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COVID-19 first appeared in December of 2019 in Wuhan, China. Since then it has become a global pandemic. A robust and scalable diagnostics strategy is crucial for containing and monitoring the pandemic. RT-PCR is a known, reliable method for COVID-19 diagnostics which can differentiate between SARS-CoV-2 and other viruses. However, PCR is location dependent, time consuming and relatively expensive. Thus, there is a need for a more flexible method which may be produced in an off-the-shelf format and distributed more widely. Paper-based immunoassays can fulfill this function. Here we present the first steps towards a paper-based test which can differentiate between different between the Spike protein of various coronaviruses, SARS-CoV-1, SARS-CoV-2 and CoV-HKU1 with negligible cross reactivity for HCoV-OC43 and HCoV-229E in a single assay which takes less than 30 minutes. Furthermore, our test can distinguish between fractions of the same Spike protein. This is done by an altered assay design with four test line locations where each antigen builds a unique, identifiable binding pattern. The effect of several factors, such as running media, immunoprobe concentration and antigen interference is considered. We find that running media has a significant effect on the final binding pattern where human saliva provides results while human serum leads to the lowest signal quality.

Lateral flow type detection is becoming interesting not only in regions with a poor medical infrastructure but also for practitioners in day-to-day clinical work or for veterinary control in case of possible epidemics. In this work, we describe the first steps of development of a multi-channel strip with potential internal calibration of multiparametric and colorimetric lateral flow assays for the simultaneous detection of the lipopolysaccharides (LPS) of Salmonella typhimurium (S. typhimurium) and Salmonella enteritidis (S. enteritidis). We structured four channels in the nitrocellulose membrane with a Yb:KGW solid-state femtosecond laser ("cold" ablation process) to form distinct tracks of porous material and used gold nanoparticles for the labeling of the antibodies. In addition, calibration curves of the spot intensities of both serovars are presented, and it was shown that no cross reactivity between the different capture antibodies and LPS occurred. Finally, we detected LPS of both Salmonella serovars simultaneously. The color changes (spot intensities of the reaction zones) were evaluated using the open-source image-processing program ImageJ. Graphical abstract Multiparametric testing, strip A was tested with LPS S. enteritidis ( c=0.01 g/L) and LPS S.typhimurium ( c=0.0001 g/L), strip B with LPS S. enteritidis ( c=0.001 g/L) and LPS S. typhimurium ( c=0.001g/L) and strip C with LPS S. enteritidis (c=0.0001 g/L) and LPS S. typhimurium ( c=0.01 g/L), and read-out.

International audience; A recent survey of the European Academy of Allergy and Clinical Immunology (EAACI) Drug Allergy Interest Group (DAIG) on how European allergy specialists deal with beta-lactam (BL) hypersensitivity demonstrated a significant heterogeneity in current practice, suggesting the need to review and update existing EAACI guidelines in order to make the diagnostic procedures as safe and accurate, but also as cost-effective, as possible. For this purpose, a bibliographic search on large studies regarding BL hypersensitivity diagnosis was performed by an EAACI task force, which reviewed and evaluated the literature data using the GRADE system for quality of evidence and strength of recommendation. The updated guidelines provide a risk stratification in BL hypersensitivity according to index reaction(s), as well as an algorithmic approach, based on cross-reactivity studies, in patients with a suspicion of BL hypersensitivity and an immediate need for antibiotic therapy, when referral to an allergist is not feasible. Furthermore, the update addresses availability and concentrations of skin test (ST) reagents, ST and drug provocation test (DPT) protocols, as well as diagnostic algorithms and administration of alternative BL in allergic subjects. Specifically, distinct diagnostic algorithms are suggested depending on risk stratification of the patient into high and low risk based on the morphology and chronology of the reaction, immediate (i.e., occurring within 1 to 6 hours after the last administered dose) or nonimmediate (i.e., occurring more than 1 hour after the initial drug administration), and the reaction severity. Regarding the allergy workup, the main novelty of this document is the fact that in some low-risk nonimmediate reactions ST are not mandatory, especially in children. For DPT, further studies are necessary to provide data supporting the standardization of protocols, especially of those regarding nonimmediate reactions, for which there is currently no consensus.

Highly multiplexed immunoassays could allow convenient screening of hundreds or thousands of protein biomarkers simultaneously in a clinical sample such as serum or plasma, potentially allowing improved diagnostic accuracy and clinical management of many conditions such as autoimmune disorders, infections, and several cancers. Currently, antibody microarray-based tests are limited in part due to cross reactivity from detection antibody reagents. Here we present a strategy that reduces the cross-reactivity between nanoparticle-bound reporter antibodies through the application of ultrasound energy. By this concept, it was possible to achieve a sensitivity 10(3)-fold (5 pg mL(-1)) lower than when no ultrasound was applied (50 ng mL(-1)) for the simultaneous detection of three different antigens. The detection limits and variability achieved with this technique rival those obtained with other types of multiplex sandwich assays.

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.

