found in the community was 8.4%. The profile of infection by chikungunya fever is composed of women (p&lt;0.204), aged between 18 and 39 years (p&lt;0.780), more than 08 years of study (p&lt;0.079), non-white reported race/color p&lt;0.871) and employed in the past 12 months (p&lt;0.927). Not residing with affective companion was statistically significant for infection by chikungunya virus (CHIKV) (p&lt;0.028). Conclusion: The study found that women are the most affected by the infection, as well as being single represented a risk factor, and risk behaviors, such as presence of larvae and breeding of mosquitos at home, reflect a low level of awareness of the disease.

AD biomarker stability for stored CSF based on immunoassay results

AbstractBackgroundDiagnosis of Alzheimer’s Disease (AD) is currently more often based on a clinical assessment combined with biomarker data. For AD, four core cerebrospinal fluid (CSF) biomarkers have been identified and levels are determined on measurements with various immunoassays (INNOTEST, Lumipulse, Elecsys, etc.). Each assay has its own concept (technology, antibodies, protocol) and performance characteristics.The Alzheimer’s Association Quality Control (AA‐QC) program lead by the University of Gothenburg aims to monitor the inter‐laboratory variation for immunoassays for CSF biomarkers using a set of CSF samples (2 round specific &amp; 1 longitudinal), periodically measured at participating sites.Biomarker levels over time of the longitudinal QC CSF sample (2016QC‐L) on the different immunoassays can be interpreted to assess long‐term stability. This is relevant given that CSF samples are often stored in a biorepository as part of a study cohort for future assessment of (new) biomarkers.MethodIn the AA‐QC program, immunoassays for the different AD CSF core biomarkers (Aβ1‐42, Aβ1‐40, total‐Tau, and pTau181) from different suppliers are tested three times per year with 2016QC‐L. The average result per immunoassay for this sample since round 22 (October 2016) till round 31 (October 2019) were analyzed, considering round 22 as start of storage.ResultFor all four CSF biomarkers, the coefficient of variation (CV) per immunoassay obtained over all round averages was below 10% (except one). The CVs ranged from 2% to 10% for Aβ1‐42, 1% to 8% for Aβ1‐40, 1% to 17% for total‐Tau, and 1% to 9% for pTau181, indicating relatively stable concentration assignment over time, despite the use of different batches. For each biomarker, no consistent trend in analyte concentration over all immunoassays in function of time was observed.ConclusionFor the CSF sample analyzed, AD core biomarkers are stable over the follow‐up period of 36 months. The results of the different immunoassays all show a comparable behavior of the analyte concentration over time. These results from Clinical Chemistry practice confirm the long‐term stability of the core CSF biomarkers, lending further support for their use in clinical research studies and therapy monitoring in clinical trials, where stored samples are often analyzed in batch at end‐of‐study.

Transient expression of gene encoding ZmLEA14A protein in Nicotiana benthamiana plant

LEA protein family includes proteins accumulated in the late stage of embryogenesis and in vegetative tissues of stress-confronted plant. These proteins have been demontrated to play a major role in plant response to abiotic stresses, such as drought and salinity stress. The genes coding for LEA proteins in maize are divided into 9 groups including LEA 1, LEA 2, LEA 3, LEA 4, LEA 5, LEA 6, SMP, dehydrin, and AtM. The application of LEA genes to improve drought tolerance for plants by genetic engineering has also been studied extensively all over the world. In this study, pCAM/35S-ZmLEA14A-35S vector and pCAM/Ubi-ZmLEA14A-35S vector contained the ZmLEA14A gene isolated from Te vang 1, these vectors were used to transient express into Nicotiana benthamiana tobacco leaves by agro-infiltration method. The results of immunoassay between cmyc specific antibodies with proteins from infected leaves revealed the expression of recombinant ZmLEA14A protein in N. benthamiana leaves. Thereby, two constructs habouring the ZmLEA14A gene work at transcription and translation levels in the model plant that could harnessed for stable transformation in plants.

Continuation of Over-the-Counter Biotin Supplements in the Inpatient Setting: An Unexpected Source of Laboratory Error.

