Development and assessment of a multiepitope synthetic antigen for the diagnosis of Dengue virus infection.

Immunodiagnostic tests for detecting dengue virus infections encounter challenges related to cross-reactivity with other related flaviviruses. Our research focuses on the development of a synthetic multiepitope antigen tailored for dengue immunodiagnostics. Selected dengue epitopes involved structural linearity and dissimilarity from the proteomes of Zika and Yellow fever viruses which served for computationally modeling the three-dimensional protein structure, resulting in the design of two proteins: rDME-C and rDME-BR. Both proteins consist of seven epitopes, separated by the GPGPG linker, and a carboxy-terminal 6 × -histidine tag. The molecular weights of the final proteins rDME-C and rDME-BR are 16.83 kDa and 16.80 kDa, respectively, both with an isoelectric point of 6.35. The distinguishing factor between the two proteins lies in the origin of their epitope sequences, where rDME-C is based on the reference dengue proteome, while rDME-BR utilizes sequences from prevalent Dengue genotypes in Brazil from 2008 to 2019. PyMol analysis revealed exposure of epitopes in the secondary structure. Successful expression of the antigens was achieved in soluble form and fluorescence experiments indicated a disordered structure. In subsequent testing, rDME-BR and rDME-C antigens were assessed using an indirect Elisa protocol against Dengue infected serum, previously examined with a commercial diagnostic test. Optimal concentrations for antigens were determined at 10 µg/mL for rDME-BR and 30 µg/mL for rDME-C, with serum dilutions ranging from 1:50 to 1:100. Both antigens effectively detected IgM and IgG antibodies in Dengue fever patients, with rDME-BR exhibiting higher sensitivity. Our in-house test showed a sensitivity of 77.3 % and 82.6 % and a specificity of 89.4 % and 71.4 % for rDME-C and rDEM-BR antigens. No cross-reactivity was observed with serum from Zika-infected mice but with COVID-19 serum samples. Our findings underscore the utility of synthetic biology in crafting Dengue-specific multiepitope proteins and hold promise for precise clinical diagnosis and monitoring responses to emerging Dengue vaccines.

Structure-based epitope prediction and assessment of cross-reactivity of Myrmecia pilosula venom-specific IgE and recombinant Sol g proteins (Solenopsis geminata).

The global distribution of tropical fire ants (Solenopsis geminata) raises concerns about anaphylaxis and serious medical issues in numerous countries. This investigation focused on the cross-reactivity of allergen-specific IgE antibodies between S. geminata and Myrmecia pilosula (Jack Jumper ant) venom proteins due to the potential emergence of cross-reactive allergies in the future. Antibody epitope analysis unveiled one predominant conformational epitope on Sol g 1.1 (PI score of 0.989), followed by Sol g 2.2, Sol g 4.1, and Sol g 3.1. Additionally, Pilosulin 1 showed high allergenic potential (PI score of 0.94), with Pilosulin 5a (PI score of 0.797) leading in B-cell epitopes. The sequence analysis indicated that Sol g 2.2 and Sol g 4.1 pose a high risk of cross-reactivity with Pilosulins 4.1a and 5a. Furthermore, the cross-reactivity of recombinant Sol g proteins with M. pilosula-specific IgE antibodies from 41 patients revealed high cross-reactivity for r-Sol g 3.1 (58.53%) and r-Sol g 4.1 (43.90%), followed by r-Sol g 2.2 (26.82%), and r-Sol g 1.1 (9.75%). Therefore, this study demonstrates cross-reactivity (85.36%) between S. geminata and M. pilosula, highlighting the allergenic risk. Understanding these reactions is vital for the prevention of severe allergic reactions, especially in individuals with pre-existing Jumper Jack ant allergy, informing future management strategies.

Assessment of crustacean allergen detection methods: cross reactivity with edible insect samples.