Introduction Food allergy affects 6-17% of Europeans and in 60% of the cases it is accompanied by inhalation allergy. This is due to two phenomena, namely co-sensitisation and cross-reactivity, which are caused by panallergens, such as lipid transfer proteins (LTP). These proteins are classified as defence proteins PR-14 and are responsible for protecting the plant from bacterial and fungal infections. Clinically, LTP allergy manifests with severe anaphylactic reactions or milder reactions, such as oral allergy syndrome. Aim To assess the incidence of specific IgE to components belonging to LTP based on molecular testing. Material and methods The study was conducted at the Department of Allergy, Clinical Immunology and Internal Diseases in Bydgoszcz, Poland. A total of 50 subjects participated in the study. Each subject had their asIgE levels determined with ImmunoCap ISAC. Results and conclusions Antigen-specific IgE antibodies to LTP components were identified in 24% of the subjects. In subjects with LTP allergy, adverse skin reactions were the most common after consumption of sensitising foods. Peach was the most common food allergen triggering these reactions. The Artemisia vulgaris component Art v 3 was the precursor of LTP allergy in our study population: the highest incidence and the highest mean levels of asIgE were demonstrated for this component.

Since the COVID-19 disease caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) was declared a pandemic, it has spread rapidly, causing one of the most serious outbreaks in the last century. Reliable and rapid diagnostic tests for COVID-19 are crucial to control and manage the outbreak. Here, a label-free square wave voltammetry-based biosensing platform for the detection of SARS-CoV-2 in nasopharyngeal samples is reported. The sensor was constructed on screen-printed carbon electrodes coated with gold nanoparticles. The electrodes were functionalized using 11-mercaptoundecanoic acid (MUA) which was used for the immobilization of an antibody against SARS-CoV-2 nucleocapsid protein (N protein). The binding of the immunosensor with the N protein caused a change in the electrochemical signal. The detection was realised by measuring the change in reduction peak current of a redox couple using square wave voltammetry at 0.04 V versus Ag ref. electrode on the immunosensor upon binding with the N protein. The electrochemical immunosensor showed high sensitivity with a linear range from 1.0 pg.mL−1 to 100 ng.mL−1 and a limit of detection of 0.4 pg.mL−1 for the N protein in PBS buffer pH 7.4. Moreover, the immunosensor did not exhibit significant response with other viruses such as HCoV, MERS-CoV, Flu A and Flu B, indicating the high selectivity of the sensor for SARS-CoV-2. However, cross reactivity of the biosensor with SARS-CoV is indicated, which gives ability of the sensor to detect both SARS-CoV and SARS-CoV-2. The biosensor was successfully applied to detect the SARS-CoV-2 virus in clinical samples showing good correlation between the biosensor response and the RT-PCR cycle threshold values. We believe that the capability of miniaturization, low-cost and fast response of the proposed label-free electrochemical immunosensor will facilitate the point-of-care diagnosis of COVID 19 and help prevent further spread of infection.

Inoculation with vaccine is the major intervention currently used to prevent influenza infections. However, it will be a challenge to produce and implement a new vaccine when a novel highly pathogenic influenza virus emerges in humans as significant infections. H7 subtype influenza viruses have similar epitopes on hemagglutinin, which can induce cross-reactive antibodies. In this study, a meta-analysis of the cross-reactivity of antibodies induced by one H7 subtype influenza vaccine against other H7 subtypes was performed. Database search was conducted in PubMed, Cochrane Library, EMBASE, MEDLINE, Chinese Biological Medicine Database (CBM), and Wanfang. A total of 9 articles comprising 811 human subjects were included in this meta-analysis. All assessed H7 influenza vaccines induced vaccine strain-specific protective antibodies [seroconversion rate (SCR) = 0.74, 95% CI (0.65, 0.82); seroprotection rate (SPR) = 0.81, 95% CI (0.78, 0.83)]. All H7 influenza virus monovalent vaccines exhibited cross-reactivity tested by hemagglutinin inhibition test (HI), microneutralization test (MN) and immunosorbent assay (ELISA) to other H7 subtype viruses. H7N1, H7N3, H7N7, and H7N9 vaccines elicited cross-reactive antibodies against other H7 subtype influenza viruses [SCR = 0.66, 95% CI (0.50, 0.82); SPR = 0.79, 95% CI (0.67, 0.91)]. The pooled SCR (95%CI) of cross-reactivity of H7N1 and H7N3 vaccines were 0.88 (0.85, 0.91) and 0.40 (0.26, 0.54), respectively. The consolidated SPR (95%CI) of H7N1 and H7N7 vaccines were 0.89 (0.86, 0.92) and 0.93 (0.81, 1.06). All H7 vaccines induced cross-reactive antibodies against H7N9 viruses [SCR = 0.69, 95% CI (0.52, 0.86); SPR = 0.85, 95% CI (0.76, 0.94)]. H7 vaccines can be used to limit influenza infection when a new highly pathogenic H7 virus appears.