Over the past decade, use of high-dose biotin has increased significantly and can lead to erroneous results on some clinical immunoassays. In collaboration with pharmacists at our institution, we discovered that high biotin doses were being administered to inpatients as a continuation of patient-reported home biotin use. This retrospective study evaluated high-dose biotin administration in 226 inpatient encounters from 2009 to 2019 and its potential impact on concurrent immunoassay testing. In 96% of cases, biotin was administered in the inpatient setting as a continuation of patient-reported home use. In total, 322 immunoassays capable of biotin interference were performed across 100 inpatient encounters with high-dose biotin administration. Troponin T and TSH were the most commonly performed immunoassays in this cohort. Even though less than 5% of all high-dose biotin orders at our institution are placed for inpatients, hospitalized patients are still at risk for mismanagement due to erroneous immunoassay results. Immunoassay testing susceptible to biotin interference was performed in approximately 45% of inpatient encounters with biotin administration. Laboratories utilizing biotin-susceptible, sensitive cardiac troponin assays should be particularly cautious. Pharmacokinetic data for biotin clearance is especially lacking for certain populations likely to be hospitalized, such as those with renal failure. Given that medical conditions requiring high-dose biotin therapy are extremely rare, we recommend restricting biotin dosing during inpatient encounters for all other patients.

Novel immunoassay for diagnosis of ongoing Clostridioides difficile infections using serum and medium enriched for newly synthesized antibodies (MENSA)

BackgroundClostridioides difficile infections (CDI) have been a challenging and increasingly serious concern in recent years. While early and accurate diagnosis is crucial, available assays have frustrating limitations. ObjectiveDevelop a simple, blood-based immunoassay to accurately diagnose patients suffering from active CDI. Materials and methodsUninfected controls (N = 95) and CDI patients (N = 167) were recruited from Atlanta area hospitals. Blood samples were collected from patients within twelve days of a positive CDI test and processed to yield serum and PBMCs cultured to yield medium enriched for newly synthesized antibodies (MENSA). Multiplex immunoassays measured Ig responses to ten recombinant C. difficile antigens. ResultsSixty-six percent of CDI patients produced measurable responses to C. difficile antigens in their serum or MENSA within twelve days of a positive CDI test. Fifty-two of the 167 CDI patients (31%) were detectable in both serum and MENSA, but 32/167 (19%) were detectable only in MENSA, and 27/167 (16%) were detectable only in serum. DiscussionWe describe the results of a multiplex immunoassay for the diagnosis of ongoing CDI in hospitalized patients. Our assay resolved patients into four categories: MENSA-positive only, serum-positive only, MENSA- and serum-positive, and MENSA- and serum-negative. The 30% of patients who were MENSA-positive only may be accounted for by nascent antibody secretion prior to seroconversion. Conversely, the serum-positive only subset may have been more advanced in their disease course. Immunocompromise and misdiagnosis may have contributed to the 34% of CDI patients who were not identified using MENSA or serum immunoassays. ImportanceWhile there was considerable overlap between patients identified through MENSA and serum, each method detected a distinctive patient group. The combined use of both MENSA and serum to detect CDI patients resulted in the greatest identification of CDI patients. Together, longitudinal analysis of MENSA and serum will provide a more accurate evaluation of successful host humoral immune responses in CDI patients.

Challenges in Detecting Clinically Relevant Heparin-Induced Thrombocytopenia Antibodies.

Heparin-induced thrombocytopenia (HIT) is an antibody-mediated hypercoagulable state featuring high thrombosis risk and distinct pathogenesis involving immunoglobulin G-mediated platelet activation. The target of the immune response is a cationic “self” protein, platelet factor 4 (PF4), rendered antigenic by heparin. A key problem is that only a minority of anti-PF4/polyanion antibodies induced by heparin are pathogenic, i.e., capable of causing platelet activation and thereby clinical HIT. Since thrombocytopenia occurs frequently in hospitalized, heparin-treated patients, testing for “HIT antibodies” is common; thus, the problem of distinguishing between pathogenic and nonpathogenic antibodies is important. The central concept is that those antibodies that have platelet-activating properties demonstrable in vitro correlate well with pathogenicity, as shown by platelet activation tests such as the serotonin-release assay (SRA) and heparin-induced platelet activation assay. However, in most circumstances, immunoassays are used for first-line testing, and so it is important for clinicians to appreciate which immunoassay result profiles—in the appropriate clinical context—predict the presence of platelet-activating antibodies (Bayesian analysis). Clinicians with access to rapid, on-demand HIT immunoassays (e.g., particle gel immunoassay, latex immunoturbidimetric assay, chemiluminescent immunoassay) can look beyond simple dichotomous result interpretation (“negative”/“positive”) and incorporate semiquantitative interpretation, where, for example, a strong-positive immunoassay result (or even combination of two immunoassays) points to a greater probability of detecting platelet-activating antibodies, and hence supporting a diagnosis of HIT. Recent recognition of “SRA-negative HIT” has increased the importance of semiquantitative interpretation of immunoassays, given that strong immunoassay reactivity is a potential clue indicating possible HIT despite a (false) negative platelet activation assay.