Increased interest in consumption of insects in recent years has led to an increased focus on associated food safety concerns, and allergy is one of the most relevant. In the United States, crustacean shellfish are regulated as a major allergenic food group per the Food, Drug, and Cosmetic (FD&C) Act. Insects and crustacean shellfish are both arthropods, and clinical cross-reactivity between the two groups has been demonstrated. The goal of this work was to establish whether that clinical cross-reactivity translates into analytical cross-reactivity with detection assays targeting crustacean shellfish allergens. Edible insect samples were analyzed using four different crustacean allergen detection methods: Multi-Analyte Profiling Food Allergen Detection Assay (xMAP FADA), enzyme-linked immunosorbent assay (ELISA), western blot, and real-time polymerase chain reaction (PCR). Results indicate that the immunoassay-based xMAP FADA, ELISA, and western blot were susceptible to cross-reactivity, while the DNA-based PCR methods had minimal reactivity with insect samples. These results confirm that edible insects show analytical cross-reactivity with the immunoassays which may result in false positive detection of crustacean allergens in insect samples. Confirmation using DNA-based PCR, which shows little to no cross-reactivity, clarifies ambiguous results.

Assessment of Co-Sensitization between Pollen and Food Allergen Sources among Bengali Population, West Bengal, India.

INTRODUCTION: Secondary food allergies due to cross-reactivity between pollen and food allergens are a significant health burden in India. Prevalence of cross-reactivity varies by region, and particularly in West Bengal, it remains unexplored. The present study investigated sensitivities of pollen-related food allergy (PFA) patients of West Bengal by empirically determining co-sensitization patterns. METHODS: The present study retrospectively analysed 1,310 pollen-allergic (PA) patients from the Allergy and Asthma Research Centre, West Bengal. A questionnaire survey was performed to record patients' demographic and clinical features and categorized into seasonal and perennial ones. All patients were subjected to the skin prick test and specific IgE for evaluation of sensitivity against 16 pollen and 16 food allergens. Co-relation and principal component analysis (PCA) was performed to determine co-sensitivity. RESULTS: Seasonal allergic patients were comparatively higher than perennial ones. PFA patients were mostly sensitive to eggplant (n = 387), while Azadirachta indica (n = 341) was predominant sensitizer among pollen allergen sources. Cynodon dactylon showed maximum significant positive correlation with banana, orange, peanut, pineapple, cucumber, eggplant, potato, tomato, and wheat. In the seasonal group, Cynodon dactylon-specific IgE in PFA patients (median = 4.60 kU/L) was significantly higher from that of PA ones (median = 3.44 kU/L). Among 32 allergens tested, 27 were placed in first two dimensions of the PCA biplot. CONCLUSION: The present study established that co-sensitivity between pollen and food allergen sources were predominant in allergic patients of West Bengal. The co-sensitization patterns among unrelated allergen sources may be due to possible expression of biologic cross-reactivity to similar allergens.

Allergenic risk assessment of cowpea and its cross-reactivity with pea and peanut.

BACKGROUND: Novel protein sources can represent a risk for allergic consumers. The aim of this study was to evaluate the allergenicity of cowpea (Vigna unguiculata), an increasingly consumed legume and potential new industrial food ingredient which may put legume-allergic patients at risk. METHODS: Children with allergy to legumes associated to peanut (LP group: n = 13) or without peanut allergy (L group: n = 14) were recruited and sensitization to several legumes including cowpea was assessed by prick tests and detection of specific IgE (sIgE). Cowpea protein extract was analyzed by SDS-PAGE and immunoblotting, IgE-reactive spots were subjected to mass spectrometry. IgE-cross-reactivity between cowpea, pea, and peanut was determined using ELISA inhibition assays. Basophil activation tests were performed to evaluate sensitivity and reactivity of patient basophils toward legumes. RESULTS: Prick tests and sIgE levels to cowpea were positive in 8/14 and 4/13 patients of the L group and in 9/13 and 10/13 patients of the LP group, respectively. Four major IgE-binding proteins were identified as vicilins and seed albumin. Cowpea extract and its vicilin fraction strongly inhibited IgE-binding to pea and peanut extract. Peanut, lentil, and pea were the strongest activators of basophils, followed by cowpea, soybean, mung bean, and lupin. CONCLUSION: A majority of patients with legume allergy were sensitized to cowpea proteins. Four novel allergens were identified in cowpea, among which storage proteins were playing an important role in IgE-cross-reactivity, exposing legume-allergic patients to the risk of clinical cross-reactivity to cowpea and thus adding cowpea to the group of nonpriority legumes that are not subjected to allergen labeling such as chickpea, pea, and lentil.