This study assessed long-term immunogenicity and safety following 3 doses of AS04-adjuvanted human papillomavirus (HPV)-16/18 L1 virus-like particle (VLP) vaccine in females 10–14 years old. Girls included in the immunogenicity subset in the primary controlled, observer-blinded, randomized study (NCT00196924) who received 3 doses were invited for a 10-year follow-up (NCT00316706 and NCT00877877). Serum antibody responses against HPV-16/18 (vaccine types) and HPV-31/45 (non-vaccine types) were measured by enzyme-linked immunosorbent assay (ELISA) using type-specific VLP as coating antigens. Serious adverse events (SAEs) and pregnancy information were recorded. At Month (M) 120, all subjects (N = 418, according-to-protocol immunogenicity cohort) were seropositive for anti-HPV-16/18 antibodies. Geometric mean titers (GMTs) were 1589.9 ELISA Units [EU]/mL (95% confidence interval [CI]: 1459.8–1731.6) for anti-HPV-16 and 597.2 EU/mL (95% CI: 541.7–658.5) for anti-HPV-18 in subjects seronegative at baseline for the type analyzed. Post hoc mathematical modeling predicted a durability ≥50 years for anti-HPV-16 and anti-HPV-18. For the non-vaccine humoral type response, all initially seronegative subjects had seroconverted at M7, with anti-HPV-31 GMT of 2030.5 EU/mL (95% CI: 1766.2–2334.4) and anti-HPV-45 GMT of 2300.8 EU/mL (95% CI: 2036.8–2599.0). At M120, 87.7% and 85.1% remained seropositive for anti-HPV-31 with GMT of 242.9 EU/mL (95% CI: 201.4–293.0) and anti-HPV-45 with GMT of 204.7 EU/mL (95% CI: 170.0–246.6). During the 10-year follow-up, no SAEs or abnormal pregnancy outcomes were causally related to vaccination. Three doses of the AS04-HPV-16/18 vaccine induced high and sustained antibody response against HPV-16,18,31 and 45 in girls aged 10–14 years during the 10-year follow-up, with an acceptable long-term safety profile.

Background Chronic hepatitis B virus (HBV) infections are a global health problem. There is a need for therapeutic strategies blocking continuous infection of liver cells. The grass pollen allergy vaccine BM32 containing the preS domain of the large HBV surface protein (LHBs) as immunogenic carrier induced IgG antibodies in human subjects inhibiting HBV infection in vitro. Aim of this study was the quantification, epitope mapping and investigation of HBV genotype cross-reactivity of preS-specific antibodies in subjects treated with different dosage regimens of BM32 Methods Hundred twenty eight grass pollen allergic patients received in a double-blind, placebo-controlled trial five monthly injections of placebo (aluminum hydroxide, n= 34) or different courses of BM32 (2 placebo + 3 BM32, n= 33; 1 placebo + 4 BM32, n= 30; 5 BM32, n= 31). Recombinant Escherichia coli-expressed preS was purified. Overlapping peptides spanning preS and the receptor-binding sites from consensus sequences of genotypes A–H were synthesized and purified. Isotype (IgM, IgG, IgA, IgE) and IgG subclass (IgG1-IgG4) responses to preS and peptides were determined by ELISA at baseline, one and four months after the last injection. IgG1 and IgG4 subclass concentrations specific for preS and the receptor-binding site were measured by quantitative ELISA. Findings Five monthly injections induced the highest levels of preS-specific IgG consisting mainly of IgG1 and IgG4, with a sum of median preS-specific IgG1 and IgG4 concentrations of >135 μg/ml reaching up to 1.8 mg/ml. More than 20% of preS-specific IgG was directed against the receptor-binding site. BM32-induced IgG cross-reacted with the receptor-binding domains from all eight HBV genotypes A-H. Interpretation BM32 induces high levels of IgG1 and IgG4 antibodies against the receptor binding sites of all eight HBV genotypes and hence might be suitable for therapeutic HBV vaccination. Funding This study was supported by the PhD program IAI (KPW01212FW), by Viravaxx AG and by the Danube-ARC funded by the Government of Lower Austria. Rudolf Valenta is a recipient of a Megagrant of the Government of the Russian Federation, grant No 14.W03.31.0024.

Bioinformatic amino acid sequence searches are used, in part, to assess the potential allergenic risk of newly expressed proteins in genetically engineered crops. Previous work has demonstrated that the searches required by government regulatory agencies falsely implicate many proteins from rarely allergenic crops as an allergenic risk. However, many proteins are found in crops at concentrations that may be insufficient to cause allergy. Here we used a recently developed set of high-abundance non-allergenic proteins to determine the false-positive rates for several algorithms required by regulatory bodies, and also for an alternative 1:1 FASTA approach previously found to be equally sensitive to the official sliding-window method, but far more selective. The current investigation confirms these earlier findings while addressing dietary exposure.

Biological drugs like therapeutic antibodies are widely used for the treatment of various diseases like inflammatory disorders and cancer. A drawback of these novel treatments is the substantial proportion of patients experiencing adverse reactions such as loss-of-drug effect or hypersensitivity reactions. These reactions are associated with pre-existing and/or developing anti-drug antibodies. Especially IgE development is a risk factor for life-threatening systemic anaphylaxis. Methods: In order to characterize the individual drug-specific serum IgE, an IgE cross-reactivity immune profiling (ICRIP) assay was developed. Individual IgG epitopes of anti-drug antibodies against adalimumab were identified by epitope mapping via peptide microarray. Results: ICRIP analyses of sera from patients treated with the therapeutic antibodies adalimumab (ADL) and infliximab (IFX) reveal individual, distinct IgE binding patterns. IgG epitopes were identified mostly located in the variable region of ADL. Conclusions: Using ICRIP and peptide microarrays for pharmacovigilance of the TNF-α blockers IFX and ADL, risk factors and biomarkers before and during therapy shall be identified. These diagnostic systems provide the basis for a safe and efficacious therapy decision for each patient in cases of adverse drug reactions mediated by different types of anti-drug antibodies.

Infection with one of the four dengue virus serotypes (DENV1-4) presumably leads to lifelong immunity against the infecting serotype but not against heterotypic reinfection, resulting in a greater risk of developing Dengue Hemorrhagic Fever/Dengue Shock Syndrome (DHF/DSS) during secondary infection. Both antibodies and T cell responses have been implicated in DHF/DSS pathogenesis. According to the T cell-based hypothesis termed “original antigenic sin,” secondary DENV infection is dominated by non-protective, cross-reactive T cells that elicit an aberrant immune response. The goal of our study was to compare the roles of serotype-specific and cross-reactive T cells in protection vs. pathogenesis during DENV infection in vivo. Specifically, we utilized IFN-α/βR−/− HLA*B0702 transgenic mice in the context of peptide vaccination with relevant human CD8 T cell epitopes. IFN-α/βR−/− HLA*B0702 transgenic mice were immunized with DENV serotype 2 (DENV2)-specific epitopes or variants found in any of the other three serotypes (DENV1, DENV3 or DENV4), followed by challenge with DENV. Although cross-reactive T cell responses were lower than responses elicited by serotype-specific T cells, immunization with either serotype-specific or variant peptide epitopes enhanced viral clearance, demonstrating that both serotype-specific and cross-reactive T cells can contribute to protection in vivo against DENV infection., Highlights • Serotype-cross-reactive CD8 T cells elicit a polyfunctional immune response similar to the response elicited by serotype-specific CD8 T cells. • Serotype cross-reactive CD8 T cells play a role in protection against DENV infection in a HLA-B*0702 Transgenic IFN-α/βR−/− mouse model. There are four major subtypes (serotypes) of the mosquito-borne Dengue virus. Infection with a first serotype is generally asymptomatic, but secondary infection with a different serotype is capable of causing severe disease. T cells previously exposed to a first serotype and which produce an immune response to a second serotype are said to be cross-reactive. Using a mouse model engineered with human T cell features, we characterized the cross-reactive T cell response to live dengue virus serotypes and viral protein fragments. Our results suggested cross-reactive T cells contribute to control of and protection against infection by a second dengue serotype, rather than leading to more severe disease.

Biomarkers may hold the key towards development and improvement of personalized cancer treatment. For instance, tumour expression of immune system-related proteins may reveal the tumour immune status and, accordingly, determine choice for type of immunotherapy. Therefore, objective evaluation of tumour biomarker expression is needed but often challenging. For instance, human leukocyte antigen (HLA) class I tumour epithelium expression is cumbersome to quantify by eye due to its presence on both tumour epithelial cells and tumour stromal cells, as well as tumour-infiltrating immune cells. In this study, we solved this problem by setting up an immunohistochemical (IHC) double staining using a tissue microarray (TMA) of rectal tumours wherein HLA class I expression was coloured with a blue chromogen, whereas non-epithelial tissue was visualized with a brown chromogen. We subsequently developed a semi-automated image analysis method that identified tumour epithelium as well as the percentage of HLA class I-positive tumour epithelium. Using this technique, we compared HCA2/HC10 and EMR8-5 antibodies for the assessment of HLA class I tumour expression and concluded that EMR8-5 is the superior antibody for this purpose. This IHC double staining can in principle be used for scoring of any biomarker expressed by tumour epithelium.

Autoimmunity is on the rise around the globe. Diet has been proposed as a risk factor for autoimmunity and shown to modulate the severity of several autoimmune disorders. Yet, the interaction between diet and autoimmunity in humans remains largely unstudied. Here, we systematically interrogated commonly consumed animals and plants for peptide epitopes previously implicated in human autoimmune disease. A total of fourteen species investigated could be divided into three broad categories regarding their content in human autoimmune epitopes, which we represented using a new metric, the Gershteyn-Ferreira index (GF index). Strikingly, pig contains a disproportionately high number of unique autoimmune epitopes compared to all other species analyzed. This work uncovers a potential new link between pork consumption and autoimmunity in humans and lays the foundation for future studies on the impact of diet on the pathogenesis and progression of autoimmune disorders., Highlights • We developed a comprehensive database of dietary-human autoimmune epitope overlap. • The Gershteyn-Ferreira index divides dietary antigens into three main groups with respect to overlap with human autoimmune epitopes. • Pig contains a disproportionately high number of unique human autoimmune epitopes.

To characterize the antigenicity of nucleocapsid proteins (NP) derived from canine coronavirus (CCoV) and canine respiratory coronavirus (CRCoV) in China, the N genes of CCoV (CCoV-BJ70) and CRCoV (CRCoV-BJ202) were cloned from swabs obtained from diseased pet dogs in Beijing and then sequenced. The recombinant NPs (rNPs) were expressed in Escherichia coli and purified by nickel-affinity column and size exclusion chromatography. Sequencing data indicated that the N genes of CCoV-BJ70 and CRCoV-BJ202 belonging to two distinctly different groups were relatively conserved within each subgroup. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that rNPs of CCoV and CRCoV were expressed efficiently and isolated with a final purity of over 95%. Western blot analysis revealed the rNP from CRCoV could cross-react with mice antisera against human coronavirus (HCoV-229E, NL63, OC43, HKU1), while rNP of CCoV had cross-reactivity with only anti-sera against viruses belonging to the same group (HCoV-229E and NL63). In summary, CCoV and CRCoV rNPs were successfully expressed in E. coli and showed antigenic cross-reactivity with antisera raised against human coronaviruses. These findings indicate that further serologic studies on coronavirus infections at the animal-human interface are needed.