Model-based assessment of Chikungunya and O'nyong-nyong virus circulation in Mali in a serological cross-reactivity context.

Serological surveys are essential to quantify immunity in a population but serological cross-reactivity often impairs estimates of the seroprevalence. Here, we show that modeling helps addressing this key challenge by considering the important cross-reactivity between Chikungunya (CHIKV) and O'nyong-nyong virus (ONNV) as a case study. We develop a statistical model to assess the epidemiology of these viruses in Mali. We additionally calibrate the model with paired virus neutralization titers in the French West Indies, a region with known CHIKV circulation but no ONNV. In Mali, the model estimate of ONNV and CHIKV prevalence is 30% and 13%, respectively, versus 27% and 2% in non-adjusted estimates. While a CHIKV infection induces an ONNV response in 80% of cases, an ONNV infection leads to a cross-reactive CHIKV response in only 22% of cases. Our study shows the importance of conducting serological assays on multiple cross-reactive pathogens to estimate levels of virus circulation.

Prospective Assessment of SARS-CoV-2 Seroconversion (PASS) study: an observational cohort study of SARS-CoV-2 infection and vaccination in healthcare workers.

BACKGROUND: SARS-CoV-2 is a recently emerged pandemic coronavirus (CoV) capable of causing severe respiratory illness. However, a significant number of infected people present as asymptomatic or pauci-symptomatic. In this prospective assessment of at-risk healthcare workers (HCWs) we seek to determine whether pre-existing antibody or T cell responses to previous seasonal human coronavirus (HCoV) infections affect immunological or clinical responses to SARS-CoV-2 infection or vaccination. METHODS: A cohort of 300 healthcare workers, confirmed negative for SARS-CoV-2 exposure upon study entry, will be followed for up to 1 year with monthly serology analysis of IgM and IgG antibodies against the spike proteins of SARS-CoV-2 and the four major seasonal human coronavirus - HCoV-OC43, HCoV-HKU1, HCoV-229E, and HCoV-NL63. Participants will complete monthly questionnaires that ask about Coronavirus Disease 2019 (COVID-19) exposure risks, and a standardized, validated symptom questionnaire (scoring viral respiratory disease symptoms, intensity and severity) at least twice monthly and any day when any symptoms manifest. SARS-CoV-2 PCR testing will be performed any time participants develop symptoms consistent with COVID-19. For those individuals that seroconvert and/or test positive by SARS-CoV-2 PCR, or receive the SARS-CoV-2 vaccine, additional studies of T cell activation and cytokine production in response to SARS-CoV-2 peptide pools and analysis of Natural Killer cell numbers and function will be conducted on that participant's cryopreserved baseline peripheral blood mononuclear cells (PBMCs). Following the first year of this study we will further analyze those participants having tested positive for COVID-19, and/or having received an authorized/licensed SARS-CoV-2 vaccine, quarterly (year 2) and semi-annually (years 3 and 4) to investigate immune response longevity. DISCUSSION: This study will determine the frequency of asymptomatic and pauci-symptomatic SARS-CoV-2 infection in a cohort of at-risk healthcare workers. Baseline and longitudinal assays will determine the frequency and magnitude of anti-spike glycoprotein antibodies to the seasonal HCoV-OC43, HCoV-HKU1, HCoV-229E, and HCoV-NL63, and may inform whether pre-existing antibodies to these human coronaviruses are associated with altered COVID-19 disease course. Finally, this study will evaluate whether pre-existing immune responses to seasonal HCoVs affect the magnitude and duration of antibody and T cell responses to SARS-CoV-2 vaccination, adjusting for demographic covariates.

Allergenicity assessment of the edible cricket Acheta domesticus in terms of thermal and gastrointestinal processing and IgE cross-reactivity with shrimp.