Anflu® is a seasonal trivalent inactivated split-virion influenza vaccine manufactured by Sinovac Biotech Co., Ltd. The objectives of this study were to evaluate the safety of Anflu® (2013–14 formulation: H1N1, H3N2 and BYAM) in infants and adults and its immunogenicity and cross-reactivity against mismatched influenza B lineage and avian influenza A(H7N9) viruses (hereafter BVIC and H7N9, respectively) in adults. In this phase IV open label trial, infants 6–35 months old (n=61) each received two injections with 28 days apart; adults 18–60 yrs old (n=60) and elderly >60 yrs old (n=61) each received one injection. Information of adverse events was collected through safety observation and follow-up visits. Pre- and post-immune blood samples (day 0 and 21) were collected from subjects ≥18 yrs old to detect hemagglutination inhibition antibody titers and calculate seroprotection rates (SPRs) and seroconversion rates (SCRs). The overall adverse reaction incidence was 1.6% (3/182), and no serious adverse event was reported during the study period. For subjects ≥18 yrs old, the SCRs, SPRs, and the geometric mean titers (GMTs) met the European criteria for all three strains. In addition, the point estimations of SCR, SPR and GMT for BVIC also met the European criteria. Six subjects were seroconverted against H7N9; however the serological results did not meet the European criteria. In conclusion, the results showed a satisfactory safety and immunogenicity profile of Anflu® and cross-reactivity against BVIC, but did not demonstrate cross-reactivity against H7N9 (Clinicaltrials.gov ID: NCT02269852).

Introduction Influenza A is known as a public health concern worldwide. In this study, a novel one-step real-time reverse transcription polymerase chain reaction (rtRT-PCR) assay was designed and optimized for the detection of influenza A viruses. Material and methods The primers and probe were designed based on the analysis of 90 matrix nucleotide sequence data of influenza type A subtypes from the GenBank database of the National Center for Biotechnology Information (NCBI). The influenza virus A/Tehran/5652/2010 (H1N1 pdm09) was used as a reference. The rtRT-PCR assay was optimized, compared with that of the World Health Organization (WHO), and its analytical sensitivity, specificity and reproducibility were evaluated. In total, 64 nasopharyngeal swabs from patients with influenza-like illness (ILI) and 41 samples without ILI symptoms were tested for the virus, using conventional cell culture, direct immunofluorescence antibody (DFA) methods, and one-step rtRT-PCR with the designed primer set and probe and the WHO’s. Results The optimized assay results were similar to the WHO’s. The optimized assay results were similar to WHO’s, with non-significant differences for 10–103 copies of viral RNA/reaction (p > 0.05). It detected 10 copies of viral RNA/reaction with high reproducibility and no cross reactivity with other respiratory viruses. A specific cytopathic effect was observed in 6/64 (9.37%) of the ILI group using conventional culture and DFA staining methods; however, it was not seen in non-ILI. Also, the results of our assay and the WHO’s were similar to those of viral isolation and DFA staining. Conclusions Given the high specificity, sensitivity and reproducibility of this novel assay, it can serve as a reliable diagnostic tool for the detection of influenza A viruses in clinical specimens and lab experiments.

Infections caused by Fasciola hepatica are of great importance in the veterinary field, as they cause important economic losses to livestock producers. Serodiagnostic methods, typically ELISA (with either native or recombinant antigens), are often used for early diagnosis. The use of native antigens, as in the MM3-SERO ELISA (commercialized as BIO K 211, BIO-X Diagnostics), continues to be beneficial in terms of sensitivity and specificity; however, there is interest in developing ELISA tests based on recombinant antigens to avoid the need to culture parasites. Of the antigens secreted by adult flukes, recombinant procathepsin L1 (rFhpCL1) is the most commonly tested in ELISA to date. However, although adult flukes produce three different clades of CLs (FhCL1, FhCL2, and FhCL5), to our knowledge, the diagnostic value of recombinant FhCL2 and FhCL5 has not yet been investigated. In the present study, we developed and tested three indirect ELISAs using rFhpCL1, rFhpCL2, and rFhpCL5 and evaluated their recognition by sera from sheep and cattle naturally infected with F. hepatica. Although the overall antibody response to these three rFhpCLs was similar, some animals displayed preferential recognition for particular rFhpCLs. Moreover, for cattle sera, the highest sensitivity was obtained using rFhpCL2 (97%), being equal for both rFhpCL1 and rFhpCL5 (87.9%), after adjusting cut-offs for maximum specificity. By contrast, for sheep sera, the sensitivity was 100% for the three rFhpCLs. Finally, the presence of truncated and/or partially unfolded molecules in antigen preparations is postulated as a possible source of cross-reactivity.