The allergenic potency of the cricket Acheta domesticus, a promising edible insect, has never been assessed. This work aims to study the immunoreactivity of Acheta domesticus, and its cross-reactivity with the shrimp Litopenaeus vannamei, assessing the effect of cooking and gastrointestinal digestion on their allergenic properties. Different cricket proteins were detected by immunoblotting with shrimp-allergic patients' sera. Tropomyosin was identified as the most relevant IgE-binding protein, and its cross-reactivity with shrimp tropomyosin was demonstrated by ELISA. While shrimp tropomyosin showed scarce stability to gastric digestion, cricket tropomyosin withstood the whole digestion process. The sarcoplasmic calcium-binding protein, specifically detected in shrimp, showed exceptional stability to gastrointestinal digestion. IgE-binding proteins in a model of enriched baked products were partially protected from proteolysis. In conclusion, the ingestion of A. domesticus proteins poses serious concerns to the Crustacean-allergic population. The high stability of tropomyosin may represent a risk of primary sensitization and clinical cross-reactivity.

Immunogenicity risk assessment for biotherapeutics through in vitro detection of CD134 and CD137 on T helper cells.

Biotherapeutics, which are biologic medications that are natural or bioengineered products of living cells, have revolutionized the treatment of many diseases. However, unwanted immune responses still present a major challenge to their widespread adoption. Many patients treated with biotherapeutics develop antigen-specific anti-drug antibodies (ADAs) that may reduce the efficacy of the therapy or cross-react with the endogenous counterpart of a protein therapeutic, or both. Here, we describe an in vitro method for assessing the immunogenic risk of a biotherapeutic. We found a correlation between clinical immunogenicity and the frequency with which a biotherapeutic stimulated an increase in CD134, CD137, or both cell surface markers on CD4+ T cells. Using high-throughput flow cytometry, we examined the effects of 14 biotherapeutics with diverse rates of clinical immunogenicity on peripheral blood mononuclear cells from 120 donors with diverse human leukocyte antigen class II-encoding alleles. Biotherapeutics with high rates of ADA development in the clinic had higher proportions of CD4+ T cells positive for CD134 or CD137 than biotherapeutics with low clinical immunogenicity. This method provides a rapid and simple preclinical test of the immunogenic potential of a new candidate biotherapeutic or biosimilar. Implementation of this approach during biotherapeutic research and development enables rapid elimination of candidates that are likely to cause ADA-related adverse events and detrimental consequences.

History of safe exposure and bioinformatic assessment of phosphomannose-isomerase (PMI) for allergenic risk.

Newly expressed proteins in genetically engineered crops are evaluated for potential cross reactivity to known allergens as part of their safety assessment. This assessment uses a weight-of-evidence approach. Two key components of this allergenicity assessment include any history of safe human exposure to the protein and/or the source organism from which it was originally derived, and bioinformatic analysis identifying amino acid sequence relatedness to known allergens. Phosphomannose-isomerase (PMI) has been expressed in commercialized genetically engineered (GE) crops as a selectable marker since 2010 with no known reports of allergy, which supports a history of safe exposure, and GE events expressing the PMI protein have been approved globally based on expert safety analysis. Bioinformatic analyses identified an eight-amino-acid contiguous match between PMI and a frog parvalbumin allergen (CAC83047.1). While short amino acid matches have been shown to be a poor predictor of allergen cross reactivity, most regulatory bodies require such matches be assessed in support of the allergenicity risk assessment. Here, this match is shown to be of negligible risk of conferring cross reactivity with known allergens.

Assessment of dengue and COVID-19 antibody rapid diagnostic tests cross-reactivity in Indonesia.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic remains ongoing around the world, including in areas where dengue is endemic. Dengue and COVID-19, to some extent, have similar clinical and laboratory features, which can lead to misdiagnosis, delayed treatment and patient's isolation. The use of rapid diagnostic tests (RDT) is easy and convenient for fast diagnosis, however there may be issues with cross-reactivity with antibodies for other pathogens. METHODS: We assessed the possibility of cross-reactivity between SARS-CoV-2 and dengue antibodies by: (1) testing five brands of COVID-19 IgG / IgM RDTs on 60 RT-PCR-confirmed dengue samples; (2) testing 95 RT-PCR-confirmed COVID-19 samples on dengue RDT; and (3) testing samples positive for COVID-19 IgG and/or IgM on dengue RDT. RESULTS: We observed a high specificity across all five brands of COVID-19 RDTs, ranging from 98.3 to 100%. Out of the confirmed COVID-19 samples, one patient tested positive for dengue IgM only, another tested positive for dengue IgG only. One patient tested positive for dengue IgG, IgM, and NS1, suggesting a co-infection. In COVID-19 IgG and/or IgM samples, 6.3% of COVID-19 IgG-positive samples also tested positive for dengue IgG, while 21.1% of COVID-19 IgM-positive samples also tested positive for dengue IgG. CONCLUSION: Despite the high specificity of the COVID-19 RDT, we observed cross-reactions and false-positive results between dengue and COVID-19. Dengue and COVID-19 co-infection was also found. Health practitioners in dengue endemic areas should be careful when using antibody RDT for the diagnosis of dengue during the COVID-19 pandemic to avoid misdiagnosis.

Classic and evolving approaches to evaluating cross reactivity of mAb and mAb-like molecules - A survey of industry 2008-2019.

Monoclonal antibodies (mAbs) and mAb derivatives have become mainstay pharmaceutical modalites. A critical assessment is to ascertain the specificity of these molecules prior to human clinical trials. The primary technique for determining specificity has been the immunohistochemistry (IHC)-based "Tissue Cross-Reactivity" (TCR) assay, where the candidate molecule is applied to > 30 tissues to look for unexpected staining. In the last few years, however, non-IHC array-based platforms have emerged that allow for screening 75-80% of the human membrane proteome, indicating a viable alternative and/or addition to the IHC methods. The preclinical sciences subcommittee of the Biotechnology Innovation Organization (BIO), "BioSafe", conducted a survey of 26 BIO member companies to understand current sponsor experience with the IHC and array techniques. In the last ten years, respondents noted they have conducted more than 650 IHC TCR assays, largely on full length mAbs, with varying impacts on programs. Protein/cell arrays have been utilized by almost half of the companies and sponsors are gaining familiarity and comfort with the platform. Initial experience with recent versions of these arrays has been largely positive. While most sponsors are not prepared to eliminate the IHC TCR assay, growing experience with these alternatives allows them to confidently choose other approaches with or without TCR assays.

Protein extraction protocols for optimal proteome measurement and arginine kinase quantitation from cricket Acheta domesticus for food safety assessment.

Insects have been consumed by people for millennia and have recently been proposed as a complementary, sustainable source of protein to feed the world's growing population. Insects and crustaceans both belong to the arthropod family. Crustacean (shellfish) allergies are common and potentially severe; hence, the cross-reactivity of the immune system with insect proteins is a potential health concern. Herein, LC-MS/MS was used to explore the proteome of whole, roasted whole and roasted powdered cricket products. Eight protein extraction protocols were compared using the total number of protein and distinct peptide identifications. Within these data, 20 putative allergens were identified, of which three were arginine kinase (AK) proteoforms. Subsequently, a multiple reaction monitoring MS assay was developed for the AK proteoforms and applied to a subset of extracts. This targeted assay demonstrated that allergen abundance/detectability varies according to the extraction method as well as the food processing method.

Elucidating the Impact of Immunogenicity Assessment Postapproval: A Targeted Analysis of Immunogenicity Postmarketing Requirements and Commitments.

Insufficient availability of data to evaluate immunogenicity incidence or clinical impact during regulatory review could require further evaluation postapproval. Through a keyword search of all postmarketing requirements and commitments (PMRs/PMCs) associated with products with their original US Food and Drug Administration (FDA) approvals between 2009 and 2018, we identified products that had PMRs/PMCs established to address concerns or uncertainty related to immunogenicity. Of the 113 relevant products, 50% had an immunogenicity-related PMR/PMC; of these, 68% were related to developing immunogenicity assays and 48% requested an assessment of clinical impact. Fifty-five percent of the products with a fulfilled PMR/PMC had a change in the immunogenicity information in their labeling immediately following fulfillment. This work highlights that there are often unknowns associated with immunogenicity incidence and/or impact at the time of approval. Earlier regulatory discussions on immunogenicity assessments in premarket development could improve the understanding and communication of the risk/benefit profile and reduce the need for some immunogenicity PMRs/PMCs.

A Survey on Knowledge Gaps in Assessment and Management of Severe Drug Hypersensitivity Reactions: Multicenter Cross-Sectional Study of Australian Health Care Providers.