Healthcare workers (HCW) were prioritized for vaccination during the 2009 influenza A(H1N1)pdm09 pandemic. We conducted a clinical trial in October 2009 where 237 HCWs were immunized with a AS03-adjuvanted A(H1N1)pdm09 monovalent vaccine. In the current study, we analyzed the homologous and cross-reactive H1N1 humoral responses using prototype vaccine strains dating back to 1977 by the haemagglutinin inhibition (HI), single radial hemolysis SRH), antibody secreting cell (ASC) and memory B cell (MBC) assays. The cellular responses were assessed by interferon-γ (IFN-γ) ELISPOT and by intracellular staining (ICS) for the Th1 cytokines IFN-γ, interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α). All assays were performed using blood samples obtained prior to (day 0) and 7, 14 and 21 d post-pandemic vaccination, except for ASC (day 7) and ICS (days 0 and 21). Vaccination elicited rapid HI, SRH and ASC responses against A(H1N1)pdm09 which cross reacted with seasonal H1N1 strains. MBC responses were detected against the homologous and seasonal H1N1 strains before vaccination and were boosted 2 weeks post-vaccination. An increase in cellular responses as determined by IFN-γ ELISPOT and ICS were observed 1–3 weeks after vaccination. Collectively, our data show that the AS03-adjuvanted A(H1N1)pdm09 vaccine induced rapid cellular and humoral responses against the vaccine strain and the response cross-reacted against prototype H1N1 strains dating back to 1977. publishedVersion

According to several recent studies, an unexpectedly high number of landmark papers seem to be not reproducible by independent laboratories. Nontherapeutic antibodies used for research, diagnostic, food analytical, environmental, and other purposes play a significant role in this matter. Although some papers have been published offering suggestions to improve the situation, they do not seem to be comprehensive enough to cover the full complexity of this issue. In addition, no obvious improvements could be noticed in the field as yet. This article tries to consolidate the remarkable variety of conclusions and suggested activities into a more coherent conception. It is concluded that funding agencies and journal publishers need to take first and immediate measures to resolve these problems and lead the way to a more sustainable way of bioanalytical research, on which all can rely with confidence.

The phenomenon of cross-reactivity between allergenic proteins plays an important role to understand how the immune system recognizes different antigen proteins. Allergen proteins are known to cross-react if their sequence comparison shows a high sequence identity which also implies that the proteins have a similar 3D fold. In such cases, linear sequence alignment methods are frequently used to predict cross-reactivity between allergenic proteins. However, the prediction of cross-reactivity between distantly related allergens continues to be a challenging task. To overcome this problem, we developed a new structure-based computational method, Cross-React, to predict cross-reactivity between allergenic proteins available in the Structural Database of Allergens (SDAP). Our method is based on the hypothesis that we can find surface patches on 3D structures of potential allergens with amino acid compositions similar to an epitope in a known allergen. We applied the Cross-React method to a diverse set of seven allergens, and successfully identified several cross-reactive allergens with high to moderate sequence identity which have also been experimentally shown to cross-react. Based on these findings, we suggest that Cross-React can be used as a predictive tool to assess protein allergenicity and cross-reactivity. Availability and Implementation: Cross-React is available at: http://curie.utmb.edu/Cross-React.html

In rabies endemic countries, funds and infrastructure are often insufficient to employ the approved gold standard for the definitive diagnosis of rabies: the direct fluorescent test. In the present study, two types (type 1 and 2) of an ICT kit were evaluated for detection of rabies. These were developed using monoclonal antibodies which recognize epitope II and III of the nucleoprotein of rabies virus. Both kits specifically detected all rabies virus strains and there was no cross reactivity with Lyssaviruses (Lagos, Mokola and Duvenhage), Rhabdovirus (VSV and Oita 296/1972) and other common canine‐pathogenic viruses. In type 1, a single type of monoclonal antibody was used. It was capable of detecting recombinant nucleoprotein and showed sensitivity of 95.5% (42/44) and specificity of 88.9% (32/36) using brain samples from rabid dogs. In contrast, type 2 which was made of two different monoclonal antibodies had a lower sensitivity of 93.2% (41/44) and higher specificity of 100% (36/36). These ICT kits provide a simple and rapid method for rabies detection. They need neither cold chain for transportation nor complicated training for personnel. This diagnostic test is suitable for rabies screening, particularly in areas with a high prevalence of rabies and where the fluorescent antibody test is not available.

Introduction : Differentiating between cross-reactivity and double sensitization is still a challenging issue in allergology. Aim : To differentiate cross-reactions accompanying latex allergy with the use of the ISAC test. Material and methods : Thirty-nine patients reporting immediate allergic reactions to latex were enrolled into the study (group A). The control group was comprised of 41 patients with allergic diseases not associated with latex (group B) and 20 healthy individuals (group C). Their history was recorded and skin prick tests were performed with latex, airborne and food allergens. Specific IgE against food allergens, latex (k82) and recombined latex allergens were determined. Immun o CAP ISAC test was performed with 103 molecules. Results : Sensitization to latex was found by means of skin tests in 16 cases and sIgE against latex was revealed in 12 cases (including 10 positive in both SPT and sIgE). In the ISAC test antibodies against recombined latex allergens were found in 8 patients with rHev β 6 as the most common. All the patients positive for rHev β 1, 5, 6, 8 had allergy or asymptomatic sensitization to food allergens cross-reacting with latex. Some reactions could not have been differentiated due to the lack of allergens in the ISAC test. Others, not related to latex-fruits syndrome were explained by cross-reactivity with other profilins or PR-10 proteins. Conclusions : Immun o CAP ISAC test could be useful in differentiating between cross-reactions and double sensitizations. However, in the case of latex its advantages are limited due to a small panel of allergens.