Severe drug hypersensitivity reactions (DHRs) are often encountered by health care professionals (HCPs). We evaluated knowledge of doctors and pharmacists in the assessment and management of severe DHRs using a structured questionnaire. A cross-sectional study was conducted in 4 metropolitan hospital networks in Melbourne, Australia. A 13-question, scenario-based multiple-choice questionnaire to assess specific knowledge domains in drug hypersensitivity syndrome recognition, causality attribution, cross-reactivity patterns, appropriate diagnostic tests, and therapy was administered to HCPs of various vocation and specialty groups. Data were analyzed according to profession, self-reported experience, and preparedness in managing severe DHRs. Two hundred thirty-eight participants (45.0% senior doctors, 24.4% junior doctors, and 30.7% pharmacists) across a range of subspecialties achieved an overall median score of 7 (IQR, 5-8)-overall 55.6% correct responses to all questions-with senior doctors outperforming junior doctors and pharmacists (P < .001). The best performance by all participants was in DHR syndrome recognition (60.9%), and the poorest was in diagnostics/therapy (52.0%). HCP group and experience level were significantly associated with better performance in the knowledge domains of cross-reactivity and diagnostics/therapy (P = .003 and < .001, respectively), but not in the domains of syndrome recognition and causality attribution (P > .05). Levels of self-reported preparedness in DHR management were not associated with performance rates in any of the knowledge domains. This study demonstrated significant knowledge gaps in the recognition and management of severe drug hypersensitivity reactions. Targeted multidisciplinary education of staff caring for these patients is needed to improve knowledge gaps.

Multicenter evaluation of two chemiluminescence and three lateral flow immunoassays for the diagnosis of COVID-19 and assessment of antibody dynamic responses to SARS-CoV-2 in Taiwan.

This multicenter, retrospective study included 346 serum samples from 74 patients with coronavirus disease 2019 (COVID-19) and 194 serum samples from non-COVID-19 patients to evaluate the performance of five anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody tests, i.e. two chemiluminescence immunoassays (CLIAs): Roche Elecsys® Anti-SARS-CoV-2 Test (Roche Test) and Abbott SARS-CoV-2 IgG (Abbott Test), and three lateral flow immunoassays (LFIAs): Wondfo SARS-CoV-2 Antibody Test (Wondfo Test), ASK COVID-19 IgG/IgM Rapid Test (ASK Test), and Dynamiker 2019-nCoV IgG/IgM Rapid Test (Dynamiker Test). We found high diagnostic sensitivities (%, 95% confidence interval [CI]) for the Roche Test (97.4%, 93.4-99.0%), Abbott Test (94.0%, 89.1-96.8%), Wondfo Test (91.4%, 85.8-94.9%), ASK Test (97.4%, 93.4-99.0%), and Dynamiker Test (90.1%, 84.3-94.0%) after >21 days of symptom onset. Meanwhile, the diagnostic specificity was 99.0% (95% CI, 96.3-99.7%) for the Roche Test, 97.9% (95% CI, 94.8-99.2%) for the Abbott Test, and 100.0% (95% CI, 98.1-100.0%) for the three LFIAs. Cross-reactivity was observed in sera containing anti-cytomegalovirus (CMV) IgG/IgM antibodies and autoantibodies. No difference was observed in the time to seroconversion detection of the five serological tests. Specimens from patients with COVID-19 pneumonia demonstrated a shorter seroconversion time and higher chemiluminescent signal than those without pneumonia. Our data suggested that understanding the dynamic antibody response after COVID-19 infection and performance characteristics of different serological test are crucial for the appropriate interpretation of serological test result for the diagnosis and risk assessment of patient with COVID-19 infection.

Validation and cross-reactivity pattern assessment of monoclonal antibodies used for the screening of donor-specific IgG antibody subclasses in transplant recipients.