BACKGROUND—Pigeon breeder's disease is an extrinsic allergic alveolitis in the lungs of sensitised people, caused by hypersensitivity reactions to inhaled pigeon antigens. Antigens from different sources of the animal are used for diagnostic purposes, with serum being the most widely used. Bloom is rarely used; very little is known of its antigenicity and diagnostic performance, particularly when used with the enzyme linked immunosorbent assay (ELISA) method, which is the most popular test as it permits measurement of the antibody response. METHODS—To (a) standardise an ELISA for the measurement of specific IgG against pigeon serum and pigeon bloom extract; (b) to establish reference values for specific IgG in 73 non-exposed controls, (c) to show the presence of specific IgG against pigeon serum and bloom in serum samples of 17 patients with bird fancier's lung and 11 asymptomatic fanciers, and (d) to study the similarity of the two antigen sources by cross reactivity experiments. RESULTS—Reference values of specific IgG were defined with the 97.5 percentile (367.9 U/ml for pigeon serum and 953.7 U/ml for pigeon bloom extract). Of symptomatic patients 100% had values higher than the cut off for both antigens. In asymptomatic fanciers values were higher than the cut off for pigeon serum in 45% and bloom extract in 54%. Cross reactivity experiments showed that the two antigens differed in antigenic content although some components may be common to both. CONCLUSION—The ELISA methods used proved to be useful tools for evaluating specific IgG antibody responses against both antigens. The diagnostic performance of both ELISA methods performed with these antigen sources was similar, showing very high sensitivity but moderate specificity. Although some antigenic similarity was found between pigeon serum and bloom extract, cross reactivity studies showed that various antigens seemed to be specific to the bloom extract. However, the antigens responsible for pigeon breeder's disease seem to be present in both antigenic sources. Keywords: antibodies; pigeons; ELISA

In the past three decades, ten H1 subtype influenza vaccines have been recommended for global seasonal flu vaccination. Some of them were used only for one year before being replaced by another H1 flu vaccine while others may be used for up to seven years. While the selection of a new seasonal flu vaccine was based on the escape of a new emerging virus that was not effectively protected by the existing flu formulation, there is limited information on the magnitude and breadth of cross reactivity among H1 subtype virus circulation over a long period. In the current study, HA-expressing DNA vaccines were constructed to express individual HA antigens from H1 subtype vaccines used in the past 30 y. Rabbits naive to HA antibody responses were immunized with these HA DNA vaccines and the cross reactivity of these sera against HA antigen and related H1 viruses in the same period was studied. Our data indicate that the level of cross reactivity was different for different viral isolates and the key mutations responsible for the cross reactivity may involve only a limited number of residues. Our results provide useful information for the development of improved seasonal vaccines than can achieve broad protection against viruses within the same H1 subtype.

AIM To determine if the diuretic spironolactone cross reacts with 17α-hydroxyprogesterone (17OHP) in an enzyme linked immunosorbent assay (ELISA) kit used for the mass screening of congenital adrenal hyperplasia. METHODS Concentrations of 17OHP on a blood filter paper disc were measured using an ELISA kit (kit C-7: ENZAPLATE N-17α -OHP-7; Chiron, Tokyo, Japan). The cross reactivity of spironolactone and its metabolites with 17OHP was determined. The concentrations of spironolactone and its metabolites in blood were measured using HPLC (high performance liquid chromatography). RESULTS Spironolactone cross reacted with 17OHP using kit C-7 (0.01%), by increasing 17OHP concentration in a dose dependent manner. The blood concentration of spironolactone and its metabolites was nearly 900 ng/ml, high enough to show an additive effect on the 17OHP concentration. About 12% of the false positive cases screened using the kit were due to the administration of spironolactone. CONCLUSIONS Spironolactone interferes with 17OHP concentrations, leading to false positive test results for CAH.

Background: Examination of IgE cross-reactivity among nuts has been limited to in vitro experiments. Cross-reactivity studies of nuts at the T cell level are difficult to interpret because of the inability to determine which cellular responses are from a true sensitization and which are due to cross-reactivity. Using a mouse model in which the sensitizing nuts are controlled may provide novel methods to investigate in vivo and T cell cross-reactivity. Methods: C3H/HeJ mice were sensitized by intraperitoneal injection of cashew alone (monosensitized mice), or cashew plus walnut, utilizing alum as an adjuvant. Both groups underwent challenges to cashew, walnut and peanut, with subsequent monitoring of anaphylactic reactions. Anaphylactic antibodies were quantified by ELISA, and protein allergens were identified by Western blotting. Cellular responses were studied via splenocyte proliferation assay and measurement of secreted cytokines. Results: The monosensitized mice reacted to cashew and walnut during challenges, with significantly weaker reactions induced on challenge with peanut. Cross-reactive IgE to walnut and peanut were detected by ELISA, and the cross-reactive allergens were identified as vicilin proteins. In cellular assays, splenocytes from the monosensitized mice proliferated and produced IL-4 and IL-5 in response to cashew, walnut and peanut. The cashew- plus walnut-sensitized mice experienced stronger clinical reactions to walnut, recognized additional walnut allergens and secreted significantly more IL-4 and IL-5 in walnut-stimulated splenocyte assays compared to the monosensitized mice. Conclusions: Cross-reactivity in vivo was found between cashew and walnut, while cross-reactivity among cashew, walnut and peanut was demonstrated at the T cell level.

Glycyrrhizic acid (GL) is a major active compound of licorice. The specific monoclonal antibody (MAb) (designated as 8F8A8H42H7) against GL was produced with the immunogen GL–BSA conjugate. The dissociation constant (K d) value of the MAb was approximately 9.96×10−10 M. The cross reactivity of the MAb with glycyrrhetic acid was approximately 2.6%. The conventional indirect competitive enzyme-linked immunosorbent assay (icELISA) and simplified icELISA adapted with a modified procedure were established using the MAb. The IC50 value and the detect range by the conventional icELISA were 1.1 ng mL−1 and 0.2–5.1 ng mL−1, respectively. The IC50 value and the detect range by the simplified icELISA were 5.3 ng mL−1 and 1.2–23.8 ng mL−1, respectively. The two icELISA formats were used to analyze GL contents in the roots of wild licorice and different parts of cultivated licorice (Glycyrrhiza uralensis Fisch). The results obtained with the two icELISAs agreed well with those of the HPLC analysis. The correlation coefficient was more than 0.98 between HPLC and the two icELISAs. The two icELISAs were shown to be appropriate, simple, and effective for the quality control of raw licorice root materials.

A complex and varied antibody response to allergens, in particular Dermatophagoides pteronyssinus, is observed in the serum of allergic patients. To examine the role of cross-reacting antigenic determinants in allergy, a strategy has been devised for isolating antibody populations that bind to individual polypeptides of an allergen source separated by sodium dodecyl sulfate-polyacrylamide electrophoresis and electroblotted to nitrocellulose. Polypeptides are identified on the blots, desorbed and coupled to CNBr-activated paper discs. The coupled discs are then used as immunoadsorbent surfaces for the binding of antibodies from allergic sera. Antibodies eluted from the polypeptide immunoadsorbents are then used to reprobe protein blots of the allergen. Results are presented which demonstrate strong cross-reaction between two major mite allergens, Der p I and Der p III, and some weaker intra-species cross-reactivities.

RAST-inhibition studies were performed by use of whole venom sac extracts to inhibit binding to purified venom allergens from various vespid wasps. Immunodiffusion studies were also performed with rabbit antisera raised against purified venom proteins. Immunologic cross-reactivity was demonstrated among the hyaluronidases, phospholipases, and antigen 5 s from both subgenera of yellow jackets, white-faced hornets, and paper wasps. The paper wasp hyaluronidase and antigen 5 were less related to those of the other three species than those of yellow jackets and hornets. It appears that immunologic cross-reactivity is the major mechanism of multiple allergic sensitivity to vespid venoms. Therapy with only the primary venom should be sufficient to protect against reactions from cross-reacting venoms.

We have used the RAST-inhibition technique in homologous and heterologous inhibitions of the Aspergillus fumigatus, Alternaria tenius and Cladosporium herbarum RAST assays followed by the immunoblot technique to assess the degree of shared allergenic determinants in these extracts. In the Aspergillus RAST, there was little or no inhibition with Cladosporium and Alternaria , but considerable cross-reactivity was found between Alternaria and Cladosporium . Inhibition by Alternaria of the Cladosporium RAST was found in a dose-dependent fashion in on serum, whereas all four sera in the Alternaria RAST were inhibited by Cladosporium in this fashion. Logit transformation of the Alternaria RAST-inhibition curves produced common slopes with Alternaria and Cladosporium in three of the four sera, indicating their immunologic identity. The immunoblot technique confirmed the degree of cross-reactivity found by RAST inhibition among the molds. This evidence of common allergenic determinants in these fungi could help to explain the observation that mold-allergic patients often have skin test reactions to several fungi.

Summary Bovine herpesvirus type 1 (BHV-1) glycoprotein gIV was purified by affinity chromatography. Purified preparations showed two distinct components of 71 K and 140 K following electrophoresis in sodium dodecyl sulphate polyacrylamide gels. The polypeptides were separated, excised from the gel and used to immunize rabbits; the resulting antisera showed a high degree of cross reactivity indicating that these polypeptides represent monomeric and dimeric forms of the same glycoprotein. Purified gIV was also used to develop a gIV-specific panel of monoclonal antibodies. Neutralizing monoclonal antibodies directed against gIV were conjugated to horseradish peroxidase and subjected to competition binding assays by ELISA. Three distinct neutralizing antigenic domains on gIV were identified. Domain 1 comprised two overlapping epitopes, whereas domain 2 was represented by a single monoclonal antibody. The third antigenic domain was made up of a complex of four identical or overlapping epitopes designated 3a, b, c, and d. Evidence is presented suggesting that domain 1 of gIV may be involved in penetration of the virus into the cell.

Summary A possible antigenic relationship between the porcine enteropathogenic coronavirus-like agent (CVLA) and 6 known coronaviruses was examined by immunoelectron microscopy (IEM) and by immunofluorescence (IF). CVLA did not show cross reactivity with infectious bronchitis virus, transmissible gastroenteritis virus (TGEV), canine coronavirus (CCV) hemagglutinating encephalomyelitis virus (HEV), neonatal calf diarrhea coronavirus (NCDCV) or feline infectious peritonitis virus (FIPV). Antigenic relationship was detected by IEM between TGEV and CCV, NCDCV and HEV and by IF between TGEV and CCV, TGEV and FIPV, HEV and NCDCV.