The screening for IgG subclass donor-specific antibodies (DSAs) in allograft recipients uses IgG1-4 subclass-specific monoclonal antibodies (mAbs) that should be mono-specific. The cross-reactivity discrepancies reported for IgG subclass-specific mAbs warranted a critical cross-reactivity pattern analysis of the IgG subclass-specific mAbs most commonly used to detect DSAs. We tested the reactivity of 2 anti-IgG1-, 3 anti-IgG2-, 1 anti-IgG3-, and 2 anti-IgG4-specific PE-conjugated mAbs against microbeads coated with IgG1-4 proteins separately. Each IgG subclass protein was coated at three densities on the beads (0.5, 1, and 2 µg of protein per 106 beads), and the PE-conjugated mAbs were titrated from 0.04 µg/mL to 5 µg/mL. The IgG subclass reactivity of the sample was acquired on the Luminex multiplex platform. Among the IgG subclass-specific mAbs, only the anti-IgG3 (clone: HP6050) mAb was mono-specific. All other mAbs tested were binding to IgG subclass proteins other than their respective immunogen, thereby being cross-reactive. IgG subclass cross-reactivity patterns were dependent on the concentration of both IgG subclass-specific mAbs and IgG1-4 protein targets coated onto the beads. With the current IgG subclass mAbs available, 3 of the 15 possible combinations of IgG1-4 subclass protein could be identified. While the remaining 12 unique combinations cannot be distinguished clearly, 6 groups that corresponded to two different unique combinations of IgG1-4 subclass protein could be identified. The dilution of serum samples and IgG subclass-specific mAbs, other than the anti-IgG3 (clone: HP6050), must be further optimized before their implementation in IgG subclass DSA screening in allograft recipients.

Lower cost alternatives for molecular diagnosis of COVID-19: conventional RT-PCR and SYBR Green-based RT-qPCR.

In March 2020, WHO declared a pandemic state due to SARS-CoV-2 having spread. TaqMan-based real-time RT-qPCR is currently the gold standard for COVID-19 diagnosis. However, it is a high-cost assay, inaccessible for the majority of laboratories around the world, making it difficult to diagnose on a large scale. The objective of this study was to standardize lower cost molecular methods for SARS-CoV-2 identification. E gene primers previously determined for TaqMan assays by Colman et al. (2020) were adapted in SYBR Green assay and RT-PCR conventional. The cross-reactivity test was performed with 17 positive samples for other respiratory viruses, and the sensibility test was performed with 8 dilutions (10 based) of SARS-CoV-2 isolated and 63 SARS-CoV-2-positive samples. The SYBR Green assays and conventional RT-PCR have not shown amplification of the 17 respiratory samples positives for other viruses. The SYBR Green-based assay was able to detect all 8 dilutions of the isolate. The conventional PCR detected until 107 dilution, both assays detected the majority of the 63 samples, 98.42% of positivity in SYBR Green, and 93% in conventional PCR. The average Ct variation between SYBR Green and TaqMan was 1.92 and the highest Ct detected by conventional PCR was 35.98. Both of the proposed assays are less sensitive than the current gold standard; however, our data shows a low sensibility variation, suggesting that these methods could be used by laboratories as a lower cost molecular method for SARS-CoV-2 diagnosis.

Aqueous two-phase system antibody confinement enables cost-effective analysis of protein analytes by sandwich enzyme-linked immunosorbent assay with minimal optical crosstalk.

An aqueous two-phase system formed from polyethylene glycol and dextran was used to uniformly coat the bottom surfaces of the wells of standard 96-well assay plates with capture and detection antibodies to improve the performance and cost-effectiveness of sandwich enzyme-linked immunosorbent assay (ELISA). Using this approach, limits of detection and linear dynamic range values comparable to those obtained for conventional sandwich ELISA were obtained using considerably lower antibody quantities due to the much lower reagent volumes required when antibodies are applied in a dextran solution beneath a polyethylene glycol overlay. Confinement of the antibody reagents to the bottom surfaces of the wells within the dextran phase also dramatically decreased the optical crosstalk present between neighboring wells when using transparent microplates. Adaptation of the conventional single sandwich ELISA for aqueous two-phase system antibody confinement was demonstrated by analysis of standard curves for C-reactive protein, transforming growth factor beta 1, and the chemokine CXCL10.

COVID-19 diagnostics in context.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the need for different types of diagnostics, comparative validation of new tests, faster approval by federal agencies, and rapid production of test kits to meet global demands. In this Perspective, we discuss the utility and challenges of current diagnostics for COVID-19.